Photophysiology. General Principles; Action of Light on Plants

Contributors to Volume I

MARY BELLE ALLEN L. R. BLINKS M. S. BLOIS, JR. W I N S L O W R. BRIGGS

STIG CLAESSON RODERICK K. CLAYTON ARTHUR C. GIESE J. WOODLAND HASTINGS STERLING B. HENDRICKS M. LOSADA A. D. MCLAREN HEMMING I. VIRGIN E. C. WEAVER F. R. WHATLEY

PHOTOPHYSIOLOGY Edited by

Arthur C. Giese Department of Biological Sciences

Stanford University, California

Volume I

General Principles; Action of Light on Plants

1964

ACADEMIC PRESS · NEW YORK and LONDON

C O P Y R I G H T © 1964, BY ACADEMIC P R E S S I N C .

ALL RIGHTS RESERVED.

NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,

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LIBRARY OF CONGRESS CATALOG CARD NUMBER: 63-16961

PRINTED IN THE UNITED STATES OF AMERICA

LIST OF CONTRIBUTORS

Numbers in parentheses indicate the pages on which the author's contribution begins.

MARY BELLE ALLEN, Kaiser Foundation Research Institute, Laboratory of Comparative Biology, Richmond, California (83)

L. R. BLINKS, Hopkins Marine Station of Stanford University, Pacific Grove, California (199)

M. S. BLOIS, JR., Biophysics Laboratory, Stanford University, Stanford, California (35)

WINSLOW R. BRIGGS, Department of Biological Sciences, Stanford Uni-versity, Stanford, California (223)

STIG CLAESSON, Institute of Physical Chemistry, University of Uppsala, Uppsala, Sweden (19)

RODERICK K. CLAYTON, C. F. Kettering Research Laboratory, Yellow Springs, Ohio (155)

ARTHUR C. GIESE, Department of Biological Sciences, Stanford Univer-sity, Stanford, California (1)

J. WOODLAND HASTINGS, Biochemistry Division, University of Illinois, Urbana, Illinois (333)

STERLING B. HENDRICKS, Mineral Nutrition Laboratory, Agricultural Re-search Service, U. S. Department of Agriculture, Beltsville, Mary-land (305)

M. LOSADA, Department of Cell Physiology, University of California, Berkeley, California (111)

A. D. MCLAREN, College of Agriculture, University of California, Berkeley, California (65)

HEMMING I. VIRGIN, Department of Plant Physiology, University of Gothenburg, Gothenburg, Sweden (273)

E. C. WEAVER, Department of Plant Biology, Carnegie Institute of Washington, Stanford, California (35)

F. R. WHATLEY, Department of Cell Physiology, University of Cali-fornia, Berkeley, California (111)

v

PHOTOPHYSIOLOGY—PHYSIOLOGY OF PHOTIC REACTIONS OF ORGANISMS

Preface

Photophysiology,* as here conceived, is a study of the physiology of action of non-ionizing radiations (ultraviolet visible and infrared) upon living things. Photobiology is a more inclusive term and has come to mean any studies on the action of non-ionizing radiations upon organ-isms (e.g., Pincussen, 1930), while the more inclusive field of radiation biology considers the action upon life of all types of radiations, both non-ionizing and ionizing (e.g., gamma and X-rays, alpha particles, elec-trons and neutrons), the latter branch sometimes being called radio-biology. The focus of the book is upon the fundamental mechanisms by which non-ionizing radiations affect the living cell, at the molecular level when such analysis is possible.

I t is self evident that not all of the subject matter in photophysiology can possibly be documented in the space of this book. Rather, the topics have been selected to illustrate the principles of photophysiology, although by this very selection some interesting subjects will have been omitted. For completeness, references to these are given whenever possible.

Intensive researches on radiation biology have resulted in appearance of many good books which treat various aspects of photobiological mate-rial. However, a need was felt for a book which introduces and develops some of the major themes of photophysiology. The original literature has now become sufficiently complex and extensive to justify such an introduction. If this account serves to stimulate others to participate in studies of photobiological problems the aims of the book will have been achieved.

The chapters in the present volume begin with an outline of the principles of photochemistry (Chapter 1), and continue with basic mechanisms which underlie action of light on chemical and biological systems (Chapters 2, 3, 4). Then are considered photochemical and

* I am indebted for the title to Professor L. R. Blinks who, during a conversation concerning a suitable title to cover the subject matter of the book, said: "We have such terms as electrophysiology and neurophysiology, why not photophysiology"? This title seemed most apt of the many considered.

vu

Vlll PREFACE

physical aspects of photosynthesis, accessory pigments in photosynthesis (Chapters 5, 6, 7), phototropism and other photoreactions in plants (Chapters 8, 9), photoperiodicity in plants and animals (Chapters 10, 12), the role of light in diurnal rhythms (Chapter 11), phototaxes (Chapter 13), photoreception and vision in animals (Chapters 14, 15, 16), action of ultraviolet radiation on animal cells (Chapter 17), muta-genic action of light (Chapter 18), photoreversal of ultraviolet damage by visible light (Chapter 19), and the photochemistry of nucleic acids (Chapter 20). The final chapter deals with the production of light by organisms (bioluminescence).*

Because this treatise on Photophysiology grew beyond its intended size it became necessary to subdivide it arbitrarily into two parts, but the two volumes must be looked upon as forming an integral unit. Volume I is concerned primarily with action of light upon plants, Volume II mainly with effects of light upon microorganisms and animals. General concepts and methods are introduced at the beginning of the treatise and are further developed in each of the volumes.

The Editor takes this opportunity to thank the contributors who cooperated in minimizing delays which accompany an extensive enter-prise of this sort and the publishing staff who facilitated its production.

ARTHUR C. GIESE

Stanford University October, 1963

* Because in the extensive literature referred to coenzymes I and II are abbrevi-ated DPN and TPN (di- and triphosphopyridine nucleotide, respectively), some of the authors contributing to this treatise prefer these abbreviations to the more recently adopted synonyms NAD and NADP (nicotinamide-adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate, respectively).

CONTENTS OF VOLUME II

ANIMAL PHOTOPERIODISM

ALBERT WOLFSON

PHOTOTAXIS IN MICROORGANISMS

RODERICK Κ. CLAYTON

THE PHOTORECEPTOR PROCESS IN LOWER ANIMALS

DONALD K E N N E D Y

VISION AS A PHOTIC PROCESS

W. A. H. RUSHTON

THE PHYSICAL LIMITS OF VISUAL DISCRIMINATION

H. B. BARLOW

STUDIES ON ULTRAVIOLET RADIATION ACTION UPON ANIMAL CELLS

ARTHUR C. GIESE

MUTAGENIC EFFECTS OF ULTRAVIOLET AND VISIBLE LIGHT

G. ZETTERBERG

PHOTOREACTIVATION OF ULTRAVIOLET DAMAGE

CLAUD S. RUPERT

PHOTOCHEMISTRY OF THE NUCLEIC ACIDS

KENDRIC C. S M I T H

BIOLUMINESCENCE—PRODUCTION OF LIGHT BY ORGANISMS

A U R I N M . CHASE

AUTHOR INDEX—SUBJECT INDEX

X l l l

HISTORICAL INTRODUCTION

Arthur C. Giese

From the beginning of time man has stood in awe of the sun. The very word radiation, in fact, stems from Aton Ra, the Egyptian sun god, and the Egyptians depicted the rays of the sun ending in hands holding the symbol of life (Menzel, 1959; Hawkes, 1962). The Persians had a sun god, the Greeks have left us the legend of Helios, and in England and Brittany are found the ruins of Druid temples to the sun. In the New World, the Aztecs and the Incas worshipped the sun, as did many primitive Indian tribes, such as the Dakotas (Oleott, 1914). I t is not surprising, therefore, that we should find an early interest in the nature of the effect of sunlight upon life.

While the ancient Greeks and Romans probably built solaria pri-marily for pleasure, a quotation from Herodotus tells us that "Ex-posure to the sun is eminently necessary to those who are in need of building themselves up and putting on weight. . . ." Jewish physicians in Arabia recommended sunbaths for health, as did Avicenna in the tenth century. Today, the cult of sunbathing persists all around the globe. The initial reddening and the subsequent tanning of the human skin after exposure to the sun is evidence to everyone that the sun has photobiological effects, while a sunburn from excessive exposure empha-sizes the destructive action of sunlight on living cells in the skin. The development of photobiology and the analysis of the effects of light upon the living cells, however, has been slow because our knowledge of the very nature of light and its action in physical systems is also of very recent date.

That nonvisible as well as visible light exists in the spectrum of the sun came to general knowledge only after Herschel in 1800 discovered light of wavelengths longer than red light (infrared light)* by the warm-ing of a thermometer placed in a spectrograph beyond the red end of the spectrum. The following year Ritter discovered ultraviolet light by its photochemical action on silver chloride placed in a spectrograph beyond the violet end of the spectrum. I t was much later—in fact, only with

1 Claims have been made that infrared rays and radio waves have specific effects upon cells, apart from heating. However, more careful studies indicate that regardless of the means by which the temperature is achieved—provided the same temperature is reached at the same rate—the effect is the same whether the cells are heated directly or by radio waves (see discussion in Giese, 1947).

1

2 HISTORICAL INTRODUCTION

the development of radio astronomy during World War II—that the sun was also recognized as a source of radio waves.1 Current rocket research indicates that some ionizing radiations also come to the earth from the sun (Menzel, 1959).

1. The Development of Photochemistry Although Vitruvius reported the bleaching of pigments by light in

30 B.C., and at the end of the Middle Ages some experiments of a photo-chemical nature were performed, it is often said that photochemical studies as such began only about two centuries ago when Scheele (1742-1786) found that the blackening of silver salts occurred most rapidly in the short end of the sun's spectrum, a study further continued in 1827 by J. H. Schulze, professor of medicine at Altdorf, Switzerland. Senebier (1742-1809) studied the bleaching of plant pigments under the in-fluence of sunlight. Berthollet observed the decomposition of chlorine water in sunlight in 1785, and de Saussure utilized this discovery in 1796 in making the first chemical actinometer for measuring light in-tensity (see Dhar, 1931).

A large number of workers in the early nineteenth century studied the effect of light on the reaction between chlorine and hydrogen, studies which were to play an important role in the development of photo-chemical concepts. Davy in 1812 studied the photochemical formation of phosgene gas from carbon monoxide and chlorine, and emphasized the reducing action of the more refrangible (shorter) wavelengths of light. Also, the discovery of practical photography as a result of the experi-ments of Niepce and Daguerre between 1814 and 1830, gave great impetus to photochemistry. In 1818 Grotthus formulated the photo-chemical absorption law which states that only the radiations which are absorbed are effective in promoting a photochemical change. This relationship, which has sometimes been called the first law of photo-chemistry, was experimentally supported by the studies of Draper in 1839—hence its name, the Grotthus-Draper law. Bunsen (1811-1899) and Roscoe (1833-1915) performed their classical investigations of the photochemistry of chlorine-hydrogen interaction and demonstrated that when the product of the intensity and the exposure time was constant, the photochemical effect was the same—a relationship called the Bunsen-Roscoe reciprocity law or the second law of photochemistry. (This rela-tionship had already been suggested without experimental proof by Senebier back in 1788.)

Vogel in 1873 showed that silver salts which are sensitive only to the shorter wavelengths in the visible spectrum (e.g., violet and blue) could be sensitized or made susceptible to longer wavelengths (e.g., green and

HISTORICAL INTRODUCTION 3

yellow) by mixing them first with a variety of coloring matters. There are only certain conditions under which this can occur and not all mix-tures are effective—the absorbing chemical species must be able to transfer the light energy to the nonabsorbing molecules (Dhar, 1931). The concept of sensitizing a substance to longer wavelengths of light, by introducing a material which absorbs these wavelengths of light, has been of much interest to biology (e.g., in photodynamic sensitization).

I t is difficult to trace the many pathways which photochemistry took after interest began to center on the mechanism of individual photo-chemical reactions. Improved techniques of study made it possible to work with light of known wavelengths, and, by measuring radiations with a photometer or actinometer, to define the laws governing light absorption.

The Lambert and Beer law of absorption, published in 1855-1859, states that the fraction of incident light which is absorbed by a substance in solution is independent of the initial light intensity and increases pro-portionally with increase in concentration of the substance. I t is usually important to determine whether a given material under study obeys this law, since a deviation usually indicates complications in the reaction under study and at the same time gives clues as to the nature of the reaction.

The quantum law, developed by Planck in 1900, states that radiation is emitted, not continuously, but in small units called quanta. The law of photochemical equivalence, subsequently formulated by Starck and Einstein (1908-1912), states that when one quantum of light is absorbed per molecule (atom, ion, etc.) of absorbing substance, one light-activated molecule (atom, ion, etc.) is produced (primary reaction). What this light-activated molecule does thereafter depends upon its nature and its environment. The molecule may re-emit the light (as resonant light at the same wavelength, or as fluorescence at longer wavelengths), or it may undergo a variety of secondary reactions such as isomerization, polymerization, oxidation, photolysis, union with some other molecule, etc., or it may pass its energy to another molecule (sensitization) which in turn may undergo a reaction because of this energy. Emil Warburg (1846-1931) emphasized the concept of quantum yield (the number of molecules altered or reacted as a result of the absorption of one quan-tum) , a concept which Bodenstein had also used in the early part of the twentieth century to determine the quantum yield of various photo-chemical reactions (see Kistiakowsky, 1928). Sometimes a chain reaction takes place, in which the excitation obtained from light is successively passed from one molecule to another, as a result of which as many as a million molecules may react. For example, the quantum efficiency of the


photochemical combination of hydrogen and chlorine (the latter ab-sorbing the light) is about 106. Since a photochemical reaction chain may be broken by the surfaces of a reaction chamber, it therefore de-pends upon the conditions under which the experiment is performed (Kistiakowsky, 1928; Daniels, 1936).

While temperature was found to have little effect on the primary photochemical reaction, it was shown to have a marked effect on the secondary reactions. By means of kinetic studies it was possible to separate the primary and secondary reactions of the numerous photo-chemical reactions studied in the early part of the present century (Dhar, 1931). I t was during this period that, by the application of spectroscopic methods to photochemical reactions, attempts were made to study the absorption changes following irradiation and to identify the absorbing substance (Noyes and Leighton, 1941).

One of the more important recent developments in photochemistry is flash photolysis, a technique which was first applied in 1949 by Porter and Norrish at Cambridge (see reviews by Porter, 1959, and Norrish, 1962). The entire radiation dosage is delivered to the absorbing chemi-cals at very high intensity in a fraction of a second and the absorption spectrum is measured as soon as possible after the flash. In this way it was possible to identify short-lived intermediates, such as some free radicals (see Chapter 3) which last only a millionth of a second (singlets), and others which last only a thousandth of a second (trip-lets). Newer instruments, which permit flashes of even a billionth of a second, allow the determination of many intermediate states of excita-tion, and the pathways whereby photochemical reactions proceed (Gross-weiner, 1960). The development of lasers offers the possibility of high intensity sources of monochromatic light (Smith, 1962).

All of these various developments in photochemistry had their biological overtones since photophysiology is, in the final analysis, largely a study of the photochemistry of the action of light on biological systems (Wald, 1959; see also Chapters 1, 3, and 20).

2. Photosensitization in Biological Systems In 1898 Raab, working in Tappeiner's laboratory in Heidelberg,

showed that protozoans placed in dilute solutions of acridine dyes (which themselves are without effect upon the cells) were killed in diffuse visible light (which by itself is also harmless). Raab also showed that the effect is not due to some change in the medium, since similar illumination of the medium and dyes in the absence of the cells did not make it toxic to the cells added later. I t was demonstrated by Tappeiner and others that all kinds of cells can be sensitized to light. If the wave-


lengths absorbed by the particular dye present in the solution are excluded by interposing a filter cuvette containing a somewhat higher concentration of the dye, the cells are unaffected even though they are in the photodynamic (photosensitizing) dye.

In most of the cases tested, atmospheric oxygen was found to be utilized during photodynamic action which indicates that some process of photooxidation is apparently involved. Experiments suggest that it is the proteins in the cell which are photooxidized during photodynamic action, and furthermore it seems that the aromatic amino acid residues in the proteins are the most readily photooxidized substances. The same molecule of dye appears to absorb and transfer the energy of light over and over again; it undergoes destruction in side reactions only (Blum, 1941).

Photosensitization in biological systems is not increased by an in-crease in temperature, although secondary reactions may be so affected (Blum, 1941).

The photodynamic action is either lethal or has only a slight effect on cell division if the cells recover. Extensive investigations, especially on the mechanism of photodynamic action on red blood cells, were car-ried out over several decades by Blum who has published a monograph on the subject (1941).

Natural photosensitization has been shown to occur in the skin cells of animals feeding upon certain plants containing pigments which are absorbed into the blood stream and which eventually reach the skin. The action spectrum of the effect in these cases corresponds to the absorption spectrum of the particular plant pigment (Clare, 1955). Natural photosensitizers have also been found in some cells, such as the pink ciliate protozoan, Blepharisma (Giese, 1946) and in carotenoidless strains of a purple photosynthetic bacterium (Stanier, 1960). In the latter bacterium chlorophyll acts as a photosensitizer to the cell while carotene (in the wild type) apparently acts to protect the cell from photooxidation during the photosynthetic process.

Although the photosensitization exhibited by many dyes (e.g., the fluorescein series) appears to be largely a surface phenomenon, recent experiments have shown that some of the acridine dyes, such as acridine orange, combine with nucleic acids and photosensitize them to light. Such dyes are mutagenic in light (Kaplan, 1948; see also Chapter 18). Acridine-sensitized yeast cells are killed by visible light in a way that is quantitatively quite similar to the action of UV radiations (Freifelder and Uretz, 1960). The nucleic acid of tobacco mosaic virus combines directly with acridine orange and is inactivated in the presence of diffuse visible light. Acridine orange apparently combines with both RNA and


DNA, giving rise to complexes of different colors (Chessin, 1960; Mayor and Diwar, 1961). This series of studies constitutes an interesting ap-proach to research in nuclear function.

Free radical formation during photosensitization after intense flashes of light is also being studied at the present time in an attempt to identify some of these intermediates (see Blois, 1961).

3. Photosynthesis Long ago Aristotle, a keen observer, called attention to the need

of sunlight for the development of the green color in plants, but it was not until almost two millenia later that Stephen Hales (1677-1721) asked, "May not light which makes its way into the outer surfaces of leaves and flowers contribute much to the refining of substances in plants?" Priestley, in 1777 recorded, ". . . The Present State of Dis-coveries Relating to Vision, Light and Colours," describing experiments which indicated that light falling upon certain plants "dephlogisticated" air (or added oxygen to it) . Scheele, the same year, attempted to duplicate Priestley's experiments but was unable to do so. I t was Ingen-housz, a Dutch physician, who resolved the difference between Priestley and Scheele. In 1779 Ingenhousz clearly demonstrated that green plants, like animals, absorb oxygen and give off carbon dioxide at all times (respiration) and in darkness respiration is the only process that could be detected. However, when exposed to light the green plant not only respires, but, as a result of action of light, it absorbs carbon dioxide and gives off oxygen to the air (photosynthesis) at a greater rate; photo-synthesis is the process then observed. Ingenhousz saw clearly the cosmic function of green plants aided by sunlight, and the relation between animal and plant nutrition (Spoehr, 1926).

In the years that followed many other scientists became interested in this relation of light to photosynthesis. In 1804 de Saussure demon-strated that equal volumes of carbon dioxide and oxygen are exchanged during photosynthesis and that often equal volumes of the gases are exchanged during respiration. The experiments of de Saussure also showed that the weight gained by a green plant during photosynthesis far exceeds the weight that could be accounted for by the uptake of carbon dioxide, and he suggested that the difference was due to the up-take of water by the plant (Spoehr, 1926). This was the first truly quantitative study of photosynthesis.

In 1882 Engelmann, observing behavior of some oxygen-sensitive microorganisms, found that they became concentrated along an alga illuminated only at the wavelengths of the spectrum corresponding to the lines of absorption by chlorophyll. These were the regions in which oxygen was being produced in photosynthesis (Fig. 1, Chapter 7). Since


the absorption spectrum of chlorophyll was known, it was thus possible to correlate the action spectrum of photosynthesis and the absorption spectrum of chlorophyll. In this way Engelmann proved that different wavelengths of light are effective to different degrees in promoting a biological reaction; the wavelengths of greatest effectiveness were pre-sumably those which were most absorbed. When the absorption spectrum of a compound active in a given photobiological reaction is unknown, the action spectrum gives a clue as to its identity. The use of action spectra in photophysiology is considered in Chapter 4.

In 1905 it was shown that a light reaction, with a low temperature coefficient (observable at low light intensities where light was the limit-ing factor), is followed by a dark or thermal reaction (the Blackman reaction, so named for one of the pioneer investigators). In 1923 Otto Warburg showed that 4 to 5 quanta of light were needed to reduce one carbon dioxide molecule. Many other workers have tried to determine the quantum efficiency of the photosynthetic process but most of them have found about double the value reported by Warburg.

In 1931 van Niel, on the basis of comparative studies with photo-synthetic bacteria, postulated that the oxygen which appeared during photosynthesis resulted from the splitting of a water molecule, a concept proved by Ruben in 1941 using tracer techniques. Hill, in 1939, showed that the photochemical reaction in photosynthesis could be isolated from the other reactions and that a reduction pool was formed during the action of light upon green plants (see Chapter 6).

I t has been shown in Arnon's laboratory during the last few years that light energy when absorbed is probably used to excite electrons in chlorophyll, which electrons later give off this energy to form high-energy phosphate bonds (photophosphorylation) and to reduce coen-zymes (see Chapter S).

During the last decade Calvin and his co-workers have demonstrated how carbon dioxide is taken up (added to a pentose). Calvin thus worked out the "dark" reactions in photosynthesis and it was largely for this work that he received the Nobel prize in 1961.

Emerson and Blinks (French, 1961) recently showed that enhance-ment of photosynthesis occurred when a long and a short visible wave-length (in the photosynthetically effective span) were combined or given in sequence. That is, the photosynthetic yield for the sum of the energy for both wavelengths is greater when given in this manner than is the photosynthetic yield for the same amount of energy given at each of the wavelengths independently of each other (see Chapter 7). An-other field of interest at present is the identification of the free radicals caused by chlorophyll excitation (Livingston, Krasnovsky and Rabino-vitch; see Blois, 1961).


4. Phototropism The concept of phototropism (or heliotropism), that is, the response

of the plant organism to light, was developed by DeCandolle in 1832. Charles and Francis Darwin, in 1881, had shown that the receptor for the phototropic response was in the tip of a plant coleoptile. Blauuw (1909-1915) showed that the blue region of the sunlight spectrum was more effective than the remainder of the spectrum. The action spectrum for phototropism has been a source of some controversy since some workers favor the view that carotenoids are the materials that absorb the light used in phototropism (e.g., Thimann and Curry, 1960), while others think that the light-absorbing substance is a flavin (e.g., Galston and Baker, 1949).

Went (1928) found that auxin, the plant growth hormone, is differ-entially distributed in higher plants after exposure to sunlight since more auxin is present on the unilluminated side. This distribution has been shown to be a result of lateral transport, not destruction of the auxin (Briggs, 1957). How light induces differential transport of auxin in the plant has not yet been ascertained.

In the fungi interesting reactions to light are also found and are strongest in the short end of the spectrum. Auxin, however, is appar-ently not involved. The mechanism of phototropism in fungi is even more uncertain than that in higher plants (for references, see Chapter 8).

5. Phototaxis Phototaxis is the directional motile response of plant and animal

cells under the stimulus of light. Movement without a direction com-ponent induced by light is called a kinesis. Phototaxes of plant zoospores and of microorganisms have been studied at various times, but the earlier literature was mostly descriptive. However, recent studies on phototaxes of microorganisms (e.g., Bendix, 1960) attempt to determine the mechanism of action of light in evoking the responses (see Chap-ter 13).

Phototactic responses of animals have also been studied, and in 1888 Loeb in "Die Orientierung der Thiere gegen das Licht" summarized some of this information. Loeb conceived of phototaxes as forced move-ments of the animals in response to asymmetric light stimuli on the two eyes. These views were criticized by others (Mast, 1911; Jennings, 1915), who found the responses not susceptible to so simple a formulation. A careful classification of phototaxes is available (Fraenkel and Gunn, 1940). Analytical studies at present deal not only with the photoreceptor process but also with the neural mechanisms following light reception (see Volume II, Chapter 14).


6. Vision I t is surely apparent to everyone that light is necessary for vision,

but studies purporting to analyze the action of light in the visual proc-esses had a relatively late start. The pioneer studies probably resulted in part from the various discoveries in photochemistry. At one time it was thought that we see because of something projected from our eyes (Galen, Leonardo da Vinci), but the astronomer Kepler (1571-1630), basing his account on studies of the anatomists of his time, gave an al-most present-day explanation of the way in which the rays of visible light are bent in the eye to form an image on the retina. A few years later, the Jesuit, Scheiner, experimentally demonstrated formation of the image on the retina of the eye by removing the opaque coats at the back of an animal eye (Mann and Pirie, 1950). Von Helmholtz' "Physio-logical Optics," published first between 1856 and 1866 (three volumes) left little doubt that the eye was a fine optical system resembling a camera, yet much more flexible. Meanwhile, as the result of histological investigation, Max Schulze, in 1866, demonstrated that the vertebrate eye had two kinds of visual receptors—rods (for dim vision) and cones (for color and form vision)—an idea developed by von Kries in 1895 to explain a wide variety of phenomena (Geldard, 1953).

In 1876 Boll published his studies on visual purple or rhodopsin (the pigment of the rods), followed in 1878 by the more extensive and detailed work of Kühne and his co-workers. The work of these investi-gators clearly established that changes in a visual pigment are asso-ciated with the act of seeing. Kühne fixed the retinas of eyes of dark-adapted animals suddenly exposed to a bright scene and showed that the optogram on the retina resembled a photograph of an object on a film, which suggested photochemical alterations of a pigment in the retina. In 1880 Holmgren showed that the incidence of light on the eye is accompanied by an electrical change in the retina. These early studies set the stage for the extensive developments in visual physiology which were to come some fifty years later (Bayliss, 1931).

The discovery of the connection between night blindness and vitamin A by McCollum and Simmonds in 1917 led to identification of rhodopsin as a conjugate of protein and a carotenoid. A few years later it was shown that vitamin A and retinene participate in the visual cycle. Wald and his co-workers, in 1934, then demonstrated that visible light causes an isomerization of the carotenoid components of rhodopsin (see Wald, 1959). These investigators also studied the chemical changes in the visual cycle in vitro. Rushton (1952) on the other hand, studied the visual cycle as it occurs in the intact eye ; these studies made it possible to determine many new properties of the visual system not easily


studied in vitro (see Chapter 15). The electrophysiology of vision, which is more effectively studied with primitive eyes, has been espe-cially explored during the last thirty years by Hartline, using the eye of the king crab Limulus (1928), in which the individual units are large and well separated from one another. This work has been extended to a variety of eyes (see Volume II, Chapters 14 and 15).

7. Photoperiodism Among the recent developments in photophysiology is the recogni-

tion of visible light as the timer in rhythmic responses of animal and plant organisms. Photoperiodism, or the control of plant or animal activities by the length of the light (or dark) period of the day, was first recorded by Garner and Alard in 1920. These investigators ob-served that some plants (spring-blooming) flowered only if subjected to a succession of long days after short ones, whereas other plants (fall-blooming) flowered only if subjected to short days after long ones, al-though some are insensitive to photoperiod. So novel was their concept and so contrary to the then-accepted view—namely, that it is the temperature which determines flowering—that they had difficulty pub-lishing their report (Borthwick et al., 1956). Since that time it has been shown that when a plant is subjected to an appropriate photoperiod, the flower-inducing hormone (florigen) presumably produced by the leaves of the plant, passes to the floral-producing organs and the plant flowers. In some plants exposure of even a single leaf suffices. That a hormone is involved in this phenomenon has been demonstrated by the following experiment: A leaf, exposed to the appropriate photoperiod and then cut off from the plant and grafted onto another, not exposed to light, is found to induce flowering in the second plant (Hamner and Bonner, 1938; see Bonner, 1959).

In 1937 Flint and McAlister showed that red light stimulates germination of certain types of seeds and that the far-red light (ap-plied later or simultaneously) has the opposite effect upon these seeds, preventing their germination.

It became clear in the following years that a whole host of other effects of light upon plants, such as flowering and morphogenetic effects, were subject to the same opposing actions of red and far-red wave-lengths. An action spectrum for the red: far-red light effects was first determined by Parker in 1946 and confirmed by others. On the basis of the action spectra for the red: far-red effect, the substance involved was predicted to be an enzymatic protein pigment, blue-green in color. Such a protein was subsequently isolated by Hendricks, Borthwick, and their associates in 1961 (see Chapter 10), after a search of several decades.


Studies of photoperiodism on animals followed rapidly those on plants. In 1924 Marcovitch published an account of his findings on aphids in which he showed that the production of sexual forms, which normally occurs late in summer or early fall, is determined by the length of day rather than by temperature. He was able to get sexually mature aphids in summer by shortening the time of illumination, and to keep the aphids in vegetative states, even in the fall, by lengthening the time of illumination. The work on insects has continued and other workers have been able to control the life cycle of several insects and mites almost at will, chiefly by manipulation of the length of daily illumination. Lees (1959) is engaged in determining the action spectrum of the photoperiodic induction in insects.

In 1925 Rowan showed that breeding in some species of birds, which normally occurs in spring when the day is getting longer, could be in-duced in dead winter when the temperature was —40°C, by lengthening the day with artificial light. In this manner in some species of birds several breeding seasons have been induced in a single year. As first shown by Bisonette in 1932, breeding in the ferret, which normally occurs in spring, can be hastened artificially by a series of long days after short ones, but manipulation of the entire reproductive cycle is more difficult. The induction of breeding cycles in birds and mammals by exposure to appropriate day-length regimes appears to be the result of the activating effect of the light upon the anterior lobe of the hy-pophysis (probably by way of the eyes and the hypothalamus). Because the photoperiodic response in vertebrates is so complex the photobiology of the light action has been less completely studied in these animals than in insects or in plants (see Volume II , Chapter 12).

8. Diurnal Rhythms and Visible Light As far back as the eighteenth century there occur, in both the

botanical and zoological literature, scattered observations on diurnal rhythms; e.g., the tendency of organisms to perform some acts in a periodic fashion, day by day. But a systematic study of such rhythms began only in the 1950's, when suddenly a large number of investi-gators became interested in the problem, and a massive literature has been produced within a few years. I t is now established that a diurnal rhythm of almost a 24-hour length is maintained in the dark for a long time after removal of the organism from the natural day-night cycle—in some cases indefinitely—suggesting a "biological clock." Such rhythms have been observed in both plants and animals, including several single-celled species, and for a large number of diverse activities. It has also been shown that the phase of the rhythm can be reset (phase shift) by manipulation of the illumination, indicating a photobiological


relationship. Some disagreement exists as to the extent of the endogenous and exogenous components in the determination of the diurnal rhythms; one group (Brown, 1960) maintains that the exogenous component is the more important while the other group emphasizes the importance of the endogenous component (Pittendrigh, 1960; Aschoff, 1963). The con-troversy has led to intensive efforts to determine the relative importance of the many variables (Biological Clocks, 1960). Few action spectra are available for induction of such rhythms and the few available do not appear to be general. The substances responsible for this phenomenon are, therefore, undetermined (see Chapter 11).

9. Medical Uses of Ultraviolet Light Much of the early information on the action of light on organisms

came from physicians. Thus Fiennius in 1735, concentrated sunlight upon a growth on a lip, supposedly with good results; Harris in 1782 used irradiated mollusk shells in alleviating a case of rickets; while in 1815 Löbel treated amaurosis of the eye with sunlight. Rollier and Poncet in 1840 claimed that patients with tuberculosis of the joints were benefited by sun treatments, and Rickli in 1855 established a clinic in Austria for treatment of such patients (Mayer, 1932).

Downes and Blunt, as early as 1877, had shown that sunlight will sterilize an infusion containing bacteria. Arloing, in 1887, had introduced a carbon arc as a more reliable source of radiations than sunlight. Ward (1893), using such an arc and a quartz lens-prism system to get mono-chromatic light, showed that the short UV wavelengths were more bactericidal than the long ones.

I t was Finsen (1860-1904; Nobel prize, 1900) who brought this study of the bactericidal effects of radiation to a focus when, with sun-light and artificial UV radiation, he successfully cured lupus vulgaris, a skin tuberculosis which was fairly widespread in Scandinavia at the end of the nineteenth century and is still found in some parts of north-ern and central Europe (Finsen, 1889). Finsen concentrated sunlight— and later, carbon arc radiations—by the use of special lenses. In order to permit longer exposures than previously tolerated, he cooled the radia-tions to which the lesions were exposed by the use of quartz cuvettes containing tap water. Also, Finsen squeezed the blood out of the skin to allow the radiations to penetrate deeper into the skin. General mild irradiation of the entire body, along with local exposure of the lesions, was found to be effective. Lupus vulgaris is now treated with chemicals, especially antibiotics, rather than by UV radiations—except for cases refractory to chemicals (Hollaender, 1959).


Finsen's discovery gave great impetus to the development of photo-physiology. At the Finsen Light Institute, erected in his honor in Copenhagen, Finsen initiated studies which sought to define whether the effect of light was on the cells or the medium, and to determine what part of the spectrum was most effective in its action on the cells of the skin and in killing microorganisms, as well as to investigate the effect of various environmental factors (oxygen, temperature, etc.) upon the sensitivity of microorganisms and other cells to light. The publications from the Institute form a valuable part of the photophysiological litera-ture (Busck, 1904; Hollaender, 1959).

The report of A. E. Hess and H. Steenbach in 1924 describing the production of active vitamin D by irradiation of inactive sterols with UV radiations began an intense investigation of the photochemistry of the sterols involved in this phenomenon. The photochemistry of the activation of provitamin D to vitamin D, which is a demonstration of a specific beneficial action of UV radiations upon animals (see Canterow and Schepartz, 1962) is now so well understood, it has become classic.

10. Effects of Ultraviolet Radiation upon Cells In 1929 Gates published an action spectrum for the bactericidal effect

of UV radiation. Following this publication much of the work on the effect of UV radiations upon cell activities has been preoccupied with action spectra and energy relationships. A variety of action spectra for different effects of UV radiations upon cells have since been described (see Volume II , Chapter 17).

Recently, attention has been focused on the mutagenic action of UV radiation because of the importance of the process and the usefulness of radiations as mutative agents. I t will be recalled that Muller in 1927 demonstrated that ionizing radiations increased the rate of mutation in fruit flies and other organisms. In 1933 Altenburg showed that UV radiations had similar action, but that mutation occurs only when the chromosomes in the living germ cells could be reached by the UV radiations—which are generally absorbed quite superficially. The action spectrum of mutation has been shown to resemble the absorption spec-trum of nucleic acid as a result of the work done by Hollaender and Emmons in 1942 and by Stadler and Über in 1942 (see Volume II , Chapter 18).

In 1949 Keiner called attention to the reversal of UV-induced injury to cells by subsequent (or simultaneous) illumination with visible light, a phenomenon called "photoreactivation" (Keiner, 1949). This field of investigation, so active in the last decade, is considered in Volume II ,


Chapter 19. Photoreactivation calls to mind the analogous reversal of the effects of red light upon plants by subsequent (or simultaneous) illumination with far-red light, considered in Chapter 10.

11. Bioluminescence Even in early historical times luminous jelly fishes, worms, fireflies,

glowworms, and fishes as well as the phenomenon of the occasional luminescence of decaying flesh and seafood were recorded (see Harvey, 1957). In 1667 Robert Boyle showed that oxygen was necessary for the luminescence of decaying wood (fungus-infected), and Spallanzani in 1797 showed that water must also be present. I t was not until 1885, however, when DuBois showed that the luminescence of a firefly was the result of an interaction between a heat-sensitive enzyme (luciferase) and a thermostable, oxidizable substrate (luciferin), that an analysis of the mechanism of bioluminescence began.

Luminescence was studied by the late E. N. Harvey during most of his lifetime; his work is recorded in four treatises on the subject. Under Harvey's influence Anderson in 1935 first partially purified luciferin; Chase in 1946 partially purified luciferase of the Japanese "water firefly" Cypridina and has studied many of the properties of the enzyme; while Johnson studied luminous bacteria, especially the thermal and pressure relations of bioluminescence in luminous bacteria and, more recently, other luminous systems.

Shinomura in 1961-1962 succeeded in crystallizing luciferase, as well as luciferin from Cypridina, and a tentative structural formula has been worked out for the luciferin by Hirata. McElroy and Strehler (1954) have been able to isolate luciferin and luciferase from luminous bacteria. The luciferin in luminous bacteria appears to be a complex between a long-chain aldehyde and a flavin coenzyme. During the past few years firefly luciferin and luciferase have been isolated, purified, and crystal-lized in McElroy's laboratory and a tentative structural formula has been worked out for luciferin. The quantum yield of the firefly luciferin-luciferase reaction is apparently unity, an example of the extraordinary efficiency in biological systems (see McElroy and Seliger, 1962). The newer trends in the study of bioluminescence are considered in Volume II, Chapter 21.

12. Reference Books on Photobiology The first recorded attempt to gather photobiological information in

a book was "Lichtbiologie" by Gunni Busck of Finsen's laboratory in 1904. This book was obviously an attempt of the physicians under Fin-sen's direction to explain the curative effects of light.


More comprehensive in scope is Ludwig Pincussen's "Photobiologie" which was published in 1930. In this book Pincussen covers the nature of light, its sources and measurements ; the photochemical effects of light absorption by pigment; fluorescence; the effect of light upon plant cells, animal cells, and microorganisms; the action of radiations on different organ systems of the mammal and the plant; vision; phototropism; and the relation of light to diseases of man.

Since 1930 several compendia have appeared, some of them covering a wide range of photobiological problems. For example, in 1933 appeared "Physiological Effects of Radiant Energy" by Henry Laurens, which is primarily medical in orientation. This book was followed in 1935 by the Symposium at Cold Spring Harbor devoted to "The Inter-action of Ourselves and Things about Us with Light," containing a number of chapters on photochemistry, photosynthesis, phototaxis and phototropism, vision, bioluminescence, photochemistry in medicine, photosensitization in living systems, and the photochemistry of vitamin D (Harris, 1935).

In 1936 appeared Benjamin Duggar's two-volume work, "Biological Effects of Radiation," prepared under the auspices of the Committee on Radiation of the National Research Council. This compendium con-tained chapters on the physics of radiation; the principles of photo-chemistry; the radiation effects upon proteins, vitamins, venoms, toxins, and antibodies; the effects of ionizing radiations on various biological systems of plant and animal organisms; motor responses of invertebrates to visible light; photoperiodism; the problem of mitogenetic rays; photosynthesis; radiation and anthocyanin pigments; the effects of radiation on bacteria and enzymes; radiation-induced mutations and chromosomal alterations; and the biological aspects of quantum theory in interpreting the effects of radiation (Duggar, 1936).

Equally wide in scope is the three-volume treatise, "Radiation Biology," edited by Alexander Hollaender and also prepared under the auspices of the Committee on Radiation Biology of the National Re-search Council in 1955 and 1956. The first volume of this treatise is devoted to the effects of ionizing radiations as discussed from the fol-lowing viewpoints: the physical aspects of radiation biology; methods and measurements; chromosomal aberrations and mutations in plants, animals and microorganisms; the effects on division, morphology, and viability of the cell; the relation of ionizing radiations to development, pathology, physiology, histology, hematology, and carcinogenesis. The second volume of the book is devoted to the effects of UV and related radiations and includes chapters on photochemistry; the sources of radiations and techniques; absorption spectroscopy; the effect of UV


radiation upon genes, chromosomes, and viruses; the effects of UV radiations upon protozoans, marine eggs, bacteria, and fungi; photo-reactivation, sunburn, and the UV induction of cancer. The third volume, devoted to the effects of visible and near-visible light, contains chapters on generation, measurement, and control of light; the photochemical changes so-induced; some problems of photosynthesis, phototropism, photoperiodism, and seed germination; the effects of visible light on viscosity, permeability, and protoplasmic streaming; invertebrate photo-receptors and electrical phenomena in vision; and finally a chapter on photodynamic action and its pathological effects (Hollaender, 1955-1956).

Attempts have been made—especially in recent years—by the Comité Internationale de Photobiologie and other photobiological groups and committees in a number of countries to stimulate interest in the field of photobiology by group discussions, meetings, and international con-gresses. Three of the latter have been held; the first in Amsterdam in 1953, the second in Turin in 1956, and the third in Copenhagen in 1959—a fourth is planned at Oxford in 1964.

There also exist three international symposia in Photobiology, the latest of which is "Progress in Photobiology" (Christensen and Buch-mann, 1961). In 1961 was published the Symposium on "Life and Light" sponsored by the McCollum Pratt Institute with support from the National Science Foundation. Each of these publications contain some general articles devoted to current problems in photobiology.

Numerous symposia on special fields of photobiology have also appeared in recent years, as, for example, the publications on photo-synthesis, photoperiodism, bioluminescence, etc. (Johnson, 1955; Gaffron, 1957; Withrow, 1959; Allen, 1960). The work described in these publi-cations will be cited and described in the text.

REFERENCES

Allen, M. B., ed. (1960). "Comparative Biochemistry of Photoreactive Systems/' Academic Press, New York.

Aschoff, J. (1963). Ann. Rev. Physiol 25, 581-600. Bayliss, W. M. (1931). "Principles of General Physiology." Longmans, Greens,

New York. Bendix, S. (1960). In "Comparative Biochemistry of Photoreactive Systems" (M. B.

Allen, ed.), pp. 107-128. Academic Press, New York. "Biological Clocks" (1960). Cold Spring Harbor Symposia Quant. Biol. 25. Blois, M. S., Jr., ed. (1961). "Free Radicals in Biological Systems." Academic

Press, New York. Blum, H. (1941). "Photodynamic Action and Diseases Caused by Light." Reinhold,

New York. Blum, H. F., Robinson, J. C, and Loos, G. M. (1951). J. Gen. Physiol. 35, 323-342.


Bonner, J. (1959). In "Photoperiodism" (R. B. Withrow, ed.), pp. 245-254. Publ. No. 55, Am. Assoc. Adv. Sei., Washington, D. C.

Borthwick, H. A., Hendricks, S. B., and Parker, M. W. (1956). In "Radiation Biology" (A. Hollaender, ed.), Vol. I l l , pp. 479-517. McGraw-Hill, New York.

Briggs, W. R. (1957). Science 126, 210-212. Brown, F. A., Jr. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 57-72. Busck, G. (1904). Lichtbiologie. Mitt. Finsens Med. Lichtinst. (Copenhagen)

8, 1-147. Canterow, A., and Schepartz, B. (1962). "Biochemistry," 2nd ed. Saunders, Phila-

delphia, Pennsylvania. Chessin, M. (1960). Science 132, 1840-1841. Christensen, B. C, and Buchmann, B., eds. (1961). "Progress in Photobiology."

Elsevier, Amsterdam. Clare, N. T. (1955). In "Radiation Biology" (A. Hollaender, ed.), Vol. I l l , pp.

693-723. McGraw-Hill, New York. Daniels, F. (1936). Photochemistry. In "Biological Effects of Radiation" (B. Duggar,

ed.), Vol. I, pp. 253-302. McGraw-Hill, New York. Dhar, N. R. (1931). "The Chemical Action of Light." Blackie & Son, London. Duggar, B., ed. (1936). "Biological Effects of Radiation," 2 volumes. McGraw-Hill,

New York. Engelmann, T. W. (1882). Onderzoek. Physiol. Lab. Utrecht 7, 191-199. Finsen, N. (1889). "Über die Bedeutung der chemischen Strahlen des Lichtes für

Medizin und Biologie." Vogel, Leipzig. Fraenkel, G. S., and Gunn, D. L. (1940). "The Orientation of Animals." Oxford

Univ. Press, London and New York. Freifelder, D., and Uretz, R. B. (1960). Nature 186, 731-732. French, C. S. (1961). In "Life and Light" (W. D. McElroy and B. Glass, eds.), pp.

447-474. Johns Hopkins Press, Baltimore, Maryland. Gaffron, H., ed. (1957). "Research in Photosynthesis." Interscience, New York. Galston, A., and Baker, R. S. (1949). Science 109, 485-486. Geldard, F. A. (1953). "The Human Senses." Wiley, New York. Giese, A. C. (1946). J. Cellular Comp. Physiol. 28, 119-128. Giese, A. C. (1947). Quart. Rev. Biol. 22, 253-282. Grossweiner, L. I. (May 1960). Flash photolysis. Sei. American 202, 135-145. Harris, R. (1935). The interaction of ourselves and things about us with light.

Cold Spring Harbor Symposia Quant. Biol. 3. Hartline, A. K. (1928). Am. J. Physiol. 83, 466-483. Harvey, E. N. (1957). "A History of Luminescence." Am. Phil. Soc, Philadelphia,

Pennsylvania. Hawkes, J. (1962). "Man and the Sun." Random House, New York. Hollaender, A., ed. (1955-1956). "Radiation Biology," 3 volumes. McGraw-Hill,

New York. Hollaender, A. (1959). In "Progress in Photobiology" (B. C. Christensen and B.

Buchmann, eds.), pp. 5-11. Elsevier, Amsterdam. Jennings, H. S. (1915). "Behavior of Lower Organisms." Columbia Univ. Press,

New York. Johnson, F. H., ed. (1955). "The Luminescence of Biological Systems." Am. Assoc.

Adv. Sei., Washington, D. C. Kaplan, R. W. (1948). Naturwiss. 35, 127-128. Keiner, A. (1949). Proc. Natl. Acad. Sei. U. S. 35, 73-79.


Kistiakowsky, G. B. (1928). "Photochemical Processes." Chem. Catalog Co., New York.

Laurens, H. (1933). "The Physiological Effects of Radiant Energy." Chem. Catalog Co., New York.

Lees, A. D. (1959). In "Photoperiodism and Related Phenomena in Plants and Animals" (R. B. Withrow, ed.), pp. 585-600. Publ. No. 55, Am. Assoc. Adv. Sei., Washington, D. C.

Livingston, R. (1955). Photochemistry. In "Radiation Biology" (A. Hollaender, ed.), Vol. II, pp. 1-40. McGraw-Hill, New York.

McElroy, W. D., and Glass, B., eds. (1961). "Life and Light." Johns Hopkins Press, Baltimore, Maryland.

McElroy, W. D., and Seliger, H. H. (1962). In "Horizons in Biochemistry" (M. Kasha and B. Pullman, eds.), pp. 91-101. Academic Press, New York.

McElroy, W. D., and Strehler, B. (1954). Baeteriol. Revs. 18, 177-194. Mann, I., and Pirie, A. (1950). "The Science of Seeing." Penguin Books, Balti-

more, Maryland. Mast, S. 0. (1911). "Light and the Behavior of Organisms." Wiley, New York. Mayer, E. (1932). "The Curative Value of Light." Appleton, New York. Mayor, H. D., and Diwar, A. R. (1961). Virology 14, 74-82. Menzel, D. H. (1959). "Our Sun," rev. ed. Harvard Univ. Press, Cambridge,

Massachusetts. Norrish, R. W. G. (1962). Flash photolysis. Am. Scientist 50, 131-157. Noyés, W. A., and Leighton, P. A. (1941). "The Photochemistry of Gases." Rein-

hold, New York. Olcott, W. T. (1914). "Sun Lore of All Ages: a Collection of Myths and Legends

Concerning the Sun and its Worship." Putnam, New York. Pincussen, L. (1930). "Photobiologie." Thieme, Leipzig. Pittendrigh, C. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 159-189. Porter, G. (1959). Radiation Research Suppl. 1, pp. 479-490. Rushton, W. A. H. (1952). J. Physiol. 117, 47-48 P. Smith, R. A. (April 1962). Endeavour 21, 108-117. Spoehr, H. A. (1926). "Photosynthesis." Chem. Catalog Co., New York. Stanier, R. (1960). Harvey Lectures 54, 219-255. Thimann, K. V., and Curry, G. M. (1960). In "Comparative Biochemistry"

(M. Florkin and H. S. Mason, eds.), Vol. I, pp. 243-309. Wald, G. (Oct. 1959). Life and light. Sei. American 201, 92-108. Withrow, R. B., ed. (1959). "Photoperiodism and Related Phenomena in Plants

and Animals." Publ. No. 55, Am. Assoc. Adv. Sei., Washington, D. C.

Chapter 1

PRINCIPLES OF PHOTOCHEMISTRY AND PHOTOCHEMICAL METHODS

Stig Claesson

Institute of Physical Chemistry, University of Uppsala, Uppsala, Sweden

1. Introduction Photochemistry is the study of reactions which are caused directly

or indirectly by radiation. It has long been known (Grotthus, 1818) that only radiation which is absorbed can lead to a photochemical reaction. Einstein has shown that the primary photochemical process is caused by one single photon (or light quantum) activating the molecule. This law forms the basis of all photochemistry. Therefore it is important to know the energy in one photon (hv) or in one einstein (one mole of photons, Nhv). This energy is given for various wavelengths in Table I.

TABLE I ENERGY AVAILABLE AT VARIOUS WAVELENGTHS

Wave Wave length number Frequency Ergs/

(Â) (cm"1) (10~12 sec"1) ev/photon 1012 photons Joules/einstein Cal/einstein

2000

2500 3000 3500 4000 5000 6000 8000 10000

50000

40000 33300 28600 25000 20000 16700 12500 10000

1500

1200 1000 857 750 600 500 375 300

6.25 5.00

4.17 3.57 3.12 2.50

2.08 1.56 1.25

9.93 7.94

6.62 5.67 4.96 3.97 3.31 2.48 1.99

598000 478000

399000 342000 299000 239000 199000 150000 120000

143000 114000

95000 82000 72000 57000 48000 36000 29000

From Table I it is evident that the energy which normally corresponds to chemical changes is to be found from about 2000 Â to 5000 Â. There-fore up to the present most photochemical research has been performed

19

20 STIG CLAESSON

in the quartz UV region of the spectrum. The extremely interesting region of shorter wavelengths in the vacuum UV has until quite recently been rather unexplored because of experimental difficulties. At present the experimental techniques in this area are being rapidly developed which means that photochemical processes leading to ionization can also be studied. This is of the greatest importance in bridging the gap between photochemistry, which deals with chemical effects of nonionizing radia-tions, and radio-chemistry, which deals with chemical effects of ionizing radiations.

In most cases a chemical change will not be induced by every quantum absorbed by a molecule. Therefore the efficiency of the photo-chemical process or the quantum yield, φ, is of primary importance. It is defined as

_ Number of molecules reacting chemically ,^ Number of photons absorbed

or

_ Number of moles reacting chemically ,^ Number of einsteins absorbed

Obviously the quantum yield will vary greatly with wavelength, type of reaction, etc., and may take values as low as 10~6 for inefficient processes in macromolecular systems and values of 104 or higher for photochemically initiated chain reactions.

Most photochemical processes consist of a long series of reaction steps following the primary photochemical process, and the overall quantum yield may give no information about the primary processes taking place. Thus the complete elucidation of a photochemical process normally means that a complex set of concurrent and successive chemical reactions has to be resolved into its individual reactions. Until quite recently the mechanism of the primary step had normally to be inferred from analysis of data from reaction kinetics. This was because the primary photochemical reaction intermediates are so short-lived that their actual concentration under normal experimental conditions is much too low to be observable. However, the situation has changed completely during the last ten years because the flash-photolysis tech-nique introduced by Norrish and Porter (1949) has come into wide use. This method employs a short but extremely intense light flash to produce intermediates at such a high concentration that they can be studied directly by fast spectroscopic methods.

It should also be noted that the flash-photolysis technique is very valuable as a means of changing the relative importance of successive

1. PRINCIPLES AND METHODS OF PHOTOCHEMISTRY 21

reactions. For instance, when the primary process is the production of free radicals, their concentration in classical photochemical experi-ments is so low that the overwhelming part of them will react with neighboring normal molecules. In flash photolysis, on the other hand, the concentration of free radicals can be so high that they will predom-inantly react with each other to produce other types of stable end-products about which more will be said later.

We will first discuss the experimental techniques employed in classical photochemistry where the intensity of the incident light is such that the number of photons absorbed during the average life time of the excited species is small compared to the number of molecules present. The actual concentration of primary reaction intermediates is then very small compared to that of the original molecules present. Then we shall return to a study of flash photolysis.

Since the energy of a photon varies with its wavelength most photo-chemical reactions are wavelength-dependent. The availability of mono-chromatic light of sufficient intensity at various wavelengths is there-fore of prime importance in almost all photochemical work.

2. Light Sources Relatively few monochromatic light sources are available and the

only one which has been widely used is the low-pressure mercury resonance lamp which primarily emits at 2537 Â. The shorter wave-lengths which are also emitted by the lamp can easily be removed with appropriate filters. In fact a very large fraction of the photochemical studies has been performed with such lamps ever since their introduc-tion. Mercury resonance lamps are available from a large number of commercial manufacturers and can be had in many different shapes; straight lamps, helical shapes for irradiation around tubes, etc. They are also available for direct immersion into the solution to be irradiated. I t should be noted that the light output from such lamps becomes much more constant if they are placed in a thermostat at room temperature. I t might also be mentioned that a microwave discharge through krypton under low pressure produces the 1236 Â resonance line as the principal radiation in the vacuum ultraviolet (Mahan and Mandai, 1962).

For wavelengths other than 2537 Â lamps are used which emit either a limited number of lines or a continuous spectrum. The spectral region of interest is then isolated either by filters or monochromators. For such work there is also available commercially a large variety of suit-able light sources which are filled with different metal vapors and which emit most of the light as line spectra. To this type of light source belong the popular medium-pressure mercury lamps from which a number of

22 STIG CLAESSON

lines can easily be isolated by filters. As the pressure inside the lamp is increased the lines get broader and at high pressures the spectrum becomes essentially continuous (Fig. 1). High-pressure mercury and

Uy

y — —1

i Λ

U 1 J

3000 4000 5000 6000 7000 A

FIG. 1. Relative spectral energy distribution of low-, medium-, and high-pressure mercury arcs (from top to bottom). Typical power ratings are 10 watts, 125 watts, and 500 watts, respectively.

xenon lamps are the most common ones used. As a continuous light source in the UV region hydrogen and deuterium lamps are also quite suitable. They can usually be obtained from manufacturers of spectro-photometers.

The most recent developments in the laser field are also of extreme importance to photochemistry. In the laser a substance, either a gas or a crystal which contains traces of photoactive ions, is brought to a higher energy level by adding energy (optical pumping) in such a way that an inversion in energy population occurs. Stimulated emission can then be achieved, and an intense, coherent, and monochromatic emission will take place. A short review is found in R. A. Smith (1962). Most lasers developed to date have emission at rather long wavelengths (red or infrared) but quite recently some progress has been made also for shorter wavelengths (Gandy and Ginther, 1962). There is every reason

1. PRINCIPLES AND METHODS OF PHOTOCHEMISTRY 2 3

to believe that the present remarkably rapid developments in the laser field will, within a few years, make photochemical applications possible which previously could have been regarded only as wishful thinking.

3. Filters and Monochromators For the isolation of a given spectral region monochromators would

seem to be the obvious choice. However, since the transmission of mono-chromators of moderate cost is usually quite low, they do not give light intensities high enough for practical irradiation times. The inefficiency of monochromators results from the fact that for short focal distances the slit width cannot be made great enough without losing resolving power and for long focal distances the optical elements must be very large to provide the required aperture. However, some excellent mono-chromators giving fairly good light intensities for photochemical work have been built in various laboratories. Both grating instruments (Monk and Ehret, 1956) and prism instruments [quartz (Heidt and Daniels, 1934) and water (Fluke and Setlow, 1954; Claesson et al, 1961)] have been described with a transmitted monochromatic light intensity of the order of 1013 to 1017 photons/second.

For most simple photochemical studies some type of filter is normally preferred to the monochromator. Both interference and ab-sorption filters are now available. Various manufacturers can now provide a large series of filters both for the UV and visible regions along with carefully determined transmission curves. Solutions with suitable transmission curves for use as filters have also been described by a large number of investigators. These are particularly helpful when a circulating thermostating fluid is required because they can be used both as filters and thermostating liquids. A useful collection of such filters is described by Scott and Sinsheimer (1955).

4. Measurement of Light Intensity For quantum yield determinations it is necessary to determine the

number of photons absorbed by the system under study. To do this it is necessary to determine both the number of incident and the number of transmitted photons.

The absolute measurement of the incident light as number of photons/cm2 sec is quite difficult. It can be done by using a black absorber (thermopile or bolometer) for measuring the radiant energy. This involves the use of a standard radiating source such as a certi-fied standard lamp which is available from the National Bureau of Standards. The measurements are quite time-consuming and require good physical instrumentation. An excellent review article dealing with

24 STIG CLAESSON

such problems is found in Withrow and Withrow (1956). A suitable bolometer of the "Venetian blind type" for such work is manufactured by H. Rörig, Berlin-Steglitz.

Therefore, in most cases quantum yield measurements are based on the use of actinometers, or solutions of compounds which decompose with known quantum yields. These may be considered intermediate standards.

For a long time solutions of uranyl oxalate have been used for such measurements because the quantum yield for its decomposition has been determined with great accuracy by Leighton and Forbes (1930) and Forbes and Heidt (1934). The decomposition can easily be followed by titration [permanganate or eerie sulfate (Claesson and Lindqvist, 1957a)]. Another advantage of this actinometer is that it is only sensi-tive to UV light (λ < 3500 Â) and therefore it can be handled without difficulty in ordinary visible light.

When the decomposition of this actinometer is followed by ordinary titration the sensitivity is about 6 X 1016 photons. However, for micro-photochemistry the sensitivity can be increased to 3 X 1016 photons if colorimetric methods are used (Pitts et al, 1955) and to 2 X 1014

photons when the carbon monoxide formed by the decomposition of the oxalic acid is determined by means of gas chromatography (Porter and Volman, 1962). This compares favorably with the sensitivity of the malachite green leucocyanide actinometer where the sensitivity has been given as 6 X 1014 photons (Calvert and Rechen, 1952).

For somewhat longer wavelengths ( λ<4800Α) the ferrioxalate ac-tinometer developed by Hatchard and Parker (1956) is also very con-venient but must be handled in red light. If colorimetric methods are used to determine the bivalent iron formed, the sensitivity of this actinometer can be as high as 3 X 1014 photons. Another advantage of this actinometer is that its quantum yield is almost constant over a very wide wavelength region which simplifies the measurements when polychromatic light sources are used.1

To facilitate the calculation of the number of photons absorbed by the system it is convenient to have the incident light parallel or ap-proximately so and to have the sample in the shape of a plane film if solid or in a plane-parallel vessel if liquid. The fraction of the light absorbed can then be calculated from the known absorption spectrum

xThe accepted values for the quantum yield of the ferrioxalate actinometer which are also consistent with the values for the uranyl oxalate actinometer have very recently been questioned. The new values are about 35% higher than the present ones (J. Lee and H. H. Seliger, Photochem. Symp., Rochester, New York, March 27-29, 1963).


of the sample by the Bouguer-Beer law [often referred to as the Lam-bert-Beer law; cf. Brode (1949) and K. S. Gibson (1949) for an excellent discussion of nomenclature]. In this connection it is important to re-member that if the photochemical reaction is dependent on the light intensity, it is necessary to work at low internal absorbance of the sample, otherwise the light intensity at the front and back sides of the sample will be very different. This unfortunately implies that only a small part of the incident photons is used for the chemical reaction. Also, an optical system which produces parallel light collects a rather small part of the radiant energy from the light source. Quite often one has to accept a less ideal optical arrangement in order to obtain sufficient light intensity during the irradiation.

If nonparallel light is used for illumination as for instance with a helical lamp around a cylindrical reaction vessel it is most practical to use the same vessel with an actinometer solution which has the same optical density as that of the sample being investigated. The actinometer values will then directly give the number of photons absorbed by the sample. This is sometimes referred to as the method of equivalent optical densities (Porter and Volman, 1962; Moring-Claesson, 1956). For such purposes it is sometimes convenient to have actinometer solu-tions with lower optical densities than those previously mentioned. Monochloroacetic acid is a suitable choice since the chloride ions formed can be easily titrated and the quantum yield is known (R. N. Smith et al, 1939).

5. Calculation of the Number of Absorbed Photons and the Quantum Yields

If the light is monochromatic and the absorption spectrum of the sample unchanged during the irradiation period, the calculation of the quantum yield according to Eq. (1) is straightforward. The accuracy will then primarily depend on the accuracy of the analytical methods used to determine the extent of the reaction. The technique to be used will completely depend on the type of systems studied and the discus-sion of such problems is outside the scope of this chapter.

However, even in the case of monochromatic parallel light the calculation of the quantum yields becomes quite complicated when changes in light absorption accompany the photochemical changes. This effect has been discussed by McLaren (1949) in the case of proteins where an increase in the internal absorbance of the sample is usually observed as irradiation proceeds. Such changes can either be part of the photochemical reaction under study so that the products of the reaction have greater absorption, or the increase can be due to a concurrent

26 STIG CLAESSON

reaction distinct from the one being studied. In general, the situation is quite complicated and a complete knowledge of all reactions taking place is necessary before a satisfactory calculation of all reaction parameters can be made. However, a few simple cases deserve to be mentioned. We denote the absorbance index (or absorbance per unit thickness) for the reactants, the products and the solvent (plus other noninteresting material) as μ,κ, /χΡ and μ8, respectively, and their con-centrations R, P, and S. Then the fraction /R of the light absorbed which is taken up by the reactants is

/ R = MRÄ/(MRÄ + μρΡ + ßsS) (2)

Obviously /R varies with the progress of the reaction and therefore the number of photons absorbed by the reactants per unit time is not con-stant in the general case even when the incident light is parallel and of constant intensity.

However, in the case of low internal absorbance of the sample (high transmittance) the absorbed flux Ia = h — / i s

7« = Jo{l - exp [ - (μΕΒ + μΡΡ + ßsS)b]} « 70(MRÄ + μΡΡ + MSS)6 (3) where b is the cell thickness. Therefore in this case of low absorbance the fraction of the light absorbed by the reactants is

Ia,n = / R / « = hßRÜb (4) and consequently is independent of the absorption of the other species present. This result could also have been written directly if it is re-membered that at low absorbance (low concentration) the various molecules are not screening each other (no inner screening). In this case the calculation of the quantum yield is straightforward and simple.

If IQ is regarded as constant and R(t) denotes the concentration of reactants at the time t, the number of molecules reacting in the time interval t to t + dt is —dR(t), and the number of quanta absorbed is fcJ0ju,RjR(£)&cï£ where k is a proportionality factor which converts I0

into number of photons per second. Then the quantum yield is

-dR(t) φ khßRR(t)bdt W

or integrated

or alternatively

Ψ khßRb(h - h) m R(t2) w

R(t) = Ä(0) exp (-φΜψηΜ) (7)


where t = 0 is the starting time when the reactant has the concentra-tion Ä(0).

In the case of high absorbance the absorbed flux is equal to I0 and then the amount absorbed by the reactants is

/ . .B = /O/R (8)

Here /R is a function of time, /R(£), and instead of the simple Eq. (5) we obtain

-dB«) . Ψ kl0fn(t)dt w

and before this equation can be integrated it is necessary to know /R(£) as a function of time (or reaction conversion). Approximate solu-tions for special cases can be found in the literature. Many are based on the assumption that the relative change in μκ and μΡ is the same, or on other similar simplifying assumptions, e.g., μκ is constant and μΡ constant. References are given to Moring-Claesson (1956), McLaren (1949), and McLaren and Pearson (1949), where corrections due to light-scattering are discussed.

If the incident light is not monochromatic the calculation of the absorbed flux (expressed as number of photons) can be somewhat labor-ious. For example, this is the case when filters are used for the isolation of a certain wavelength region (λχ < λ < λ2). If the number of quanta emitted per unit time from the light source in the region λ to λ + d\ is denoted by S(\)d\, the transmittance of the filter at this wavelength by T(\) and the absorptance of the sample by

α(λ) = —j— = Y

then the number of absorbed photons per unit time is

Q = β £ S(X)T(\)a(\)d\ (10)

where β is a constant which depends among other factors on the geometry of the system. If a corresponding experiment is made with an actinometer solution with absorptance α^01(λ) and quantum yield φαοΐ(λ), the number of decomposed molecules per unit time will be

N = β £* S(X)*(XKct(X)0act(X)dX (11)

and thus β can be eliminated between these two expressions as all other factors are known. Thus the proportionality factor between Q and N

28 STIG CLAESSON

is obtained. In an analogous way the proportionality factor for a monitoring phototube can be determined.

The curve S(k)T(X)a(\) which gives the spectral distribution of the light absorbed by the sample is often very much sharper than the curve Τ(λ) alone. I t is therefore quite important to remember the shape of the curve α(λ) when a filter with suitable curve Τ{λ) is sought out.

In this connection it should be pointed out that the quantum yield can also be determined directly by microcalorimetric measurements. The heat developed when the light is absorbed in an inert solution and in the sample is measured and compared. Less heat is developed in the sample than in the inert solution if the reaction taking place requires energy; therefore the quantum yield can be calculated from the values obtained. Such measurements, for instance, have been made on chloro-phyll (Tonnelat, 1945). The accuracy of such measurements can be improved if the radio balance principle (Mann, 1954) using peltier-cooling is applied.

6. High Intensity Photochemistry and Flash Photolysis

The flash-photolysis technique was introduced by Norrish and Porter (1949) as a means of studying the intimate nature of photochemical reactions. I t has proved particularly powerful in giving direct informa-tion about free radicals and other short-lived intermediates (see Chapter 2). Because of the short lifetime of such species the steady-state con-centration built up during ordinary irradiations is much too low to make them directly observable. However, if the ordinary light source is re-placed by a flash lamp (gas-discharge) through which a capacitor is rapidly discharged, an intense and very short light pulse is obtained. In ordinary photochemistry a light intensity corresponding to 1017

photons/ml sec is representative. In flash experiments intensities as high as 1024 photons/ml sec have been reached during 10 to 100 /xsec —or 107 times higher. This is quite sufBcient to cause a substantial part of the sample to be converted into intermediates. The properties of these intermediates and the subsequent reactions can then be studied by different methods provided that these are rapid enough to follow the reactions which may have half-lives from ten to a few hundred microseconds. The most powerful method up till now has been kinetic spectroscopy as originally introduced by Norrish and Porter, but also other methods like the time-of-flight mass spectrometry have been tried.

Since it would be impossible to mention all the applications of the


flash-photolysis method here, only a few typical examples will be given: adiabatic reactions, explosive processes, free radicals and iso-thermal gas reactions, energy transfer, properties of triplet states in solution, and atom recombinations. A recent review article by Norrish (1962) gives further references. However, some applications have also already been made in the field of macromolecules. Transients in oval-bumin solutions have been studied by Grossweiner (1956) and Gross-weiner and Mulac (1959) and the photochemistry of heme proteins by Q. H. Gibson and Ainsworth (1957). As already mentioned another very important advantage of flash photolysis is easier interpretation of the reaction. At these very high light intensities reactions which are themselves second order with regard to the intermediates are domi-nant, consequently the entire reaction scheme is simplified. In this way it is often possible to get much more information about the primary process than in ordinary photochemistry even if only the stable end-products are analyzed and no kinetic measurements are made. Some photochemical reaction systems have already been studied in this manner, e.g., ketones and aldehydes (Wettermark, 1961) and azoethane (Roquitte and Futrell, 1962). At the same time a decrease in quantum yield is often observed in flash-photolysis studies as compared to the same amount of light continuously applied. This decrease can easily be explained in the following way. If the primary process produces a short lived intermediate A -> A* which can either decompose to products A * - * P or deactivate back to the ground state A* + A*-»2 A by a collision process, then an increased concentration of A* will lead to a greater proportion of deactivation with a corresponding decrease in quantum yield.

Flash-photolysis units have been built for varying amounts of en-ergy from a few hundred up to several hundred thousands of joules per flash, emitting from 0.0001 einstein to 0.1 einstein per flash in the region 2000Ä-4800Ä. For most purposes units producing about 1000 to 5000 joules are most practical and they are relatively easy to make. Several quite detailed descriptions of flash-photolysis units can be found in the literature (Norrish et al., 1953; Claesson and Lindqvist, 1957a; Lind-qvist, 1960), therefore only a few points of experimental interest need be mentioned here. In addition, complete flash-units of moderate size are now becoming commercially available since they are widely used as pumping sources in laser work.

o.l The Discharge Unit

I t is desirable to make the discharge time as short as possible for a given amount of energy. In a flash-photolysis apparatus the discharge

30 STIG CLAESSON

time is primarily dependent on the self-inductance L and the capacity C in the circuit. The resistance of the lamp plays a minor part, in fact it is often difficult to get it large enough to approach critical damping. The period of the oscillation frequency T = 2π \/LC can be used as an approximation for the flash duration time and both L and C should be made as small as possible. The energy in the condensers is 1/2 CV2

where V is the voltage and it is not convenient to make V very high (working below 20 kV is easy, above 50 kV difficult). Therefore C can-not be made too small and L will have to be reduced as much as pos-sible. This is done by using coaxial connections wherever possible and also by having the return leads from the lamp passing as close to and parallel to the lamp as possible (Fig. 2). In this way the self-inductance

FIG. 2. A typical flash-photolysis apparatus for 8000 joules.


can easily be reduced to about 0.01-0.1 microhenry. In a realistic ex-ample with an apparatus for 5000 joules, C may be 100 microfarads, V is 10,000 volts and L is 0.1 microhenry. Then

T =2TT VO.l X 10"6 X 100 X 10-6 20 jusec which is a typical figure for the flash duration time in modern installa-tions.

Flash lamps. Straight quartz tubes with heavy electrodes at the ends are used as flash lamps. Typical dimensions are: length 10-60 cm, diameter 1-3 cm, wall thickne&s 1-3 mm, depending on the energy used. For special projects other types of lamps have been developed; for in-stance, completely coaxial lamps where the discharge takes place in an annular space around the reaction vessel (Claesson and Lindqvist, 1957b). They have produced the highest irradiation intensities reported but have the disadvantage that they discolor rather rapidly due to deposition from the electrodes.

For large energies tungsten is the obvious choice for the electrode material of a flash-tube, but for smaller energies other metals can be used. If the electrodes are placed in suitable metal fittings they can easily be cemented to the outside wall of the flash tube (Claesson and Lindqvist, 1957a). The discharge is normally started by triggering a high-voltage spark from a third electrode and it has been observed that the delay between triggering the spark and discharge is larger for tung-sten than aluminium electrodes (R. L. Strong, personal communication).

The lamps can be filled with different gases, the inert gases like krypton giving a slightly higher light output than do other gases. Oxy-gen can also be used since it has the advantage that the lamps stay cleaner owing to the oxidation of impurities. Also, if an inert gas is used for filling and high energies are used, some oxygen is produced by decomposition of the quartz walls and this will change the firing char-acteristics of the lamp; this is avoided by oxygen filling.

Because of the high peak power during the flash a large part of the electrical energy is converted to radiation, about 15% in the UV and visible region, and the emission spectrum is almost continuous. Also the light output from flash to flash is extremely reproducible after the first few flashes. In fact the limiting factor seems to be the accuracy with which the voltage of the condenser is measured, and therefore these light sources compare favorably with other sources developed for pre-cision photometry.

The light output from flash lamps can be measured by means of chemical actinometer solutions. It has been demonstrated that their quantum yields are unchanged up to very high intensities. The only limiting factor seems to be that the actinometer should not be depleted

32 STIG CLAESSON

of reactive material and furthermore if the degree of conversion is too high, gas bubbles may be formed at the surface of the actinometer ves-sel which will affect the transmittance and destroy the accuracy of the measurements.

6.2 Monitoring System

When kinetic spectroscopy is used to follow a fast reaction mecha-nism, the limiting time factor depends not only upon the flash duration time, but also on the properties of the kinetic spectrometer. In all flash studies some stray light from the photolysis flash will enter the record-ing system. Therefore the recording system will not work well until the stray light intensity has decayed to a value which is low compared to the monitoring light intensity (Fig. 3). If the latter is high, the flash

flash profile (stray light)

N i i g h monitoring light

^ l o w

time

FIG. 3. The influence of monitoring light intensity on flash-photolysis experi-ments.

duration time appears short and measurements can be made when the transients are still present in rather high concentrations. Therefore the photometric accuracy need not be very high. On the other hand, if the monitoring light intensity is low, the flash duration time appears long and measurements cannot be made until a large portion of the transients has disappeared. Their concentration is then low and high photometric accuracy is needed. However, intense monitoring light sources (xenon arcs, zirconium arcs etc.) are less stable than low-intensity sources and the choice is not obvious. It can therefore be said that greater progress in flash-photolysis work can be made by improving the monitoring sys-tem (Rand and Strong, 1960; Witt et αΖ., 1959; Zieger and Witt, 1961) than by improving the flash sources which have already reached a cer-tain degree of perfection.

REFERENCES

Brode, W. R. (1949). J. Opt. Soc. Am. 39, 1022. Calvert, J. G., and Rechen, H. J. L. (1952). / . Am. Chem. Soc. 74, 2101.

1. PRINCIPLES AND METHODS OF PHOTOCHEMISTRY 3 3

Claesson, S., and Lindqvist, L. (1957a). Arkiv Kemi 11, 535. Claesson, S., and Lindqvist, L. (1957b). Arkiv Kemi 12, 1. Claesson, S., Nyman, B., and Wettermark, G. (1961). Proc. 5th Intern. Symposium

on Free Radicals, Inst. of Phys. Chem., Univ. of Uppsala, Uppsala, 1961, p. xxiii. Almqvist and Wiksell, Stockholm, 1961. Partly unpublished.

Fluke, D. J., and Setlow, R. B. (1954). / . Opt. Soc. Am. 44, 327. Forbes, G. S., and Heidt, L. J. (1934). J. Am. Chem. Soc. 56, 2363. Gandy, H. W., and Ginther, R. J. (1962). Appl Phys. Letters 1, 25. Gibson, K. S. (1949). Natl. Bur. Standards (U. S.) Cire. 484. Gibson, Q. H., and Ainsworth, S. (1957). Nature ISO, 1416. Grossweiner, L. I. (1956). J. Chem. Phys. 24, 1255. Grossweiner, L. I., and Mulac, W. A. (1959). Radiation Research 10, 515. Hatchard, C. G., and Parker, C. A. (1956). Proc. Roy. Soc. (London) A235, 518. Heidt, L. J., and Daniels, F. (1932). / . Am. Chem. Soc. 54, 2384. Leighton, W. G., and Forbes, G. S. (1930). J. Am. Chem. Soc. 52, 3139. Lindqvist, L. (1960). Arkiv Kemi 16, 79. McLaren, A. D. (1949). Advances in Enzymol. 9, 75. McLaren, A. D., and Pearson, S. (1949). J. Polymer Sei. 4, 45. Mahan, B. H., and Mandai, R. (1962). / . Chem. Phys. 37, 207. Mann, W. B. (1954). / . Research Natl. Bur. Standards 52, 177. Monk, G. S., and Ehret, C. F. (1956). Radiation Research 5, 88. Moring-Claesson, I. (1956). Arkiv Kemi 10, 21. Norrish, R. G. W. (1962). Am. Scientist 50, 131. Norrish, R. G. W., and Porter, G. (1949). Nature 164, 658. Norrish, R. G. W., Porter, G., and Thrush, B. A. (1953). Proc. Roy. Soc. (London)

A216, 165. Pitts, J. N., Pitts, J. N., Jr., Margerum, J. D., Taylor, R. P., and Brim, W. (1955).

J. Am. Chem. Soc. TJ, 5499. Porter, K., and Volman, D. H. (1962). J. Am. Chem. Soc. 84, 2011. Rand, S. J., and Strong, R. L. (1960). J. Am. Chem. Soc. 82, 5. Roquitte, B. C , and Futrell, J. H. (1962). J. Chem. Phys. 37, 378. Scott, J. F., and Sinsheimer, R. L. (1955). In "Radiation Biology" (A. Hollaender,

ed.), Vol. II, p. 119. McGraw-Hill, New York. Smith, R. A. (1962). Endeavour 21, 108. Smith, R. N., Leighton, P. A., and Leighton, W. G. (1939). J. Am. Chem. Soc. 61,

2299. Tonnelat, J. (1945). Thesis, Paris. Wettermark, G. (1961). Arkiv Kemi 18, 1. Withrow, R. B., and Withrow, A. P. (1956). In "Radiation Biology" (A. Hollaender,

ed.), Vol. I l l , p. 125. McGraw-Hill, New York. Witt, H. T., Moraw, R., and Müller, A. (1959). Z. physik. Chem. (Frankfurt)

[N.S.] 20, 193. Zieger, G., and Witt, H. T. (1961). Z. physik. Chem. (Frankfurt) [N.S.] 28, 273.

Chapter 2

ELECTRON SPIN RESONANCE AND ITS APPLICATION TO PHOTOPHYSIOLOGY

M. S. Blois, Jr. and E. C. Weaver

Biophysics Laboratory, Stanford University, and Department o] Plant Biology, Carnegie Institution of Washington, Stanford, California

1. Introduction Photophysiology, to the extent that it attempts a continuous under-

standing of the sequences leading from the arrival at an organism of an incident photon to an ultimate biological result, is an interdisciplinary subject of high ambition. In the overall sequence of events it would appear that frequently one's knowledge of the earliest and very last steps is the most advanced. Thus, in the acquisition of a suntan, the physics of sunlight and the synthesis of melanin are at least partially understood. About the intervening events, our knowledge is less de-tailed. It may also be remarked that the first and last stages of a photophysiological process are experimentally the most accessible.

In the hope that information regarding the more obscure intermedi-ate events may be obtained, there has been considerable interest in a relatively new technique which involves neither light nor biology but, oddly enough, magnetism. The relationship of magnetism to photo-physiology and the possibilities of using a magnetic technique, electron spin resonance, to study these intermediate processes, will be considered in this chapter. No attempt will be made comprehensively to review electron spin resonance (ESR) studies in photophysiology, but it is proposed to discuss the application, usefulness, and limitation of the method for those who are unacquainted with it.

The first observation of electron spin resonance was by Zavoisky (1945), and its first application to biological studies was due to Com-moner and his associates (1954). During the years since the discovery of the phenomenon there has arisen a vast literature describing its appli-cation to physics, chemistry, and biology. For a discussion of the physical and instrumental details of this method, the reader is referred to the treatises on electron resonance.1

1 Electron spin resonance and many of its applications are described in Ingram (1955, 1958), by Varian Associates (1960), in Blois et al. (1961a), and Androes and Calvin (1962).

35

36 M. S. BLOIS, JR. AND E. C. WEAVER

2. Principles of Atomic and Molecular Magnetism

2.1 Diamagnetism, Para magnetism, and Ferromagnetism

In order to observe magnetic resonance of any sort it is necessary that one have a sample possessing the requisite magnetic properties. Un-like the magnetism associated with flowing currents or traveling elec-tromagnetic waves, the magnetism of a sample of matter which one may observe by a resonance method originates in its fundamental con-stituents. The two sources of atomic and molecular magnetism are the paramagnetic atomic nuclei and the electrons. Both of these, because of their spin angular momentum ("spin") behave like tiny bar magnets; they have two distinguishable directions of orientation under appro-priate conditions, and their magnetism is permanent. While all electrons have a magnetic moment, the nuclei may or may not possess magnetic moments depending upon the particular isotope. If the nuclear spin of a given isotope is not zero, it will then have a given magnetic moment with the same general properties as the electron magnetic moment, al-though about 2000 times smaller. The direct observation of these nu-clear magnetic moments may be carried out in a nuclear magnetic resonance (NMR) experiment; we will not be concerned here with this phenomenon. The existence and distribution of these magnetic nuclei may, however, be inferred, somewhat indirectly, from an electron spin resonance experiment and the mechanism for this will be discussed be-low.

Since every electron has a magnetic moment, one might suppose that all matter would then be magnetic. This is not so, because accord-ing to the Pauli principle each energy level in an atom or molecule can accommodate a maximum of two electrons and these must have oppo-site spins. Thus, two electrons in the same energy level will have zero total spin and zero magnetic moment, and are spoken of as being "paired off." One would expect hydrogen to have a magnetic moment cor-responding to its single orbital electron. This is true for monatomic hydrogen, but for the case of molecular hydrogen it is found that the two electrons are paired off and there is no net electronic magnetism. With certain important exceptions, those atoms or molecules with an even number of electrons will not display electronic magnetism since the electrons will be paired. Atoms or molecules with an odd number of electrons cannot have them all paired so there should be at least one unpaired electron left over. The number of such atoms or molecules in nature is less common than might be supposed, and for the same reason that monatomic hydrogen does not occur naturally. It is energetically

2 . ELECTRON SPIN RESONANCE 37

so favorable for electrons to pair off that monatomic, odd atoms of gaseous elements invariably do so by forming diatomic molecules. In the reverse process (homolysis), when a covalent bond in an even mole-cule is broken, the electron pair which formed this bond becomes dis-rupted and the two molecular fragments may now each have one un-paired electron. Each of these fragments, which by definition is a free radical, has a resultant magnetism conferred by the odd electron.

Before the electronic structure of matter was known, and long before magnetic resonance was discovered, substances were divided into three classes on the basis of their gross response when placed in a steady in-homogeneous magnetic field. If the substance in question is placed in a container of uniform cross section and then suspended from a balance into a magnetic field as shown in Fig. 1, one of three types of behavior

o o I

o I

DIAMAGNETISM PARAMAGNETISM FERROMAGNETISM

FIG. 1. The macroscopic classes of matter as defined in terms of a static mag-netic susceptibility measurement.

will be noted: 1. If there is a weak repulsive force tending to push the sample out

of the magnet gap, the sample is said to be diamagnetic. 2. If there is a weak force tending to pull the sample into the gap,

the sample is said to be paramagnetic. 3. If there is a strong force of the order of a 1000 times that in the

paramagnetic case drawing the sample into the gap, and certain addi-tional properties are displayed, the sample is called ferromagnetic.

For a single atomic or molecular species it is possible to have com-binations of diamagnetism and paramagnetism, or diamagnetism and ferromagnetism, in the same sample. With a mixed, contaminated, or complex sample all three effects may be simultaneously present. I t may be relatively difficult to separate these since in such an experiment one measures the sum of these effects. An understanding of the mechanisms responsible for these effects is most helpful.

Diamagnetism may be considered the atomic scale consequence of

38 M. S. BL0IS, JR. AND E. C. WEAVER

Lenz's law. When an atom or molecule is placed in a magnetic field, its electrons (regardless of number, or oddness-evenness) may be considered to alter their orbits in such a way that the new orbits—which may be thought of as tiny circulating currents—set up a magnetic field op-posed in direction to the original applied field. Since this magnetic field induced in the sample is opposite to the applied one, the energy of the system can be reduced by the sample moving out to a region of space when the applied magnetic field is weaker. Since too, this induced magnetic field arises regardless of the configuration or number of elec-trons, one would expect to find this effect in all atoms or molecules. This is found to be so, and diamagnetism is a property of all matter, but observable only in response to an externally applied magnetic field.

Paramagnetism, on the other hand, derives from the magnetic mo-ment of the unpaired electron or electrons of an atom or molecule (or from the magnetic moment of the nucleus in the case of nuclear para-magnetism). Such unpaired electrons therefore are the sine qua non for paramagnetism, and, if present, give the material permanent magnetic properties. When such a material is placed in a magnetic field, the in-dividual magnetic moments tend to line up with the external magnetic field. In this orientation the total energy of the system is reduced if these magnetic moments (and the sample itself) can move to a region of greater magnetic intensity, and the sample is thus drawn into the magnet gap. While these individual magnetic moments attempt to line up with the external magnetic field, they are subjected to the buffetings of the random thermal motions of the system. At any combination of magnetic field strength and temperature an equilibrium will be reached between these orienting and disorienting factors so that the average alignment (or more properly, the paramagnetic susceptibility) is pro-portional to β~μΗ/1ίΤ when μ is the magnetic moment of the atom or mole-cule, H is the applied field, k is Boltzmann's constant, and T is the absolute temperature. The phenomenon of paramagnetism thus involves the temperature, and under experimental conditions in which μΗ <^kT (the usual case) this dependence is approximately 1/27. Note that in diamagnetism there is no such effect and the induced magnetic moment is exactly opposite to the external field and of characteristic magnitude regardless of temperature. Finally, there is a third type of behavior pos-sible with atoms or ions which individually are paramagnetic, as for example in solution or in some of their compounds. If these are appro-priately accommodated in the solid phase so that they may act coopera-tively instead of individually, it is found that the individual magnetic moments may line up parallel with one another. This gives rise to a much greater total magnetic moment than the simple sum of magnetic

2. ELECTRON SPIN RESONANCE 39

moments, the net force acting toward the magnet gap is increased, and the phenomenon is called ferromagnetism. Note that the existence of this imposes special spatial requirements on the system such that the so-called "exchange interaction" permits complete alignment of magnetic dipoles against thermal disordering effects. This phenomenon is restricted to limited temperature ranges and for any ferromagnetic material there exists a temperature—the Curie temperature—above which thermal dis-orientation takes over and the material alters its behavior from ferro-magnetic to paramagnetic. Ferromagnetism, since it is a solid state property, might not appear of much biological interest but we shall later have occasion to reintroduce it. Our main preoccupation will be with paramagnetism and the electron spin resonance due to individual, independent, unpaired electrons.

2.2 The Origin of Paramagnetic Species in Organic Systems

Paramagnetism occurs when an atom or molecule has one or more unpaired electrons. One may consider now the various systems which contain such electrons.

2.2.1. TRANSITION ELEMENTS

As electrons are successively added to the valence shells of atoms in building up the periodic table, those of odd atomic number will have an odd number of electrons. However, in nature, as has already been discussed, such odd atoms are usually found in diatomic form so the molecule will have an even number of electrons. This is the case with the diatomic gases, H2, N2, and Cl2, none of which is paramagnetic. Other elements, such as the alkali metals, which have an odd number of electrons, exist as the monovalent, diamagnetic ions, or as the solid metal in which the valence electrons are paired off in the conduction bands. In general, atoms with an odd number of electrons in their valence shells are not found naturally in this form, but will have under-gone some process to lower the energy of the system by pairing off these electrons. This, however, is not the case of the transition ele-ments. Here there is an unfilled inner shell, which if it contains an odd electron cannot pair it off by entering into the solid state. For example, V, Cr, Mn, Fe, Co, and Ni, all with incompletely filled 3d shells, are all paramagnetic as isolated atoms and frequently so as ions. Since most, if not all of these, occur in biological preparations it frequently be-comes of importance to be able to distinguish between the ESR signals of the transition elements or their ions, and those of organic free radi-cals.


2.2.2. FREE RADICALS

In contrast to the ionic bond which undergoes dissolution in such a manner that the electron pair passes intact to one or the other fragment (heterolysis), the breaking of a covalent bond commonly finds the elec-tron pair separated, with the result that each fragment has an unpaired electron (homolysis). Because of the reactivity associated with the free valence, the free radicals so formed tend quickly to undergo fur-ther reaction to pair off the odd electron and achieve stabilization. However, the lifetime of an organic free radical may, under especially favorable experimental conditions, be prolonged so that its observation may be accomplished. This may be achieved in a number of ways: Many gaseous free radicals may be stabilized at liquid helium tem-peratures almost indefinitely (Bass and Broida, 1960) ; organic free radicals which enter into polymerization reactions are sometimes found trapped in the resulting polymer and prevented from further reaction (Bamford et al., 1955) ; and aromatic free radicals may possess a degree of resonance stabilization that makes them relatively long-lived. As an example, hexylphenylethane in benzene solution spontaneously forms two triphenylmethyl radicals to the extent of a few per cent, with which it remains in equilibrium. Organic free radicals thus arise from covalent bond breakage (radiolysis, pyrolysis, photolysis, etc.), univalent oxida-tion or reduction of an even molecule, and under appropriate circum-stances, the ionization of an even molecule.

2.2.3. BI-RADICALS

In a highly conjugated molecule one might expect to find, at most, one unpaired electron. Thus, if the naphthalene molecule becomes singly ionized by reaction with an electron donor such as metallic sodium, it will have one unpaired electron and be paramagnetic. If, however, it becomes doubly ionized by the addition of a second electron, it turns out that the two electrons which were added will be paired off and the molecule will be diamagnetic. For this pairing off to occur, however, it is necessary that the electrons in question occupy molecular orbitals which extend essentially over the entire molecule. A molecule consisting of two aromatic groups separated by a long saturated aliphatic chain would be an example of a species in which an unpaired electron could be present at each end. Such a molecule, while having an even number of electrons, is known as a bi-radical and will be paramagnetic since the unpaired electrons cannot communicate so as to pair off. This mechanism could be conceptually extended to the case of an irradiated


solid polymer in which a given molecule could have several highly localized unpaired electrons, and thus qualify as a poly-radical.

In the case of the Chichibabin hydrocarbons, which are frequently used as examples of bi-radicals, the electron mobility is not as restricted as in the foregoing examples and they could equally well be considered as having undergone excitation to a triplet state.

2.2.4. TRIPLET STATES

In the preceding discussion of paramagnetic systems it has been assumed that the atoms or molecules were in the unexcited, or ground state, and that the magnetism was independent of any excitation proc-ess. Another source of paramagnetism is illustrated by a number of photosensitive fluorescent compounds which upon illumination undergo an excitation such that the two electrons—originally associated with a particular bond and antiparallel—become unpaired and aligned with their spins parallel. For this to be possible, one electron must be in a different energy level from the other, although they still interact. The total spin of these coupled electrons can then take up three different orientations in an applied magnetic field, each of different energy, so that the ESR spectrum should consist of two lines. Such a molecular state is termed a "triplet state." Since such a molecule has two unpaired electrons, it is occasionally referred to as a bi-radical, although it seems preferable to restrict the latter term to molecules with two relatively uncoupled electrons in Kramers' doublet state.

Although most triplet states result from an excitation process, and at physiological temperatures must decay back to a diamagnetic ground state, there is one noteworthy exception. The normal state of molecular oxygen is 3Σ, so that while it has an even number of electrons, its low-est energy electronic configuration is that with two of them unpaired. Molecular oxygen is thus strongly paramagnetic. Molecular sulfur (S2), owing to its similarity to oxygen, might also be expected to exist in a triplet state. Magnetic susceptibility measurements, however, have shown that while sulfur vapor is paramagnetic, sulfur in the liquid or solid phase is diamagnetic.

The experimental study of triplet states has been carried on for many years with the magnetic susceptibility balance. When the ESR technique became available, offering several orders of magnitude more sensitivity, it was quickly applied to the study of triplet states. Many experiments were conducted in the attempt to observe the ESR of 02, and of intensely illuminated fluorescent compounds, under conditions in which it was known that the number of unpaired electrons should give


resonance signals with signal-to-noise ratios of 1000 or so. These at-tempts uniformly failed. The reason for this difficulty was that the samples had been prepared as glasses or frozen solutions, in which form they are satisfactory for static susceptibility measurements, but the random orientation of individual molecules results in such excessive line broadening that they are unobservable by ESR. This limitation was first overcome by Hutchison and Mangum (1958) who grew mixed crystals of naphthalene in durene. The oriented naphthalene molecules were then excited to the triplet state by UV illumination and the elec-tron spin resonances were observed. The details of the mechanism leading to this absorption line broadening in polycrystalline or amor-phous samples are discussed by Ingram.

The need for orienting (and perhaps diluting) molecules, if their triplet state is to be observed, raises obvious difficulties in the case of photobiological systems. The great increase in the amount of informa-tion which ESR may give (Whiffen, 1961) in such oriented systems, not only with respect to triplet states but of the hyperfine interaction as well, renders this difficult task more attractive.

3. Electron Spin Resonance Method

3.1 The Magnetic Resonance Phenomenon

In order to observe an electron spin resonance, it is a necessary but not always sufficient condition that one have a sample containing un-paired electrons, usually in one of the forms described above. If such a set of electrons is placed in a steady magnetic field, its behavior may be described as follows. The magnetic moment of the electron (Fig. 2a)

„ H, + f

ΧϊηέΚΓ»« iP<J Ho

(a) (b) (c)

FIG. 2. The Larmor precession of an electron in a magnetic field, (a) The magnetic moment μ, associated with an isolated electron, (b) the precessional motion of μ in an applied magnetic field H0, and (c) the introduction of a second, oscillating magnetic field Hly at perpendicular to H0.

may be represented by the dipole y, a vector quantity since it possesses both magnitude and direction. We may represent the magnitude and direction of the applied (steady) magnetic field by a second vector H0.

■/


The magnetic field H0 which may be produced by a laboratory magnet, will be assumed to be stationary, but the orientation of μ need not be. It is known, as a matter of fact, that the dipole moment t* will precess about H0 as shown in Fig. 2b for the analogous dynamical reasons that a spinning top precesses about the vertical, due to the gravitational field of the earth. This precessional motion of the magnetic dipole may be characterized by its angular velocity ω and it is found that ω is proportional to H0

ω = yHo (1) where the constant of proportionality, γ, is known as the gyromagnetic ratio. Thus, as the intensity of the magnetic field is increased, so is the precessional velocity of the dipole. If the value of the gyromagnetic ratio for the free electron is substituted into the preceding expression, and one solves for the frequency instead of the angular velocity

v = (megacycles/second) = 2.80261?o(gauss) (2) This Larmor precession, as it is called, is a purely passive process

and by itself does not allow us to detect unpaired electrons. If, how-ever, a second, alternating, magnetic field is provided at right angles to H0 (which in Fig. 2c is denoted as Ht), one may disturb the motion of the dipole in an informative way. It is found that for quite small values of Ht—that is, where ίΖΊ <ξ Η0—if the frequency of the alternating field is just equal to the precessional frequency of the dipole, a resonance transfer of energy occurs between the energy source of H1 and the sys-tem of precessing dipoles. Since the low energy configuration of the dipoles is that in which their direction is generally along the direction of Ho, one may think of them as absorbing energy from the alternating field Ητ and then reversing their direction to point oppositely to H0. This resonance absorption of energy is in fact observable, and its detec-tion is the function of the ESR spectrometer.

It should not be concluded that one may indefinitely supply energy at the resonance condition to a set of unpaired electrons and find it to be absorbed. After a specified fraction of the electrons have been raised to the upper energy state (the antiparallel one) no further resonance absorption can take place and the system is said to be saturated. The electrons raised to the upper state do not remain there indefinitely, how-ever, since they are coupled magnetically to the other nearby electrons of the sample and they will revert to their lower state by giving up their energy to the bulk of the sample in the form of thermal energy. The rate at which the unpaired electrons will absorb the applied microwave energy and thus give an observable signal is limited by the rate at which these


upper level electrons can drop back to their original lower level and provide vacancies in the upper level for other electrons to occupy. This rate is a property of the individual molecule and is related to its relaxa-tion time. It is clear then that once the applied microwave power is sufficient to raise all the available electrons to their upper state, further increases in power will have no effect upon the intensity of the resonance absorption.

Instead of visualizing the Larmor precession of dipoles as described by Eq. (1), one may consider the ESR phenomenon in a form resembling conventional spectroscopy. The separation of the two energy levels in-volved in the transition is AE — gßH0 where g is the spectroscopic splitting factor (for a free electron g = 2.00229), ß is the Bohr magneton, and H0 is the d.c. magnetic field. The quantum energy of the microwave field is hv, so that in place of Eq. (1) one may write as the condition for resonance:

hv = gßH0 (3) Since g is a property of the unpaired electron in its particular

environment, it is a characteristic of the free radical or paramagnetic system being observed.

The experimental approach then consists in placing the sample in a steady magnetic field H0 (the magnitude of which we may choose at will) and arranging a second, alternating magnetic field at right angles to the first. Resonance will then be observed when H0 and v satisfy Eq. (2) or Eq. (3). Although in principle one can use an arbitrarily small H0 and correspondingly low-frequency alternating magnetic field, it is found that for the best sensitivity—in order to detect the least number of unpaired electrons—the frequency should be as high as practicable. Since the sensitivity increases with the square root of the frequency it proves convenient to operate in the microwave region; X-band (^9500 mc/sec) being most commonly employed. The sensitivity gain obtained by going to still higher frequencies, say K-band (^23,000 mc/sec), is somewhat offset by the expense and inconvenience of the small micro-wave components required. As one increases the microwave frequency, the field H0 required for resonance also increases, so that while at X-band one needs a uniform field of about 4000 gauss, which is easily obtained, at K-band this has become 10,000 gauss which is less con-venient. Since the operating frequency, v, is so high, a klystron oscillator is ordinarily employed as the source of energy and it is a property of this device that while it is easily tuned over narrow frequency ranges it cannot be tuned over broad ones and hence it is used most effectively at a fixed frequency. Of the two parameters v and H0 it is thus eus-


tomary to fix the value of the former and then vary the value of H0

in order to satisfy the resonance condition. In its simplest form, the experimental problem consists in varying the ratio of H0 to v until a resonance is observed. In contrast with this conceptually simple process, the experimental realization involves considerable detail.

3.2 Experimental Methods

Since several types of ESR spectrometers are commercially available, it is much more probable that the biologist will purchase rather than construct such an instrument. Nevertheless, the most effective use of a spectrometer requires more than a casual acquaintanceship with its mode of operation.

The essential elements of a typical ESR spectrometer are shown in Fig. 3. The magnet, which provides the field H0, is most conveniently

FIG. 3. The components of an ESR spectrometer. The console at left houses the controls, modulation equipment, klystron power supply, detection, and display equipment. In the center is the electromagnetic unit, with the klystron and microwave bridge unit on the shelf. The waveguide is seen extending into the magnet gap where it is coupled to the sample-containing cavity. At the right is the magnet power supply. (Courtesy of Varian Associates.)


an electromagnet in order that this field may be varied over wide limits to provide the resonance conditions for odd electrons having quite differ-ent g values. The range 0-6000 gauss is typical. In addition to variability, the magnet field must accurately maintain the value to which it is set, despite line voltage and temperature fluctuations. This is provided for by a suitably regulated power supply. In order to vary the field H0

slowly and smoothly over a present range, an automatic scanning device is employed, ordinarily with a choice of several scanning speeds. Finally the field H0 should have the same value over all parts of the sample— so that unpaired electrons in all portions of the sample will come into resonance simultaneously. Sample sizes in ESR (at X-band) are of the order of 1 inch in length, so the magnet must provide a region of at least an inch in diameter, over which the change in H0 is less than some predetermined value. If the difference of H0 over the sample is greater than the ESR line width (expressed in gauss), the resolution of the line will be degraded. Since ESR line widths in biochemical systems are usually from tenths of a gauss to perhaps a hundred or more the necessary magnetic field homogeneity can be specified. For a given gap between the pole pieces—necessary to accommodate the sample cavity and waveguide—and for comparable precision of work-manship, the field homogeneity increases with pole face diameter. Most ESR work is carried out with magnets having pole faces about 6 inches in diameter. This is contrasted with NMR which, because of the very narrow lines (milligauss) requires a larger (and more expensive) magnet.

The source of microwaves is a klystron tube which in turn requires its own highly stable power supply, and a waveguide structure to lead the electromagnetic energy to the sample. The latter is placed in a suitably designed cavity which in turn is coupled to the waveguide. The microwaves, as with all electromagnetic waves, consist of coexisting electric and magnetic fields, but it is only with respect to the latter that the ESR phenomenon is concerned. The cavity is therefore designed in such a way that the magnetic field of the microwaves is a maximum at the sample, and that the electric component is a minimum or zero. An experimentally unfortunate feature of X-band microwaves is their di-electric absorption by water, or other polar substances. Since, however, this dielectric loss takes place through the interaction of the sample with the electric field of the microwaves, it is minimized, but never eliminated, by using a cavity designed as above. A feature of the cavity which leads to increased sensitivity is the so-called "Q" which, among its several interpretations, can be thought of as a measure of the number of times the microwaves are reflected back and forth through the sample,


thus increasing the opportunity of interaction. Most cavities, before introduction of the sample, will have a Q of as high as several thousand. If a dry sample, such as a protein, is introduced this may fall to 80-90% of its original value. When a solution made up in a nonpolar solvent is introduced, the Q may fall to several hundred; if the solvent is water the Q may fall to a hundred or less. The signal-to-noise ratio of the apparatus is proportional to the square root of Q.

The usual cylindrical sample tube has dimensions of about 3 cm X 1-2 mm, in the cavity, and is located so that the axis lies along the ff-field maximum. Since the sample has finite diameter, much of the sample volume will lie in a region where the electric field is not exactly zero, and it is because of this that the dielectric loss (and decrease in Q) occurs. In order to increase the sample volume—upon which the ESR absorption intensity will depend—and not proportion-ately increase the losses, sample holders have been devised wherein the sample is maintained in the form of a thin sheet. In general, sample holder design is one of the experimental variables which is determined by the nature of the system being observed, the purpose of the experi-ment, and ingenuity of the user.

A number of modifications to provide for particular experimental conditions have been designed. For photobiological or photochemical studies it is necessary to provide a means of illuminating the sample. This ordinarily requires additional openings in the cavity, which must be located in such a manner that the flow of high frequency electric currents in the cavity walls is not excessively disturbed. Such cavities are commercially available.

The control of sample temperature can be achieved over the range from 77°K (liquid nitrogen) to perhaps 400°K with the use of fairly inexpensive accessories. To extend the temperature range downward requires more complicated Dewar flasks, and the use of liquid helium or other low boiling gases. Provision of sample temperatures above 100°C require special provisions and are probably not of great interest for biological purposes. For observation at fixed temperatures, e.g., that of liquid N2 or frozen C02, small Dewar flasks can be constructed which will contain the refrigerant and sample and fit directly into the cavity. If it is desired to provide a continuously variable temperature, a gas flow system may be arranged in which dry nitrogen is passed through a heat exchanger which is immersed in a refrigerant (e.g., liquid nitrogen) and is then allowed to flow around the sample. By regulating the gas flow rate, and using auxiliary resistance heating elements in the gas stream, if necessary, the temperature may be brought to a predetermined


value. A thermocouple may be inserted in the sample tube in order to measure its temperature and, if it does not extend too far into the cavity, it will not interfere with the operation of the spectrometer.

3.3 Application to Biological Systems

From what has been said previously regarding the dielectric loss of water or polar solvents it follows that the observation of biological samples involves a compromise. One may observe dried materials, accepting a reduction in biological relevance, with high ESR sensi-tivity. A fully hydrated biological sample or an aqueous solution, how-ever, implies a degradation in spectrometer performance. This may be offset by two approaches: (1) The use of low temperatures, since ice has a smaller dielectric loss than liquid water, or (2) the use of sample holders which restrict the sample in or very near the electric field node of the cavity. The latter is of most general applicability and is crudely approximated by the usual capillary sample holder. A better technique is to employ a flat-sided cell which is carried in a mounting providing for careful positioning in the cavity. I t has been shown by Feher (1957) that the maximum sensitivity is obtained when the sample is of such size or shape that the cavity Q is reduced to two-thirds of its original (unloaded) value. The compromise is between the two competing factors; the dielectric loss of water and the filling factor (essentially the amount of paramagnetic material in the cavity). By making the sample volume smaller the loss of signal intensity due to water is reduced, but then so is the filling factor.

Assuming a given spectrometer sensitivity (ordinarily about 2 X 1011

AH unpaired electrons, where AH is line width in gauss, for commercially available instruments), and a sample suitably prepared to optimize the chances for observing a resonance, what kinds of information will an ESR observation yield?

The very appearance of an absorption indicates the existence of unpaired electrons in the sample, and the intensity of the absorption is a measure of their number and/or state. The inverse conclusion can-not be drawn; failure to observe resonance does not strictly imply an insufficiency of unpaired electrons ("spins") in the sample since it is possible that the local molecular environment may so broaden an absorption line that it becomes undetectable, as discussed in the case of the triplet state. This is more of a theoretical than practical limita-tion since the conditions for this are rather special and in most experi-ments do not prevail. I t should be noted that, in this instance, classical magnetic susceptibility measurements may disclose the presence of paramagnetism in such a system.


In order to determine the number of unpaired electrons in a given sample, the usual procedure is to calibrate the spectrometer with standard samples having known spin concentrations. Since the graphical display of an ESR resonance is frequently in the form of the first derivative of the actual absorption line, it becomes necessary to perform a double integration. These may be performed graphically, or by appropriate integrating devices. The result of the first step is to give the actual absorption line, and the second then gives the area under this curve which is proportional to the number of unpaired electrons. An approxi-mate method, which is applicable directly to the derivative curve, is to use the product of the peak-to-peak height and the square of the half-width of the absorption line (equal to the peak-to-peak displace-ment along the abscissa). For successive measurements on the same sample, or on any samples having the same line shape and width, the peak-to-peak height is proportional to the number of spins. An alternative method of determining the integrated absorption, which is more precise than the graphical double integration method, is dis-cussed by Köhnlein and Müller (1962).

It is convenient to choose standards for spin density measurements, having line-widths comparable to those of the experimental system, and of these, a,«-diphenyl-/?-picryl hydrazyl (DPPH) as a solid or in solution, gadolinium or vanadium salts in solution, and organic chars or pitch have been used.

The g value of the free electron is close to 2.00229, but the g values of free radicals or paramagnetic ions will differ slightly from this. The experimental determination of the g value requires the simultaneous measurement of the magnetic field H0 (at the position of the sample), and the microwave frequency, at resonance. Since these two quantities may be determined with considerable precision (Blois et al, 1961b), knowledge of the g value of a signal may be helpful in identifying its source or in eliminating certain alternative choices. The g value is not a unique property of a given paramagnetic species, however, but, as with melting points or absorption spectra, may be useful supportive evidence in identifying a molecule.

Another type of information revealed by ESR which diagnostically is much more helpful in identifying the origin of an absorption is the hyperfine interaction. The origin of the hyperfine structure seen in appropriate ESR spectra may be understood qualitatively in the follow-ing way. We may consider the semiquinone of p-benzoquinone (Fig. 4). When prepared by alkaline oxidation of the hydroquinone, this free radical will have lost both hydroxyl hydrogens—one through ionization, leaving the electron pair undisturbed, and the other by univalent oxida-


l I I I I

f t M tttl Ml! t i l l t i l l Hit Ulf Ulf m l π π li lt I I I ! t i l l l i l t UM Mil t i l l

" Γ 4 6 4 1

FIQ. 4. The modification of the applied magnetic field H0 by nuclear magnetic moments present in a molecule gives rise to the ESR hyperfine spectrum.

tion so that there remains an unpaired electron on the molecule. This unpaired electron may then be regarded as not residing at a particular site, but associated with the molecule as a whole and interacting with each of the atoms present. The four ring hydrogens each have a mag-netic nucleus, (the proton) and these magnetic moments will be also affected by the magnetic field Ηϋ. Specifically, a given proton moment will be oriented either parallel or antiparallel with H0. In the semi-quinone molecule of Fig. 4 all four proton moments are shown oriented parallel to Ηϋ. Since they act as tiny bar magnets they will produce a small magnetic field of their own, and this field, together with H0, comprises a modified magnetic field actually experienced by the unpaired electron. In the configuration sketched, these small magnetic fields may be considered as additive to H0) in which case the unpaired electron will find itself satisfying the resonance condition at an apparently lower applied field than otherwise. If two of the proton moments are parallel to H0, and the other two are antiparallel, their individual mag-netic fields will cancel and the unpaired electron will be acted on only by H0. The effect of the other combinations is obvious, and it will be seen that in a sample containing a great many such molecules, each of which will have its proton moments in one of the five possible arrange-ments, there will be five individual resonance lines, as molecules having these arrangements satisfy in turn the resonance condition. Since these arrangements of proton moments are equally probable (for our present purposes), it follows that the numbers of molecules in each configuration will be proportional to the number of permutations possible in each arrangement. These permutations are enumerated in the figure, and it is found experimentally that the absorption lines in the spectrum of this


free radical enclose areas in the ratio 1:4:6:4:1. In a case such as this, in which the hyperfine splitting arises from electron interaction with protons, and the protons are equivalent, it is found that the hyperfine spectrum consists of (n-\-l) equally spaced lines where n is the number of protons. The nuclei of other atoms may be magnetic as well, but for organic compounds, nitrogen with spin 1 is the principal other nucleus of concern. The general expression for the number of hyperfine components is (21+1) where i" is the nuclear spin. The displacement of the hyperfine lines from the position of symmetry of the spectrum depends upon the coupling of the unpaired electron to the magnetic nuclei. In the previous example the four ring protons interact equally with the unpaired electron and they were described as being equivalent. In the case of the semiquinone of o-quinone this is no longer true and the two protons nearer the oxygens interact differently with respect to the unpaired electron than the other two protons. One thus has two sets of two equivalent protons and the number of hyperfine components is predicted to be {n+1) X {n + 1) = (2 + 1) X (2 + 1) or nine components. Since the number of hyperfine components increases with the number of magnetic nuclei and with their degree of non-equivalence, it is obvious that free radicals of moderate molecular weight may have a very complex spectrum.

A final word of caution regarding the general application of ESR to biology seems appropriate. This is with regard to the contamination of samples by ferromagnetic particles, e.g., microscopic chips of iron or its alloys. In the use of ESR in physics and chemistry, this type of contamination seems not to have been much of a problem since the samples ordinarily studied produce intense, narrow absorption lines. However, when biological tissues are manipulated (homogenized, cut, or handled), they are frequently in contact with metallic iron at some point, and if extracts so prepared are later studied with the maximum ESR sensitivity, it will be found that they frequently show broad ferromag-netic resonances. When these signals are studied at low temperatures they will be found to be essentially independent of temperature, unlike the 1/27, or Curie, dependence of a paramagnetic system. The magnitude of the absorption due to a microscopic flake of iron, invisible to the naked eye, is somewhat surprising, though perfectly accounted for by the greatly increased magnetic susceptibility of iron in ferromagnetic form.

3.4 The Application of Electron Spin Resonance to Photobiology

Inasmuch as the study of a photobiological system by ESR is primarily a biological investigation, it may be useful first to comment


on the application of ESR to biological research as a whole. The most significant contribution of this technique has probably been the un-equivocal confirmation of Michaelis' prediction that biological oxida-tions are one-electron transfers; that is, that free radicals normally occur in living systems. A second application of ESR having an in-creasing importance is the study of the transition element ions in their biochemical environments.

In addition to the unpaired electrons associated with single electron transfers, it had long been suspected that photolytic and photoexcitation processes involve electron unpairing and it was natural to examine these mechanisms in organisms by means of ESR. As in other biological experi-ments, ESR has been applied to a wide range of systems from intact living organisms to single, purified biochemicals.

In the field of photobiology itself the major application has been to the study of photosynthesis and it is with this problem that we will be mainly concerned. However, the general usefulness of the spin resonance technique is shown by its application to observation of the effects of UV and visible light on amino acids, peptides, and proteins (B. T. Allen and Ingram, 1961 ; Gill and Weissbluth, 1962) ; on photo-dynamic systems (Smith et al, 1961) ; in various enzyme reactions (Commoner and Lippincott, 1958) and the light-induced paramagnetism in the melanin granules of the beef eye (Sever et al., 1962).

3.5 Electron Resonance Studies in Photosynthesis

The biophysics and biochemistry of photosynthesis form a vast and complex field of research. A survey of the present status of this field is to be found in the present volume (Chapters 5, 6, and 7) to which the reader is referred for the context in which any electron spin reso-nance studies must be placed. The interpretation of observations made with this instrumental technique should be in harmony with mechanisms proposed on the basis of other types of studies, and will, it is hoped, eventually enable one to distinguish between alternative theories of photosynthetic systems involving single electron transfers.

Considerable effort to make electron resonance a useful tool in photosynthetic studies followed the early observations on lyophilized material by Commoner et al. (1954). Its potentiality is revealed by the fact that one does observe a resonance in photosynthesizing cells or chloroplasts and that it changes with illumination. However, by itself this is neither surprising nor useful information. For this purpose it is necessary to identify the origin of the resonance, to characterize its behavior under various environmental conditions, and to establish its generality or uniqueness.


The modern electron resonance spectrometer is sensitive enough to detect, and gives a basis for estimating, unpaired electron concentra-tions in living material under circumstances approaching physiologically normal conditions. A typical procedure in using this instrument is as follows: The material to be examined (whole cells, chloroplasts or chloroplast fragments, photosynthetic bacteria, etc.) is pipetted into a flat quartz cuvette, which is then placed in the microwave cavity of the spectrometer and in the region of maximum magnetic field. The photosynthetic material is illuminated through a slotted opening in the cavity, the magnetic field is varied (swept), and a signal approximating the first derivative of an absorption is recorded by some suitable means. Typically, this light-induced resonance is centered at g = 2.0025, is 7 to 9 gauss from peak to peak, and shows no hyperfine structure. At room temperature the signal rises to a maximum amplitude from the dark level in a few seconds, and, after the light is turned off, decays in about the same time. Both slow and fast components have been reported for rise and decay times. A signal with these characteristics has been de-scribed by a number of groups (Commoner et al, 1956, 1957; Sogo et al, 1957, 1961; M. B. Allen et al, 1962) and although it has been variously designated, it seems safe to assume that the phenomenon is a general one, and that the same signal is seen in every case. I t will be referred to herein as the R (rapidly decaying) signal.

A residual signal is observed in green plant material when the light is turned off, and may remain for minutes or hours. There is good reason to believe that it is a quite different resonance, since it is centered at about g = 2.005, is about 20 gauss wide, and exhibits partially resolved hyperfine structure, with five or six equally spaced peaks 5 or 6 gauss apart (Commoner et al, 1957; M. B. Allen et al, 1962). Figure 5 illus-trates the two signals, as observed in living Chlamydomonas reinhardi. Both R- and S-signals appear superimposed under illumination, but only the S-signal, with its slow decay rate, is seen a few seconds after the light has been turned off. Although the S-signal has been termed the "dark" signal, it has been demonstrated in at least one case (in Chlamydomonas) that light is essential to its appearance. I t decays completely in the dark, but can be reinduced by a short exposure to a low level of light, even the stray light in a semidarkened room. There has been one report of an S-signal in Chlamydomonas which did not decay at room temperature, even after several hours in the dark (Levine and Piette, 1962). However, even in this instance, light was necessary for its initial appearance.

These two light-induced signals behave differently when various environmental factors are altered, and there are a number of reasons


FIG. 5. The two ESR absorption lines of Chlamydomonas reinhardi at room temperature. The R-signal is obtained upon illumination and the S-signal is present in the dark.

to believe that they represent two different, and perhaps quite unrelated, paramagnetic systems. Table I summarizes some of the observations which have been reported.

The conclusion seems inescapable that the R-signal is associated with chlorophyll. I t is found wherever there is chlorophyll, not only in living material and in fresh chloroplasts, but in methanol extracts (Calvin, 1961), in crystalline chlorophyll, and in dried chloroplasts (Sogo et al., 1957; Smaller, 1961). Mutant species or etiolated leaves which lack chlorophyll have no light-induced signal (Bubnov et al., 1960; Weaver and Weaver, 1962). A dark-grown mutant of Chlamydomonas (which is similar to the wild type in the light, but which forms no chlorophyll if it is grown in the dark) has been employed for a


TABLE I A SUMMARY OF THE OBSERVATIONS MADE BY ESR ON PHOTOSYNTHESIZINQ

ORGANISMS AND SYSTEMS DERIVED FROM THEM

Observation

Occurrence

Probable source of

Shape of first derivative

Half-width Half-width with D20

substitution g Value Microwave saturation Light saturation Time constants (time to

reach one-half maximum amplitude)

Rise at 25°C Decay at 25°C

Rise—low tempera-tures

Decay—low tem-peratures

Temperature effects on signal amplitude

Effect of Mn deficiency

Effect of DCMU Effect of water washing Effect of aging Action spectrum

R-signal

All photosynthetic material examiried, chlorophyll extracts," dried chloro-plast films,6 crystals0

Chi a / P700,' pyrrole nucleus*

Simple, unstructured

7-9 gauss 3 gauss7'

2.00251 Saturates at low levels7

Saturates at high levels* 0.2 sec (fresh

L chloroplasts)0*"*

2 sec (dialyzed chloroplasts) 10-15 sec (washed chloro-

plasts' 25 sec (dialyzed chloroplasts

Unchanged"

Usually slowed"

Increases to —15°, de-creases*1 with lower tem-peratures

Decreases0·2' Enhanced* Enhanced* Enhanced*·* Enhanced* Maximum corresp. to Chi

maximum,d,m or is at longer wavelength*'*

S-signal

Absent in purple bacteriad,e,°

Chi b / plastoquinone*·"*

Partially resolved hyperfine structure

About 20 gauss 9 gauss

2.0046 Saturates at high levels7

Saturates at low levels*

l

Minutes to hours*

No change noted

Decreased*

Unchanged* Decreased* Decreased* Induced preferentially by

short wavelengths (no action spectrum reported7

« Calvin (1959). * Tollin ei aZ. (1958). «Smaller (1961). d Androes et al. (1962). «Calvin (1961). ' M. B. Allen et al (1961a). « Beinert et al. (1962). A Smaller (1962).

* Bishop (1961). » Commoner (1961). * Weaver and Weaver (1962). 1 Commoner et al. (1957). m Weaver (1962b). » Calvin (1959). * Tanner et al. (1960). * M. B. Allen et al. (1962).


decisive test. The dark-grown culture has no R-signal, but successive aliquots exposed to light for lengthening periods of time develop both chlorophyll and an R-signal (Androes and Calvin, 1962).

Further evidence that the R-signal is associated with chlorophyll is provided by action spectra for the amplitude of this signal. There have been a number of reports of action spectra in which the maximum lies on the long wavelength side of the absorption maximum for chlorophyll, which is about 680 ηΐμ. There is a strong possibility that the reason for this shift lies in self-absorption effects. If the material to be examined is diluted sufficiently, an action spectrum is obtained in which the maximum corresponds to that of the absorption spectrum. The problem of achieving a signal-to-noise ratio which is good enough for getting accurate measurements limits the degree to which the dilution can be made, and necessitates many determinations of each point. Nevertheless, An-droes et al. (1962) have recently published action spectra for Rhodo-spinllum rubrum chromatophores, and for quantasomes (pigment-con-taining particles too small to scatter light) derived from spinach chloroplasts in which the wavelength of light most effective in producing

lo*2xl014 Q/sec

o < 3.0 or z LÜ O

O Ü

2.0

Q_ CO

E 1.0 ω _J

O tu

500 600 700 800

λ (mjj)

FIG. 6. The action spectrum for unpaired electron production in spinach quan-tasomes, taken with samples of different optical density. The optical path length is 0.25 mm, and the incident light contains the same number of quanta/second and is nonsaturating. (From Androes et al, 1962.)

1

PD-678

°·°·678 '

0°678

^ ^ j ^

1

s* ■ 2.0

•1.0

"7" »0.5

1

Η^"™

^" ~~

-1

ι 1

1 \ '-.V 1 \ *'

J J


an electron resonance signal was the wavelength most strongly absorbed. The optical density in the cuvette was about 0.5 at 679 m/x for these measurements (Fig. 6). A similar result has been achieved with whole Chlamydomonas cells, in a suspension transmitting approximately 50% of the light at 680 τημ (Weaver, 1962b). It must also be borne in mind that comparisons between the effects of light at different wavelengths can only be made if the comparisons are made on the basis of equal energy or equal number of photons. The amplitude of the signal is proportional to the amount of light at low light levels; however, the slope of the linear portion of the amplitude vs. intensity curve is wave-length dependent, and the amount of light needed to saturate the signal is also wavelength dependent, being at a minimum for the strongly absorbed wavelengths. The amplitude of the maximum signal obtain-able is not wavelength-dependent, however, for wavelengths shorter than those corresponding to the minimum light energy required to induce a photo effect. Ideally then, the best procedure is to plot a signal-amplitude vs. light-intensity curve for each wavelength, making mul-tiple determinations for each point of the curve. Since successive traces of the same signal may vary, frequent checks with a standard are necessary to monitor the state of the sample. The relative efficiencies of each wavelength in generating an electron resonance signal may then be determined.

Recently some circumstantial evidence has been obtained, based on the number of unpaired electrons and on the relative concentration of active centers in chlorophyll (1 to every 300-400 chlorophyll mole-cules), that the R-signal is due to a pigment identified with the active centers and absorbing at 700 ηΐμ (Ρ700) (Beinert et al, 1962). This hypothesis may perhaps prove to be correct despite the seeming con-flict of the recent action spectrum results. Another difficulty in assign-ing the R-signal to P700 springs from the fact that various factors, to be described below, greatly alter the amplitude of the R-signal, which would not be likely a priori to alter the concentration of P700. Although the unpaired electron concentration in photosynthesizing material is generally reported as being far less than one per chlorophyll molecule, Smaller (1961) has reported that nearly 100% of chlorophyll molecules can be converted to paramagnetic centers, using flashing high-intensity light on a system of crystalline chlorophyll in organic solvents at 77°K.

It may be concluded that the R-signal is associated in an intimate way with some form or state of chlorophyll.

The source of the slowly decaying signal is less well defined. Various lines of evidence implicate a quinone as an electron acceptor. Among these may be mentioned the model system consisting of phthalocyanine


and o-chloronil, in which photoconductivity and electron resonance signals were demonstrated in a solid matrix (Kearns et al., 1960) ; the work of Tollin and Green (1962) who have shown by electron resonance spectroscopy that in a deoxygenated solution of organic solvents a single electron transfer takes place between chlorophyll and a quinone upon illumination, and that a reverse transfer takes place in the dark; light-induced absorbance changes in spinach chloroplasts at 255 m/x, which is the absorption maximum of plastoquinone (Klingenberg et al., 1962). Moreover, it has been determined that one quinone, Q-255 (plasto-quinone) is of apparently universal occurrence in aerobic systems (Lester and Crane, 1959). It is relatively abundant, and is found to be in-timately associated with chlorophyll (Crane, 1959). Bishop (1959) has established the fact that it, along with manganese, is necessary for oxygen evolution and has suggested (1961) that it is the factor which is seen in electron resonance spectra. Another bit of suggestive evidence is the observation of Levine and Piette (1962) that no S-signal appeared in suspensions of Chlamydomonas mutants which had a plastoquinone level only about 20% that of wild type and which showed negligible Hill reactivity (oxygen production) (Levine and Smillie, 1962).

Wild-type Chlamydomonas, grown on a medium deficient in man-ganese, were also almost free of the S-signal, although they displayed a large R-signal; these, too, evolved almost no oxygen. That the broad signal is not due simply to oxygen evolution can be demonstrated by treating the system with 10~5Af, 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), an herbicide which totally prevents production of oxygen at this concentration but whose effect may be reversed by washing (Gaffron, 1960). The S-signal is apparently unchanged by this treatment of Chlamydomonas cells (Weaver and Weaver, 1962), although it can be abolished in Chlorella by 0.01 M KCN (M. B. Allen et al, 1961b). In general, the S-signal is present in an oxygen-evolving system (green plants and their derivatives) and absent in a system which cannot evolve oxygen (photosynthetic bacteria and green material in which this part of the mechanism has been removed). The differences in behavior between these two classes of photosynthetic material suggest a correla-tion with the intrinsic differences in photosynthetic mechanism between plants in the two phyla (Arnon et al., 1961; Losada et al., 1961).

The g value of purified plastoquinone, determined by J. E. Maling (unpublished) is 2.0044, matching, within the limits of experimental error, the g value of the S-signal—2.0046. The spacing of the peaks is greater, in the S-signal, and the peaks are not nearly so well-defined as in the purified quinone. These effects are possibly a consequence of the


fact that the compound is bound in a lipid matrix. I t has also been suggested that the S-signal is due to chlorophyll b, to some other accessory pigment, or simply to respiratory processes (M. B. Allen et al., 1962).

If one assumes that chlorophyll is responsible for the rapidly-decaying signal, and that plastoquinone accounts for the more stable radical, some interpretation is possible of those observations which have been made on different variations of the photosynthetic system. I t is plausible to assume that at least some of the electrons resulting from the initial excitation chlorophyll in oxygen-evolving organisms proceeds eventually to plastoquinone as the electron acceptor, as many have suggested. Commoner and his co-workers (1957) early concluded from their electron resonance data that electrons are transferred from the R- to the S-system.

The temperature at which observations are made reveals some interesting relationships. Commoner (1961) has reported that warming living Chlorella to 29.5°C abolished the R-signal, while not affecting the S-signal. His earlier observations, however, (Commoner et al., 1956) were made at 35°C on spinach chloroplasts, which did display the rapid-decaying, light-induced signal. Cooling Chlorella to 12.8°C en-hanced the R-signal, without affecting the residual component (S-signal). Calvin and Sogo (1957; Calvin, 1961) have carried out low temper-ature experiments, on chloroplasts and chromatophores, from —15°C to —160°C. The rise time for the signal (presumably only the R is being observed) is temperature-independent. The amplitude of the signal is maximal at — 15°C, and decreases thereafter. Decay times are temper-ature-dependent in the green material, becoming slower with decreasing temperature. Chromatophores derived from the purple bacteria, Rhodo-spinllum rubrum, displayed instantaneous decay at — 160°C, with somewhat slower decay times at intermediate temperatures. There has also been a report of signal production in a crystalline chlorophyll sys-tem at —196°C which is absolutely stable at that temperature (Smaller, 1961).

The facts that the R-signal can be observed in every chlorophyll containing system, and that it can be produced at very low temperatures provide evidence for the view that this signal coincides with the primary, purely physical process of photosynthesis. A plausible interpre-tation of this primary event is that it corresponds to a semi-conductor, with some sort of charge separation taking place to prevent immediate recombination of electrons.

Kinetic studies have shown that decay curves for the R-signal show

60 M. S. BLOIS, JR. AND E. 0. WEAVER

at least two time constants, indicating that more than one electron path-way is involved. It has also been observed that the rise and decay rates are dependent on the spin concentration (Sogo, et al., 1961 ; M. B. Allen et al., 1961b) and on the intensity, and perhaps on the wavelength, of the exciting light. They are also dependent on the age and condition of the photosynthetic material, on the treatment to which it has been sub-jected, on the suspending medium, etc. There remains a great deal of information to be extracted from such studies when the variables are more completely controlled.

Determinations of signal intensity, which is a function of both amplitude and width of the resonance, reveals that in Chlamydomonas a large proportion of the unpaired electrons may be attributed to the S-signal, even when the R-signal has a far greater amplitude (Weaver, 1962b). The largest R-signals are generated by light when the photo-synthesizing system is operating under conditions which presumably block some part of the electron flow. This has been demonstrated by adding DCMU, which prevents the production of oxygen; the R-signal builds up to large amplitudes, leaving the S-signal unaffected. Cells which are crowded and anaerobic also produce a greatly enhanced R-signal, as do manganese-deficient cells. On the other hand, cells observed under conditions approximating physiologically favorable ones have a relatively small R-signal; presumably electrons are flowing along quite unobstructed (see Arnold and Clayton, 1960). Aside from these demon-strations on the effect of a block, there has been no determination of what size and ratio of signals is to be expected from an optimally photo-synthesizing system. Enhancement of photosynthetic rate has been reported by use of two wavelengths of light, which give a highest rate of oxygen production when they are combined than the simple sum of the rate with each alone (Emerson, 1958). There has been one pub-lished example of a two-wavelength electron resonance experiment. M. B. Allen and her associates (1962) observed on one occasion that when Chlorella was illuminated simultaneously with 540 τημ, and 694 m/x, light the R-signal was smaller than the signal from 540 πΐμ light alone.

If deuterium is substituted for hydrogen in the compound responsible for a signal, a narrower resonance should be observed, provided the signal is due to an electron interacting with a proton. Taking into account the reduced magnetic moment of deuterium, and its increased nuclear spin, a reduction of the order of three is to be expected, all other things being equal. Commoner (1961) performed the experiment of growing Chlorella in 99.9% D20, and found the predicted effect in both


signals. Androes et al. (1962), prepared both quantasomes and chromato-phores in D20 buffer solutions, but found no difference between these and the usual H20 preparations. They concluded that the principal line-width-producing protons are not labile ones which would be exchanged for deuterons in the preparative procedure.

A further possible application to photosynthetic studies of electron resonance which has scarcely been touched upon in the foregoing ac-count is the exploration of the effects of different nutritional elements. The question of whether manganese plays a role in the synthesis of plastoquinone can perhaps be solved with the aid of electron resonance; only Mn^· has been observed as the typical six-line resonance so often seen in photosynthetic material; Mn+ and Mn+++ are not seen. Growth and decay of manganese signals have been reported (Tanner et αΖ., 1960) which may be associated with the role of this metal in photo-synthesis. The role of other trace elements vital to growth may be better understood if the effect of their lack or superabundance could be directly observed on the electron transport system. Vanadium, for in-stance, is present in some marine algae and may be observable as the vanadyl ion which presents a characteristic eight-line resonance, centered not far from the free radical absorption.

The number of organisms in which photosynthetic reactions have been observed with electron resonance is relatively small. Natural selec-tion has provided many variations of functioning photosynthetic pig-ment and enzyme combinations. Mutants whose photosynthetic mech-anism has been altered in a variety of ways are also easily available.

Electron spin resonance analysis of judiciously chosen material may provide an insight into the photosynthetic process not attainable by other methods now in use.

4. Summary The phenomenon of electron spin resonance has been introduced in a

somewhat schematic but physical form. The physicochemical systems in which such a phenomenon might be observed were shown to include certain metallic ions, free radical intermediates, and certain molecular excited states.

If the appearance of an ESR signal in a biological system depends upon its prior or concurrent exposure to light then clearly one is observing a photo effect, though perhaps a remote one. The valid interpre-tation of magnetic resonance results—and consequently the experimental usefulness of the method—will depend ultimately upon an appropriate overall design (both biologically and physically) of the experiment,


and a close acquaintanceship with the total system. Inasmuch as ESR yields essentially unique information, it may be concluded that the effort to develop such an acquaintanceship will be well worthwhile.

REFERENCES

Allen, B. T., and Ingram, D. J. E. (1961). In "Free Radicals in Biological Systems" (M. S. Blois, Jr., et al., eds.), pp. 215-225. Academic Press, New York.

Allen, M. B., Piette, L. H., and Murchio, J. C. (1961a). Biochem. Biophys. Research Communs. 4, 271-274.

Allen, M. B., Piette, L. H., and Murchio, J. C. (1961b). In "Progress in Photo-biology" (B. C. Christensen and B. Buchmann, eds.), pp. 170-171. Elsevier, Amsterdam.

Allen, M. B., Piette, L. H., and Murchio, J. C. (1962). Biochim. et Biophys. Acta 60, 539-547.

Androes, G. M., and Calvin, M. (1962). Biophys. J. 2, Suppl. No. 2, Pt. 2, 217-258. Androes, G. M., Singleton, M. F., and Calvin, M. (1962). Proc. Natl. Acad. Sei.

U. S. 48, 1022-1031. Arnold, W., and Clayton, R. K. (1960). Proc. Natl. Acad. Sei. U. S. 46, 769-776. Arnon, D. I., Losada, M., Nosaki, M., and Tagawa, K. (1961). Nature 190, 601-606. Bamford, C. H., Ingram, D. J. E., Jenkins, A. D., and Symons, M. C. R. (1955).

Nature 175, 894-895. Bass, A. M., and Broida, H. P., eds. (1960). "Formation and Trapping of Free

Radicals." Academic Press, New York. Beinert, H., Kok, B., and Hoch, G. (1962). Biochem. Biophys. Research Communs.

7, 209-212. Bishop, N. I. (1959). Proc. Natl. Acad. Sei. U. S. 45, 1696-1702. Bishop, N. I. (1961). In "Ciba Foundation Symposium on Quinones in Electron

Transport" (G. E. W. Wolstenholme and C. M. O'Connor, eds.), pp. 385-404. Churchill, London.

Blois, M. S., Jr., Brown, H. W., Lemmon, R. M., Lindblom, R. O., and Weissbluth, M., eds. (1961a). "Free Radicals in Biological Systems." Academic Press, New York.

Blois, M. S., Jr., Brown, H. W., and Maling, J. E. (1961b). In "Free Radicals in Biological Systems" (M. S. Blois, Jr., et al., eds.), pp. 117-131. Academic Press, New York.

Bubnov, N. N., Krasnovskii, A. A., Umrikhina, A. V., Tsepalov, V. F., and Shliapintokh, V. I. (1960). Biophysics (U.S.S.R.) (English Translation) 5, 145-151.

Calvin, M. (1959). In "Biophysical Science—A Study Program" (J. L. Oncley, ed.), pp. 157-161. Wiley, New York.

Calvin, M. (1961). In "Light and Life" (W. D. McElroy and B. Glass, eds.), pp. 317-355. Johns Hopkins Press, Baltimore, Maryland.

Calvin, M., and Sogo, P. B. (1957). Science 125, 49&-500. Commoner, B. (1961). In "Light and Life" (W. D. McElroy and B. Glass, eds.),

pp. 356-377. Johns Hopkins Press, Baltimore, Maryland. Commoner, B., and Lippincott, B. B. (1958). Proc. Natl. Acad. Sei. U. S. 44,

1110-1116. Commoner, B., Townsend, J., and Pake, G. E. (1954). Nature 174, 689. Commoner, B., Heise, J. J., and Townsend, J. (1956). Proc. Natl. Acad. Sei. U. S.

42, 710-718.


Commoner, B., Heise, J. J., Lippincott, B. B., Norberg, R. E., Passoneau, J. V., and Townsend, J. (1957). Science 126, 57-63.

Crane, F. L. (1959). Plant Physiol. 34, 128-131. Emerson, R. (1958). Science 127, 1059-1060. Feher, G. (1957). Bell System Tech. J. 36. Gaffron, H. (1960). In "Plant Physiology—A Treatise (F. C. Steward, ed.), pp.

3-277. Academic Press, New York. Gill, J., and Weissbluth, M. (1962). Proc. 6th Ann. Meeting Biophys. Soc, Wash-

ington, D. C. (Abstr. WC 6). Hutchison, C. A., Jr., and Mangum, B. W. (1958). J. Chem. Phys. 29, 952. Ingram, D. J. E. (1955). "Spectroscopy at Radio and Microwave Frequencies."

Butterworths, London. Ingram, D. J. E. (1958). "Free Radicals as Studied by Electron Spin Resonance."

Academic Press, New York. Kearns, D. R., Tollin, G., and Calvin, M. (1960). J. Chem. Phys. 32, 1020. Kessler, E., Moraw, R., Rumberg, B., and Witt, H. T. (1960). Biochim. et Biophys.

Acta 43, 134. Klingenberg, M., Müller, A., Schmidt-Mende, P., and Witt, H. T. (1962). Nature

194, 379-380. Köhnlein, W., and Müller, A. (1962). Phys. in Med. Biol. 6, No. 4, 599. Lester, R. L., and Crane, F. L. (1959). J. Biol. Chem. 234, 2169-2175. Levine, R. P., and Piette, L. H. (1962). Biophys. J. 2, 369-379. Levine, R. P., and Smillie, R. M. (1962). Proc. Natl. Acad. Sei. U. S. 48, 417-420. Losada,M., Whatley, F. R., and Arnon, D. I. (1961). Nature 190, 606-610. Maling, J. E. (1962). Personal communication. Sever, R. J., Cope, F. W., and Polis, B. D. (1962). Science 137, 128-129. Smaller, B. (1961). In "Free Radicals in Biological Systems" (M. S. Blois, Jr.,

et al., eds.), pp. 315-323. Academic Press, New York. Smaller, B. (1962). Personal communication. Smith, D. E., Santamaria, L., and Smaller, B. (1961). In "Free Radicals in Bio-

logical Systems" (M. S. Blois, Jr., et al, eds.), pp. 305-310. Academic Press, New York.

Sogo, P. B., Pon, N. G., and Calvin, M. (1957). Proc. Natl. Acad. Sei. U. S. 43, 387-393.

Sogo, P. B., Carter, L. A., and Calvin, M. (1961). In "Free Radicals in Biological Systems" (M. S. Blois, Jr., et al, eds.). Academic Press, New York.

Tanner, H. A., Brown, T. E., Eyster, C , and Treharne, R. W. (1960). Biochem. Biophys. Research Communs. 3, 205-210.

Tollin, G., and Green, G. (1962). Biochim. et Biophys. Acta 60, 524-538. Tollin, G., Sogo, P. B., and Calvin, M. (1958). Ann. N. Y. Acad. Sei. 74, 310-328. Varian Associates (1960). "NMR and EPR Spectroscopy" (Staff, eds.). Pergamon

Press, New York. Weaver, E. C. (1962a). Arch. Biochem. Biophys. 99, 193-196. Weaver, E. C. (1962b). Carnegie Inst. Wash. Yearbook 61, 353-365. Weaver, E. C , and Weaver, H. E. (1962). Paper presented at 6th Ann. Meeting

Biophys. Soc, Washington, D. C, 1962 (Abstr. WC 5). Whiffen, D. H. (1961). In "Free Radicals in Biological Systems" (M. S. Blois, Jr.,

et al., eds.), pp. 227-238. Academic Press, New York. Zavoisky, E. (1945). J. Phys. (Uß£.R.) 9, 211.

Chapter 3

PHOTOCHEMICAL ACTION OF LIGHT ON MACROMOLECULES

A. D. McLaren

College of Agriculture and Miller Institute for Basic Research in Science University of California, Berkeley, California

1. Kinetics of Loss of Activity of Enzymes, Nucleic Acids, and Viruses

By the first law of photochemistry, in order for a photochemical reaction to take place, quanta of light must be absorbed by the reactant molecules. All proteins and nucleic acids absorb ultraviolet light (UV). By the second law, although all absorbed quanta do not necessarily in-duce a chemical reaction, when reaction occurs, one quantum is involved per molecule. Other absorbed quanta may lead to fluorescence and heat (Livingston, 1955). In other words, molecules E react by a "one-hit" process which may be described by the equation

Ε + ^->Ρχ + Ρ 2 + · . - (1) where q is a quantum and Pi are products. This equation is oversimpli-fied since ions, gases, and solvent may also participate in the reaction. In describing this reaction kinetically, one must take into account the fact that, as soon as products are formed, they too can absorb quanta. If we are assaying for the loss of E, these latter quanta are wasted. Now, in order to find the effective fraction of quanta absorbed we reason as follows: The probability that molecules of E will absorb light in a beam will be proportional to their number, and if reactants and products absorb alike the fraction absorbed by E will be [E]/([E] + [2Pi]) where [E] and [Pi] are concentrations. It usually happens, in a photo-chemical reaction, that E and P do not have equal absorbing capacities and the solvent may also absorb. A measure of absorbing capacity is the extinction coefficient, defined by Beer's law as

log6 h/I [E]

65

(2)

66 A. D. MCLAREN

for unit path length, where I0 is the intensity incident on a cell of 1-cm depth, in quanta/cm2, I is the transmitted intensity, and [E] is the concentration of, for example, the reactant in moles/cm3. Thus the general form of the fraction sought, /, is given by

ft[E] De ^ J ße[E] + ßp\P] + ß.[8] De + Dp + D8

w

where [S] is the concentration of solvent and the D's are the familiar optical densities or absorbances (expressed as log10Io/I).

On this base we can now write a general rate equation for the disappearance of a reactant, be it an enzyme or a virus, in the form

-^^-^m+m (4)

Here Jabs = h — I and Φ, the quantum efficiency (or yield) is the ratio of molecules reacting to quanta absorbed by E. We have assumed that water is the solvent; it has virtually no absorption above 2000Â. In general, the solution of this equation is difficult and requires graphical methods, but there is an approximation which holds true for moderate degrees of inactivation (McLaren, 1951). If the amount of Pi is small or if ßp nearly equals ße, Eq. (4) simplifies to

- 4 Ε ] Μ = Φ ^ 8 | | (5)

and on integration we obtain the useful form

φ = [Eo]log6[E0]/[E] ( 6 )

This equation for a given system involves constants all of which may be lumped together to give the classical equation used by Northrop and others, namely [E] = [Eoje-**, where t is the time of irradiation. The constant K is inversely proportional to the initial concentration as may be seen from Fig. 1.

Equation (6) is sufficient to describe the inactivation of enzymes, antibodies, and ribonucleic acid from tobacco mosaic virus (McLaren, 1957; Kleczkowski, 1954). The small departure from this first-order equation, in the form log [E]/[E0] = —Kt, reported for ribonuclease is within the experimental errors of enzyme assay (Brighenti, 1962). Also, although it was suggested that disulfide compounds protect the enzyme from UV of 2537 Â, when absorption by disulfide is taken into account [Eq. (3)], the quantum yield, 0.016, is within experimental error of the yield for solution of pure enzyme under Brighenti's conditions.

Viruses are large enough to scatter some of the incident light and /

3 . PHOTOCHEMISTRY OF MACROMOLECULES 67

2.0

1.8

t 1.6

5 1.4 h

1.2

r

s o

o\^ ^^

1 1

1 1 1

x^JXJmg ficin/ml 1

NP;5 mg ficin/ml

1 1 1 1

2 4 6 8 Time of irradiation (min)

10

FIG. 1. Inactivation of ficin by UV light at 2537 Â. The lower concentration shows the greater inactivation rate constant. (Mandl and McLaren, 1949.)

must be multiplied by a factor (1 ■— ßR/ße) which gives the fraction of the light absorbed by the reactant which is not scattered; ßR is the scattering coefficient of the reactant (Claesson, 1956). The factor is about 0.6 for tobacco mosaic virus. An example of the degree of scatter by very large molecules is shown in Fig. 2. X-protein from plants in-

2 3 0 2 5 0 2 7 0 2 9 0 3 , 0 3 3 0 3 5 0 3 7 0 3 9 0

Wavelength, m/x

FIG. 2. UV-absorption spectra of tobacco mosaic virus (1.054 mg/ml), X-protein at pH 7.3 and at pH 5.3 (1 mg/ml), and nucleic acid (0.054 mg/ml). Uppermost curve (U) sums that of RNA and a hypothetical curve for X-protein fully poly-merized to rods of 2710 Â length. (McLaren and Takahashi, 1959.)

68 A. D. MCLAREN

fected with the virus is of low molecular weight at pH 7.3 but poly-merizes to give rods commensurate in length with the rodlike molecules of the virus if the pH is adjusted to 5.3. The difference in optical density for these two situations is shown in the figure as a function of wave-length. Other more general methods are available for scattering correc-tions for viruses and phages (Zelle and Hollaender, 1954; Shugar, 1960).

It happens with the nucleic acids, and with some enzymes requiring activation such as urease, that not all molecules present initially may be active, or that not all molecules (such as transforming nucleic acids) may have some selected marker. Nevertheless, Eq. (6) still holds for the bioassay selected, provided that all molecules present are of the same size and have the same optical properties (McLaren and Takahashi, 1957). Here [E0]/[E] is replaced by [100%]/[% activity at time t].

2. Photochemistry of Ammo Acids, Proteins, and Nucleic Acids

2.1 Amino Acids

Although the subject of photochemistry of the amino acids is exten-sive, it is probably not of importance in biology except insofar as the subject is useful in interpreting the photochemistry of proteins. There are only small amounts of free amino acids in cells and these amounts can absorb only vanishingly small fractions of incident UV light. In Table I are listed some known products from the photolysis (at 2537 Â) of the strongly absorbing amino acids. Only a few quantum yields are known. With cystine under oxygen-free conditions the quantum yields for the products nearly total to the known value for loss of identity (0.13). Cleavage of —SS— bonds is not greatly influenced by oxygen (Dose and Rajewsky, 1962).

In order to account for the formation of cysteine sulfinic acid under oxygen-free conditions, Dose and Rajewsky suggest the following reactions:

2 Cy— S—S—Cy + 2 q -* 2 /Cy—S—S—Cy\ * The excitation step

\ H—OH / 2 /Cy—S—S—CyX* -> 2 CySH + 2 CySOH The dissociation step

\ H—OH / 2 CySOH -» CySH + CyS02H The disproportionate step

On irradiation of solutions at pH about 5, cystine undergoes —CS— fission to a far greater extent than —SS— fission, however (Forbes and Savige, 1962).

No quantitative results for products of photodecom£>osition are

3. PHOTOCHEMISTRY OF MACROMOLECULES 69

TABLE I PRODUCTS FROM THE ACTION OF UV LIGHT ON SOME AMINO ACIDS AND PEPTIDES"

Substance

Amino acids Cystine

Phenylalanine Tryptophan Tyrosine

Peptides Acetylalanine

Glutathione (sans 02)

Acetyltryptophan Phenylpropionylalanine

Quantum yield for total

destruction (2537Â)

0.13

0.013 0.004 0.002

—

—

— —

Products

Alanine Cysteine Cysteine sulfinic acid NH3 H2S S — — —

NH3 Acetaldehyde Pyruvate Alanine Glutamylalanylglycine Reduced glutathione Tryptophan NH3 Alanine

Quantum yield for product

0.001 0.05 0.01 0.02

? ?

— — —

0.05 0.03 0.02 0.004 0.06 0.02 0 0.0002 0.004

α McLaren and Luse, 1961; Estermann et αΖ., 1956; Dose and Rajewsky, 1962.

available on the other amino acids. The yields for acetaldehyde plus pyruvate equal the quantum yield for ammonia liberated from acetyl-alanine, and as with other hemipeptides and peptides, the liberation of free amino acids is very low or nil. Peptide-bond cleavage is thus very inefficient, whereas destruction of some of the amino acid side chains seems to be the more important photochemical step. These reactions, once studied qualitatively, are now being studied in great detail with modern techniques (Forbes et αΖ., 1962; Dose and Rajewsky, 1962). Free-radical intermediates are definitely involved in photolysis.1

2.2 Proteins and Nucleic Acids

Quantum yields for photoinactivation of enzymes are found to be generally (a) independent of concentration and of the presence of oxygen, and (b) dependent on pH and temperature. They are of the same order of magnitude in aqueous media or in vacuum (McLaren,

aThe most recent reviews of this subject are by Doty and Geiduschek (1953), Claesson (1956), Beaven and Holiday (1952), and McLaren and Shugar (1963).

70 A. D. MCLAREN

1957; Setlow and Doyle, 1957). As yet no adequate explanation is available for the pH dependence of photochemical inactivation of en-zymes. The products of irradiation are different in the presence or ab-sence of oxygen. Chymotrypsin irradiated at low temperature and having lost some of its activity (as measured at low temperature) loses addi-tional activity on being warmed to a temperature somewhat above room temperature; thus, unstable intermediates can exist at low temperature. At room temperature loss of activity parallels roughly a loss of solubility in a salt solution in which the active enzyme dissolves. Clearly the photo-chemical inactivation is complex. In Table II are listed quantum yields

TABLE II ESTIMATION OP QUANTUM YIELDS FOR ENZYME INACTIVATION FROM QUANTUM

YIELDS FOR AMINO ACID DESTRUCTION (2537Â)a

Ammo acid, φ

Cystine, 0.13 Histidine, <0.03 Phenylalanine, 0.013 Tryptophan, 0.004 Tyrosine, 0.0020 —CONH— as in

acetylalanine, 0.05

Calculated Known

Chymotrypsin p*

U{

5 2 6 7 4

200

= 23,000

ηφίφι

175 <0.015 11 80 3

1

0.01 0.005

Enzyme

Lysozyme ß ~-

m

5 1 3 8 3

130

= 18,000

Πίβίφί

175 0.007 5

92 2

1

Ribonuclease ß =

Ui

4 4 3 0 6

130

= 4,400

Πίβίφί

140 <0.03

5

4

1 Φ for enzymes

0.01 0.024

0.03c

0.027

Trypsin ß --

Ui

6 1 3 4 4

200

= 15,500

Πχβίφί

210 0.007 5

46 3

1

0.01 0.015e

α McLaren and Luse, 1961. 6 In this table β is the molecular extinction coefficient corresponding to concentrations

in moles per liter. c These numbers were incorrectly printed in the original paper.

for the inactivation of some enzymes irradiated near the pH of maximum (photochemical) stability. Yields of the order of 10"2 to 10~3 are common.

In an effort to explain these quantum yields we have proposed that the inactivation of enzymes involves, as the primary chemical reaction, photolysis of disulfide and aromatic resides. If this be true, then the rate is

-d[E]/dt = Jabe &[Eo] (7)


where Ui is the number of residues per molecule of enzyme and φ% is the quantum yield for destruction (loss of chemical identity) of each kind of residue. Equating Eqs. (5) and (7) we find that

i Φβηζ = X rtißrfi/ße (8)

In evaluating this equation, we have assumed that φι is the same for an amino acid residue as for the amino acid monomer, which is also saying that the amino acid side chains are the reactive sites and that peptide bonds are unimportant. We have also assumed that βι is the same for an amino acid free or combined. Although we have no way of knowing, a priori, whether ψι for the amino acid side chain is the same whether free or combined, ßi for these residues are only approximately the same for free and combined amino acids (Beaven and Holiday, 1952). All available information for evaluating Eq. (8) is in Table II. Agreement between calculated and known quantum yields for inactiva-tion is obtained within a factor of two, which is surprisingly good. The two most important residues are cystyl and tryptophanyl. That breakage of hydrogen bonds is also involved is suggested by the above-mentioned temperature dependence of the yield for chymotrypsin and the solu-bility change. Although chemical cleavage studies of —SS— in some of the enzymes has revealed that not all —SS— groups need be intact for enzyme activity, it must be remembered that photochemical cleavage results in breakage at —CS— as well as at —SS— (Forbes and Savige, 1962) and that inactivation is accompanied by a kind of denaturation as well.

Analysis of irradiated enzymes revealed that —SS— and tryptophanyl groups were altered by UV. Peptide bonds and other aromatic groups are photolyzed appreciably only after ca. 99% of the enzymes are in-activated.

Assuming quantum yields, φίι close to the values we have found, Setlow has been able to account for the action spectra for inactivation over a wide range of wavelengths for several enzymes. Before discussing the spectra obtained by this author however, we need equations for action spectra. Equation (6) reduces to a simple form if a solution is very dilute, i.e., optically thin:

[EQ] loge [EQ]/[E] [EQ] log. [Ep]/[E] _ 2.3 log10 [E0]/[E] ha J0(l - e~^)t ~ I0ßt w

Upon substituting Beer's equation in Eq. (9) there results

log, [Eo]/[E] = g j 10^/0/7 = 1 ^ (10)

72 A. D. MCLAREN

where Q is the incident energy at wavelength λ and N, h, and c are Avogadro's number, Planck's constant, and the velocity of light, re-spectively. Calling log10 [E0]/[E] the action, it is clear that the action is proportional to the absorbance for a given initial reactant concentra-tion, provided Φ is independent of wavelength and I0t is kept constant. As an example, we plot both action and absorbance versus wavelength in Fig. 3. (Often 1/Q or 1/QX is plotted as a function of wavelength for

i.o

0.8 o

8Ό.6 c Ό

"υ < 0.4

0.2

2 3 0 0 2 5 0 0 2700 2 9 0 0

Wavelength, Â

FIG. 3. Action and absorption spectra of infectious nucleic acid (RNA) from TMV. (McLaren and Claesson, 1961.)

a given action. The resulting figure is also an action spectrum.) The quantum yield varies from 3.4 χ 10~3 to 3.8 X 10"3 in the wavelength range between 2804 and 2300 Â with infectious nucleic acid from tobacco mosaic virus. No light scatter corrections are involved for this linear molecule (molecular weight = 2 χ 106). The correspondence between these spectra fulfills the requirements rather well.

We return now to Setlow's action spectra for enzymes. In his labora-tory much study has been devoted to the irradiation of enzymes in thin, dry layers. Setlow therefore chose to plot the action spectra he obtained in terms of inactivation cross sections and absorption in cross sections, s. The latter cross section is defined in terms of an alternate form of Beer's law, namely

s = l^M. (11)

nx K J


where n is the number of particles per cubic centimeter and x is the path thickness. I t has the dimensions of square centimeters and is con-venient in thinking of the thin dry films of enzyme undergoing irradia-tion. The inactivation cross section is defined in an alternate form of Eq. (9), namely

[E]/[Eo] = exp (-a/ t f ) (12) for dry thin films. Here a is in square centimeters/erg, or better, square centimeters/quantum. Incidentally, Φ = a/s. Spectra for trypsin are shown in Fig. 4. Clearly these spectra do not agree well and the reason

6 0 0

3 0 0

100 6 0

3 0 CM <* 10 ■E 6

I 3 o <D w I

8 0.6

ύ 0.3

0.1 0.06 0.03

0.01 2 0 0 0 2 4 0 0 2 8 0 0 Incident wave length in A

FIG. 4. The absorption spectrum and the action spectrum of dry trypsin irradiated at 300°K and 90°K. (Setlow and Doyle, 1957.)

is that trypsin has maxima in quantum yields at about 2500 Â and above 3000 Â and below 2000 Â. Setlow and Doyle (1957) were, however, able to account for the inactivation cross section for trypsin in terms of as-sumed values for the quantum yield of cystine (0.07) and the rest of the molecule (2*7^0* = 24w* 0.002) as a function of wavelength (com-pare Table I I ) .

So far we have considered the inactivation of enzymes and nucleic acids by UV. These studies have been possible because some proteins and nucleic acids have an ability to "turn over" a large amount of sub-strate or else to become amplified by an infectious process. Thus photo-chemical changes in dilute solutions can be followed as loss of identity

— i — r — ι — i — i — r

Γο Dry trypsin 1 o

■ ■

Γ v °° 1 >■

— i — 1 '—1 '—1—'—1

irradiated at o300°K"i ■ 90°K-j

°o

■

h Absorption-^ v

[(mjlti^ly scale by 1)

_

1 i

-|

•-Action -/(multiply leftJ

oscale by I0"5)j o

ΧΛ°° J V -1

\ °

J i I 1 1 lAJ

74 A. D. MCLAREN

of reactants in terms of biochemical or biological assay. These changes seem to involve relatively few or perhaps single photochemical events, i.e., a change in perhaps one or two chromophores. In this connection it is interesting to note that as UV inactivation of chymotrypsin proceeds in dilute buffer solution, it loses its catalytic activity at equal rates toward natural and synthetic ester and amide substrates. Also, as already men-tioned, there is a parallel loss in solubility observed if the chymotrypsin is transferred to 0.56 saturated ammonium sulfate. Trypsin loses its ability to combine with a protein, soybean-trypsin-inhibitor following inactivation, which suggests that UV does not necessarily destroy the active site of the enzyme: the enzyme may just not be able to form a Michaelis-Menten complex with substrate (Estermann and McLaren, 1962). Similarly, chymotrypsin loses its ability to dimerize (McLaren, 1957). We are thus led to conclude that inactivation involves three possibilities all of which may be involved at the same time (McLaren, 1957; McLaren and Luse, 1961; Augenstine and Ghiron, 1961); namely, (1) destruction of side chain residues, (2) cleavage of disulfide linkages, and (3) denaturation, which may be closely associated with (2) in some cases and which may be a consequence of loss of intramolecular hy-drogen bonds.

In an extensive study of hemocyanin, Claesson found that it split into halves with a quantum yield of about 10~5. At a pH near its iso-electric point, hemocyanin also undergoes aggregation. Similar results have been obtained with urease. With bovine serum albumin it has been concluded that one photochemically activated molecule combines with an unactivated molecule during aggregation (Claesson, 1956). Free-radical formation may be responsible (Rideal and Roberts, 1951).

During the inactivation of infectious nucleic acid from TMV, de-polymerization does not take place. A slight change in absorbance at 2600 Â has been interpreted in terms of photochemical changes in pyrimidine residues, which are about a hundred times as sensitive as are purine bases (see Table III). An application of Eq. (8) is not feasible since the quantum yield is somewhat dependent on concentra-tion. Also, nucleic acids decrease in optical density much less than do nucleotide mixtures during irradiation (Rushizky et al., 1960; Rushizky and Pardee, 1962; Wierzchowski and Shugar, 1962). Absorbance changes have thus far been the sole analytical procedure applicable to both nucleotides and nucleic acids. Unlike the situation with enzymes, the number of kinds of monomers in nucleic acids is so small that photolysis of the, perhaps one or two, bases involved in inactivation would be difficult to detect and identify by chemical methods.

On prolonged irradiation, depolymerization of the nucleic acid

3. PHOTOCHEMISTRY OF MACROMOLECTJLES 75

TABLE III QUANTUM YIELDS FOR ALTERATION OF NUCLEOTIDES AND RELATED COMPOUNDS

AT 2537Âa

Quantum yield Compound (in solution)

Uracil Uridylic acid Uridine Cytidylic acid Adenine Guanine Thymidylic acid

Thymine Thymidine Uracil Uridylic acid

0.005 0.022 0.02 0.017 0.00006 0.0002 0.001

(in ice) 0.2 0.004 0.055 0.002

« Sinsheimer, 1957; Shugar and Wierzchowski, 1958; Wang, 1961, 1962.

does take place, but with, of course, a much lower quantum yield than that calculated for inactivation (Coahran et al., 1962).

In addition to infectious ribose nucleic acids from viruses, trans-forming desoxyribose nucleic acids (DNAs) from bacteria have also been studied in some detail (see e.g. Stuy, 1962; Marmur et al., 1961). It is probable that during UV inactivation dimer formation can occur between pyrimidine residues in complementary, double-stranded DNA molecules with the formation of chemical cross linkages. This possi-bility was suggested by the fact that dimers are formed in ice during irradiation of pyrimidine molecules which have α,β-unsaturated ketone configurations (see Table III) (Beukers and Berends, 1960; Wang, 1961), and Marmur et al. present an impressive amount of evidence in favor of this interpretation with DNA isolated from D. pneumonias and B. subtilis. Perhaps 50% of biological inactivation is due to thymine dimer formation (Setlow and Setlow, 1962). That some such process is involved is also suggested by the fact that survival curves for loss of transforming markers do not follow Eq. (6) (Marmur et al., 1961). The DNA seems to become more resistant with increasing doses of irradia-tion. Alternately, as an explanation, transforming DNA has a greater sensitivity to UV the smaller the molecular weight and the survival curves may simply reflect the heterogeneity of the preparations. Mech-anisms other than simple dimer formation between strands are un-doubtedly involved in DNA inactivation, including irreversible destruc-

76 A. D. MCLAREN

tion of pyrimidine bases. For details see Chapter 20 in Volume II. As with infectious RNA, quantum yields are low with DNA's (see Section 3).

Inactive DNA can be partially reactivated by exposure to light (3000-4000 Â) in the presence of an enzyme from baker's yeast. It therefore seems probable that photoreactivation with the aid of enzymes is one of the mechanisms whereby irradiation-damaged cells can use sunlight to repair damage to DNA in vivo (Rupert, 1961).

2.3 Some Comments on Biological Systems

Depending on wavelength, the significant biological effects of UV may be attributed to protein and nucleic acid moieties (Giese, 1950). The biological influence of UV action on protein in cells is not understood, and clues are few. Haurowitz and Turner (1949) suggested an "amplification reaction" on a cellular level; since it is known that irradiated, de-natured proteins are more easily hydrolyzed by proteolytic enzymes, essential structures may be digested by cathepsins following irradiation. Since the product of βφ for enzymes roughly equals this product for nucleic acids, it is equally probable in a 50:50 nucleoprotein that the nucleic acid is affected. The latter reaction can be amplified enormously in terms of biological mutations. In Fig. 5 is shown an action spectrum for gene alteration. Stadler and "über determined a quantity a, which

2500 2800 Ä

FIG. 5. Action spectrum for deficiency rates compared with the absorption spectrum of nucleic acid. (Stadler and Über, 1942.)


is proportional to φΌ for endosperm deficiencies in maize (the term deficiency was applied to all losses of effect of dominant marker genes), with doses of 2000 ergs/mm2 of energy incident at the surface of pollen grains [see Eq. (10)]. These values agreed within the limits of sampling error with the relative absorption coefficients of nucleic acid for the same wavelengths. Here important corrections for effective quanta absorption had to be made for the random orientations of nuclei within pollen grains. In contrast, the action spectrum for the paling of chromo-somes parallels the absorption spectrum of a tyrosine-containing pro-tein (Zirkle and Uretz, 1963).

3. Inactivation of Viruses by Visible and Ultraviolet Light

One of the most frequently studied groups of viruses are those from tobacco mosaic diseases. The common strain was the first to be purified and, as a nucleoprotein, has become the best understood both physically and chemically. Through the remarkable efforts of Schramm and of Fraenkel-Conrat it has been possible to "take it apart" study each part, and reassemble the parts to give virus again. As already noted, the RNA moiety alone is biologically active. Early work by Hollaender and Duggar (1936) revealed that the action spectrum for the virus did not look like the absorption spectrum of virus or any other known substance. Since a light-scatter curve for the virus is monotonous, this factor cannot account for the difference. The difference, it turns out, follows from the fact that quantum yields for inactivation of viruses, like those for proteins, are wavelength-dependent. Oxygen has no in-fluence on yield at 2537 Â and the inactivated virus is unchanged in size or shape from the original.

An analysis of inactivation of the virus is summarized in Table IV.

TABLE IV QUANTUM YIELDS FOR THE INACTIVATION OF TMV, Φ, AND FOR RNA

INCORPORATED IN THE VIRUS, Φβ β

Wavelength,

2804 2652 2537 2483 2300

Ä Rate constants for inactivation X 105

1.8 3 .9 4.3 4 .0 8 .7

Φ X 105

(virus)

2 .3 4 .9 5 .8 5.9

—12

Φ. (RNA

r>j

X 105

in virus)

9 .3 11 12 14

200

α McLaren and Claesson, 1961.

78 A. D. MCLAREN

As already indicated, quantum yield calculations are complicated by the necessity of correcting apparent light absorption of the virus for light scattered by the virus. Also, in dilute solution reabsorption of scattered light by the virus will be small, whereas in concentrated solutions, reabsorption may be appreciable. Neglecting the reabsorption, for which we have no means of evaluation, we can correct absorbances for scattering by means of Fig. 2 and compute quantum yields for the inactivation of TMV from the rate data of Rushizky et al. (1960). As may be seen from Table IV, quantum yields for TMV are not inde-pendent of wavelength and therefore the action spectrum cannot parallel the absorption spectrum. To find the quantum yields, Φ8 for RNA in-corporated in the virus particle, assuming that only light absorbed by the RNA moiety leads to inactivation, Φ must be corrected further for the fraction of the absorbed light absorbed by RNA. These values are also tabulated. It becomes clear that from 2483-2904 Â the sensitivity of RNA in the virus is fairly independent of wavelength, although much less than that of the free RNA (devoid of protein). The quantum yield for free RNA is essentially constant within the experimental variability of the tests. On the other hand, Φ8 is much larger at 2300 Â than in the range of 2483-2804 Â, and we suspect that the quantum yield calculated in this way is without physical meaning at 2300 Â. In other words, since Φ for free RNA is independent of wavelength (in the wavelength range under consideration), we can conclude that the high value of Φ at 2300Â really means that the virus is rendered nonviable because light absorbed by both protein and the RNA moieties can lead to inactivation (at 2300 Â most of the light is absorbed by protein). The host plant cannot extract active RNA from a coat of protein which has become denatured by UV light, and therefore the quantum yield is higher than would be expected if the RNA was equally available to the plant after irradia-tion at any wavelength. This conclusion was reached in another way by Siegel and Norman (1958).

Different strains of virus inactivate at different rates when intact, although their separate nucleic acids are equally sensitive (Siegel et al, 1956). Reconstituted virus, however, inactivates at the same rate as the original (Rushizky et al., 1960).

An important consideration in the above experiments is that RNA from TMV can be photoreactivated by visible light after irradiation at 2537 Â and applied to leaves (Bawden and Kleczkowski, 1959). With or without photoreactivation, inactivation is approximately first order [Eq. (6) ] ; photoreactivation is equivalent to halving the dose of radia-tion in this instance. The type of bonding between RNA and protein in TMV is sufficient to prevent photoreactivation of inactivated TMV per se, but six other plant viruses studied by Bawden and Kleczkowski


showed photoreactivation, with potato X virus showing this property most markedly. If uracil residues dimerize at all in the irradiation of the RNA, such dimerization either does not affect infectivity or is not photo-reversible (Kleczkowski, 1963).

Poliovirus ribonucleic acid has been compared with TMV-RNA and found to be about equally sensitive to UV (Norman, 1960). Both RNA's are similar in molecular weight and base composition. The role of pro-tein is very important, however, as poliovirus is some 18 times more sensitive to UV than TMV.

RNA from TMV causes a color shift when combined with acridine orange, and in the presence of small amounts of the dye visible light causes inactivation (Chessin, 1960). A photosensitized inactivation of TMV by acriftavine has been found to proceed parallel with the destruc-tion of the dye; this action was found to be dependent upon adsorbed dye (Oster and McLaren, 1950). Just how the absorbed energy reaches the RNA is not known, but free radicals resulting from photochemical changes in the dye-H20 system may be responsible. Energy in the form of absorbed quanta apparently is not transmitted readily from protein to RNA in TMV (Shore and Pardee, 1956).

At this point we must agree with Kleczkowski that the photo-chemistry of inactivation of viruses is still largely vague. The chemical changes observed are more obscure than those found with enzymes.

Practically speaking, Taylor et al. (1957) have explored the utility of combining formaldehyde inactivation with UV as a method for the inactivation of poliovirus for the production of vaccines with success.

As a parallel to RNA, the bacterial virus φΧ174 (φ here does not mean a quantum yield) contains a single strand of DNA. This virus has been studied at different wavelengths by Setlow and Boyce (1960). The shapes of the action spectra at pH 2, 7, and 12 were analyzed in terms of the effects of UV on the pyrimidines and purines [see Eqs. (8) and (12)]. In the interpretation of these authors the pyrimidines were at least two to three times more sensitive than purine bases in the polymeric DNA; the quantum yield for inactivation of the virus at 2650 Â and pH 7 was found to be 0.006 and the apparent quantum yields were of this order of magnitude, which for pyrimidines is lower than known values for the bases in monomeric form. Following a rather in-volved argument Setlow concludes that action spectra for single- and double-stranded polynucleotides should have minima at different wave-lengths (the former at 2400 Â and the latter at 2300 Â), and that this difference may be used to distinguish between these configurations in vivo. The theory was applied by them as evidence for the existence of a single-stranded stage of T2 bacteriophage during replication (Setlow and Setlow, 1960).

80 A. D. MCLAREN

Bacteriophages are so complicated from a photochemical point of view that they will not be considered further in this chapter. Recent detailed reviews on viruses include those of Kleczkowski (1960), Shugar (1960), Taylor (1960), and a monograph by McLaren and Shugar (1963).

4. Concluding Remarks The photochemical inactivation of enzymes and some viruses pro-

ceeds via first-order kinetics, and quantum efficiencies can be calculated unambiguously. These yields can be more or less successfully interpreted in terms of photolysis of side-chain residues, disulfide cleavage, and perhaps intramolecular dimer formation, as the case may be. Depoly-merization of proteins by splitting of —CONH— or of nucleic acids by splitting the phosphorus-sugar chain can occur, but the efficiencies are entirely too low to be involved in the primary steps of inactivation at 2537 Â.

A photochemical reaction within a macromolecule probably takes place at the chromophore whereat a quantum of active light is absorbed. There is no need to postulate energy migration within macromolecules to account for biochemical inactivations. I t must be remembered, how-ever, that most of the absorbed quanta do not take part in photo-chemical reactions and some evidence exists for energy migration prior to molecular fluorescence in a few cases, not discussed here (cf. Shore and Pardee, 1956).

Recent results with transforming DNA in vitro and in vivo point to the happy conclusion that we are on the way to resolving some of the complex events associated with UV injury and mutation in cells and to describing them in chemical terms.

ACKNOWLEDGMENT

Support of this study by the U. S. Atomic Energy Commission during the past decade is greatly appreciated, and the initiation of the work by my teacher, Profes-sor Fred M. Über is acknowledged.

REFERENCES

Augenstine, L. G., and Ghiron, C. A. (1961). Proc. Natl. Acad. Sei. U. S. 47, 1530-1547.

Bawden, F. C , and Kleczkowski, A. (1959). Nature 183, 50&-504. Beaven, G. H., and Holiday, E. R. (1952). Advances in Protein Chem. 7, 320-386. Beukers, R., and Berends, W. (1960). Biochim. et Biophys. Acta 41, 550-551. Brighenti, L. (1962). Biochim. et Biophys. Acta 59, 376-388. Coahran, D. R., Buzzell, A., and Lauffer, M. A. (1962). Biochim. et Biophys. Acta

55, 755-767. Chessin, M. (1960). Science 132, 1840-1841.


Claesson, I. M. (1956). Arkiv Kemi 10, 1-102. Dose, K., and Rajewsky, B. (1962). Photochem. Photobiol. 1, 181-190. Doty, P., and Geiduschek, E. P. (1953). In "The Proteins" (H. Neurath and K.

Bailey, eds.), Vol. 1. Academic Press, New York. Estermann, E. F., and McLaren, A. D. (1962). Photochem. Photobiol. 1, 109-116. Estermann, E. F., Luse, R. A., and McLaren, A. D. (1956). Radiation Research 5,

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97-108. Giese, A. C. (1950). Physiol. Revs. 30, 431-458. Haurowitz, F., and Turner, A. (1949). Enzymologia 13, 229-235. Hollaender, A., and Duggar, B. M. (1936). Proc. Natl. Acad. Sei. U. S. 22, 19^24. Kleczkowski, A. (1954). Brit. J. Exptl. Pathol. 35, 402. Kleczkowski, A. (1960). Rept. Rothamst. Expt. Sta. pp. 234r-245. Kleczkowski, A. (1963). Photochem. Photobiol. 2, in press. Livingston, R. (1955). In "Radiation Biology" (A. Hollaender, ed.), Vol. II, pp. 1-

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Christensen and B. Buchmann, eds.), pp. 573-575. Elsevier, Amsterdam. McLaren, A. D., and Luse, R. A. (1961). Science 134, 836-837. McLaren, A. D., and Shugar, D. (1963). "Photochemistry of Proteins and Nucleic

Acids." Pergamon Press, Oxford, in press. McLaren, A. D., and Takahashi, W. N. (1957). Radiation Research 6, 532-541. McLaren, A. D., and Takahashi, W. N. (1959). Biochim. et Biophys. Ada 32,

555-557. Mandl, L, and McLaren, A. D. (1949). Arch. Biochem. 21, 408-415. Marmur, J., Anderson, W. F., Matthews, L., Berns, K., Gajewska, E., Lane, D.,

and Doty, P. (1961). / . Cellular Comp. Physiol. 58, Suppl. 1, 33-56. Norman, A. (1960). Virology 10, 384-386. Oster, G., and McLaren, A. D. (1950). / . Gen. Physiol. 33, 215-228. Rideal, E. K , and Roberts, R. (1951). Proc. Roy. Soc. A205, 397. Rupert, C. S. (1961). / . Cellular Comp. Physiol. 58, Suppl. 1, 57-68. Rushizky, G. W., and Pardee, A. B. (1962). Photochem. Photobiol. 1, 15-20. Rushizky, G. W., Knight, C. A., and McLaren, A. D. (1960). Virology 12, 32-47. Setlow, J. K , and Setlow, R. B. (1960). Proc. Natl Acad. Sei. U. S. 46, 791-798. Setlow, R. B., and Boyce, R. (1960). Biophys. J. 1, 29-41. Setlow, R. B., and Doyle, B. (1957). Biochim. et Biophys. Acta 24, 27-41. Setlow, R. B., and Setlow, J. K. (1962). Proc. Natl. Acad, Sei. U. S. 48, 1250-1257. Shore, V. G., and Pardee, A. B. (1956). Arch. Biochem. Biophys. 62, 355-368. Shugar, D. (1960). In "The Nucleic Acids" (E. Chargaff and J. N. Davidson, eds.),

Vol. I l l , pp. 39-104. Academic Press, New York. Shugar, D., and Wierszchowski, K. L. (1958). / . Polymer Sei. 31, 269. Siegel, A., and Norman, A. (1958). Virology 6, 725. Siegel, A., Wildman, S. G., and Ginoza, W. (1956). Nature 178, 117-118. Sinsheimer, R. L. (1957). Radiation Research 6, 121. Stadler, L. J., and Über, F. M. (1942). Genetics 27, 84^118.

82 A. D. MCLAREN

Stuy, J. H. (1962). Photochem. Photobiol 1, 41-48. Taylor, A. R. (1960). Ann. N. Y. Acad. Sei. 83, 670-683. Taylor, A. R., Kay, W. W., Timm, E. A., Hook, A. E., and McLean, I. W. (1957).

J. Immunol. 79, 265-275. Wang, S. Y. (1961). Nature 190, 690-694. Wang, S. Y. (1962). Photochem. Photobiol. 1, 37-40, 135-146. Wierzchowski, K. L., and Shugar, D. (1962). Photochem. Photobiol. 1, 21-36. Zelle, M. R., and Hollaender, A. (1954). / . Bacteriol. 68, 210. Zirkle, R. E., and Uretz, R. B. (1963). Proc. Natl. Acad. Sei. U. S. 49, 45-52.

Chapter 4

ABSORPTION SPECTRA, SPECTROPHOTOMETRY, AND ACTION SPECTRA

Mary Belle Allen

Kaiser Foundation Research Institute Laboratory of Comparative Biology

Richmond, California

1. Introduction—Measurement of Absorption Spectra of Scattering Materials

The absorption spectrum of a substance is often a useful property for its characterization in biological systems. Measurement of absorption spectra provides a nondestructive method of both qualitative and quanti-tative analysis that can frequently be carried out on very small samples. Since the general principles of absorption spectroscopy in the ultraviolet, visible, and infrared have been discussed in several recent treatises (Hiskey, 1955; Scott, 1955; Clark, 1955; West, 1960; Gibson, 1949), it will be assumed that the reader is familiar with these principles, and the emphasis in this chapter will be on the special problems of measure-ment of absorption spectra of biological materials. The most serious of these problems is that the spectra must often be measured in turbid suspensions, rather than in clear solutions or homogeneous solids.

When light passes through a homogeneous medium there is no attenuation of the beam unless the light is of a wavelength correspond-ing to the difference between two energy levels of the molecules com-prising the medium. In this case, the light is absorbed by the molecules; the pattern of light absorption obtained as one varies the wavelength is the absorption spectrum. In a turbid suspension, however, the light is also reflected from the surface of the particles and refracted by passage through them, as shown in Fig. 1. This results in an attenuation of the light beam in addition to that due to absorption of light.

Light scattering is a complex phenomenon which depends on the size of the scattering particles, their shape, and the refractive index difference between the particles and suspending medium. The simplest relation is found for optically isotropic particles small compared to the wavelength of the light used. For all colorless particles

J = Ιφ-τΐ

83

84 MARY BELLE ALLEN

FIG. 1. Reflection, refraction, and transmission of light by a suspension of particles.

where / is the transmitted intensity, l0 the initial intensity, τ the turbidity, and I the path length. For small independent isotropic par-ticles

32π%Μη02 in - n0

T " 3iVX4 \ c

where c is the concentration of particles in gm per milliliter, M the molecular weight, N Avogadro's number, n0 the refractive index of the medium, and n the refractive index of the solution (cf. Oster, 1948).

As the dimensions of the particles approach the wavelength of light, each portion of the particle scatters according to the Rayleigh equation [Eq. (1) shown above]. Since the positions of these scatterers are fixed relative to each other, the scattered wavelets will interfere. This results in a greater scattering of light in the direction of the incident beam than in other directions. The proportion of forward scattering depends on the shape of the particles, being greatest for spheres and smallest for thin rods (cf. Oster, 1948). Moreover, for particles large compared to

4. SPECTROSCOPY : SPECTROPHOTOMETRY 85

the wavelength of light, the scattering is no longer proportional to λ-4, but to A-4·*·2*, where B = 1.0 for rods, 1.74 for coils, and 2.0 for spheres (Oster, 1949). For particles close to the wavelength of light in size, diffraction effects may also appear. For the polydisperse suspensions of different-sized particles found in many biological systems, the situation is extremely complex. The general theory of light scattering developed by Mie (1908) applies, but is too complicated to be very useful for absorption spectrum calculations.

The most obvious effect of light scattering on the experimental measurement of absorption spectra is a decrease in the definition of the absorption bands. This effect is most evident when an instrument that collects only a small cone of light is used. Several workers have used devices that diffuse the light transmitted by the samples so as to obtain a greater effective solid angle of collection of light. These have included filter paper (Lundegardh, 1951), opal glass (Shibata et ai., 1954), and fluorescing solutions (Amesz et al., 1961). Such diffusing materials do appreciably increase the resolution of spectra obtained with some spectrophotometers, as shown in Fig. 2a. However, when instruments that collect light from a larger solid angle are used, good resolution is obtained without the use of diffusing materials, as shown in Fig. 2b. Some useful details of the absorption and scattering curve are lost when diffusers are employed (Murchio and Allen, 1962).

Another method of minimizing the effects of scattering has been proposed by Barer (1955), who placed cells in protein solutions of a refractive index matched to that of the cell contents by microscopic examination. The relation between scattering and refractive index will be discussed more fully below, but it should be mentioned here that the Barer method has also something in common with the diffusion plate methods, since the protein solutions act as Rayleigh scatterers.

Another effect of light scattering is the shifting of the apparent position of absorption bands. It will be recalled that the scattering of light depends on the difference in refractive index between the scattering particle and the medium. In the neighborhood of an absorption band the refractive index changes, due to the phenomenon of anomalous dispersion (cf. Wood, 1934). The changes in refractive index are given by the equations

and

nk-ÜS* "'" (2)

86 MARY BELLE ALLEN

I . . i^Z . Λ 200 300 400 500 600 700 800

λ in πιμ

(a)

FIG. 2. (α) Absorption spectrum of Chlorella pyrenoidosa measured with (B) and without (A) a diffusing plate in a spectrophotometer with a diverging beam (from Shibata et al., 1954). (6) Absorption spectrum of Chlorella pyrenoidosa measured without a diffusing plate in a spectrophotometer with a collimated beam.

where n is the index of refraction, k the absorption coefficient, N Avo-gadro's number, q the number of electrons contributing to the dispersion, e the charge of the electron, m the mass of the electron, v the frequency, v0 the frequency of the absorption maximum, and V the frequency differ-ence between the two frequencies corresponding to the half-width of the absorption band. The magnitude of the effect is thus dependent on the shape and intensity of the absorption band and on the type of electronic transition involved. The changes in refractive index have been both measured and calculated for a number of dyes in solution (Söderborg,

4. SPECTROSCQPYISPECTROPHOTOMETRY 87

O.D.

4000 5000 6000 7000 &

FIG. 2. (b)

1913; Wood, 1934). Theory and experiment agree in showing that the differences for these dyes are small, in the second decimal place. On the other hand, n for solid cyanine varies from 2.35 at 640 m/x to 1.1 at 530 m/i (Wood, 1934).

The influence of scattering on the measurement of absorption spectra has been extensively investigated with the chlorophylls in living organ-isms. Chlorophyll a in the living cell has its absorption maximum at a wavelength 10-15 ηΐμ longer than that of chlorophyll a in organic solvents. Moreover, the band in vivo is broadened and flattened relative to that in vitro. These changes in spectrum, if they are not optical artifacts, can provide information on the state of chlorophyll in the living cell. The extent to which scattering influences the measurement of spectra has therefore been of interest. Latimer (Latimer and Rabinowitch, 1956, 1957, 1959; Latimer, 1959) measured the wavelength dependence of scattering in suspensions of the green alga Chlorella pyrenoidosa. A scattering maximum was observed on the long wave-length side of the red absorption band of chlorophyll. The position of the red maximum measured with an ordinary spectrophotometer was shifted toward the red compared to that measured in an integrating

88 MARY BELLE ALLEN

sphere (cf. Section 2 of this chapter). This shift, which was ascribed to scattering, was removed by introducing a diffusing plate into the system.

Charney and Brackett (1961) determined the magnitude of the shift in the chlorophyll absorption band that could be caused by selective scattering, and found it to be no more than 4-5 ηΐμ,. The remaining difference between the position of the chlorophyll bands in vivo and in vitro must therefore be ascribed to complexing or to other changes in the environment of the chlorophyll molecules.

Murchio and Allen (1962) showed that with a spectrophotometer collecting light from a moderately large solid angle (0.0194 steradian) the position of the chlorophyll absorption peak in C. pyrenoidosa was the same as that reported by Latimer (1959) using the integrating sphere. They also measured the scattering of Chlorella suspensions from 10,000 A down and found, rather surprisingly, that the scattering de-pended on the inverse fourth power of the wavelength. This could be taken to imply that most of the scattering is due to small particles within the cell rather than the cell envelope. The physical situation is probably actually more complex, however. In very dilute suspensions the scattering becomes independent of wavelength (diffractive scatter-ing?). Moreover, Jaycock and Parfitt (1962) have shown that for certain values of the refractive index, including those expected for cell material, the errors involved in using the Rayleigh equation for particles con-siderably larger than those to which it should apply are not significant.

In addition to being present in scattering material, light-absorbing materials in living cells are usually inhomogeneously distributed through-out the cell (chlorophyll in chloroplasts or chromatophores, cytochromes in mitochondria, etc.). In some parts of the cell the pigment concentra-tion may be very high. This results in a flattening of the absorption bands relative to those observed in a homogeneously dispersed pigment. I t should be emphasized that this effect is the result of concentration of the pigment in packets, and has nothing to do with scattering. Duysens (1956) has treated this problem and developed methods for correcting the measured spectra for these flattening effects and for the measure-ment of the optical density of a pigmented particle. His methods would appear to be of particular value for dealing with materials that cannot readily be dispersed by chemical or mechanical means.

In spite of these difficulties of scattering and flattening, quantitative spectrophotometry of materials in living cells is often possible. Although Beer's law does not in theory strictly hold for particles, in practice it is found to apply to cell or particle suspensions if the appropriate con-centration range is chosen (Lundegardh, 1960; Murchio and Allen,


1962). Lundegardh found a linear relation between the concentration of a yeast suspension and light absorption in the pyridine nucleotide band at 340 m/* and in cytochrome bands at 400-450 ηΐμ,, 544-550 ταμ, 562-570 m/A, and 590-604 m^. Murchio and Allen found similar relations in the red absorption band of chlorophyll when the spectra were corrected for scattering by extrapolation of the scattering curve measured in the 7500-10,000 A region. Although the correction should, strictly speaking, be based on the inverse fourth power wavelength dependence, a simple linear extrapolation was found to be satisfactory in practice.

2. Instrumentation for Absorption Spectra Although it should be evident from the preceding section that the

measurement of absorption spectra of biological materials is often not a simple matter, there are commercial instruments available that are satisfactory for most purposes. The best spectrophotometer that the budget can afford is a worthwhile investment for anyone engaged in photobiological work.

Among the points to be considered in choosing an instrument are its sensitivity, its resolution, its freedom from stray light, and the con-venience of its data presentation. When scattering material is being meas-ured, the collimation of the light beam and the relative position of sample and detector should be added to the above requirements. These features determine the cone of light falling on the detector. The ability to perform well at high absorbance is a distinct advantage when absorp-tion must be measured on top of a background of scattering. If exten-sive use is to be made of the instrument, a recording type is definitely recommended. A complete discussion of available instruments will not be given here, but a few remarks on readily available American spectro-photometers may be in order.

The author has found the Cary Model 14R spectrophotometer to be an excellent instrument for the measurement of absorption spectra of living cells. This instrument contains a double monochromator and has a range from 1750 A to 26,000 A. Its resolution is between 1 and 3 A in the visible and near infrared, and it is exceptionally free from stray light. Its light beam is well collimated, so that a large cone of light reaches the detector. The manufacturers have recently introduced an accessory for the measurement of spectra of scattering materials in which the detector is moved into the sample compartment, next to the sample cuvette, so that a very large cone of light is collected. Model 14R differs from the standard Model 14 in having a tungsten light source of higher intensity and in having a lead sulfide cell in the forward as well as the reverse direction of the light beam. The first modification makes

90 MARY BELLE ALLEN

it possible to work at high absorbance with a narrow slit width and consequent good resolution; the second has been found essential for measurement of absorption spectra in the far red and for determination of scattering curves, which must be continued well past the wavelength of pigment absorption.

A less expensive instrument, manufactured by Perkin-Elmer, has a similar optical system to the Cary, but does not present its data on a linear wavelength scale.1

The Beckman DK-2 recording spectrophotometer uses the optical system of the DU instrument, which has been a reliable performer in many laboratories for a number of years. It has appreciable amounts of stray light at certain wavelengths. It also has a diverging light beam, the effect of which is more marked in the DK-2 than in the DU be-cause of the greater distance between sample and detector in the former. Collimating lenses can be added in the sample compartment to correct this disadvantage (Bailey, 1961).

Theoretically, the best method for measuring absorbance in the presence of scattering is by the use of an Ulbricht sphere. This is a hollow sphere coated on the inside with magnesium oxide. Light enters the sphere through a window and is then diffusely reflected by the coated walls, so that the sphere is ideally uniformly filled with light. A photo-cell or other detector located in a portion of the sphere shielded from the incident beam measures this light. When an absorbing sample is introduced into the sphere, any light scattered or reflected by the sample is again diffusely reflected, so that the quantity of light in the sphere is diminished only by the amount absorbed. Errors due to scattering are thus eliminated. The sphere, however, has a number of other possible sources of error, including stray light, specular reflections from the sample or its container, and the difficulties of maintaining a perfectly reflecting MgO surface. It has been used in a number of critical inves-tigations (e.g. Rabideau et al., 1946; Latimer, 1959; Charney and Brackett, 1961), but is not necessarily the instrument of choice for most purposes. Several spectrophotometer manufacturers supply diffuse reflectance accessories (usually half Ulbricht spheres) which may be useful for some photobiological purposes.

3. Measurement of Mixtures of Absorbing Substances Most absorption spectra of biological materials are due to a mixture

of substances rather than a single material. Unless there are interactions between the absorbing substances, the total absorbance at each wave-

*Note added in proof: This company is now manufacturing an instrument with a linear wavelength scale.

4. SPECTROSCOPY:SPECTEOPHOTOMETRY 91

length will be the sum of the absorbancies of the components of the mixture. If the absorption coefficients of the individual components are known, the mixture can be analyzed by solving a set of simultaneous linear equations. In theory, only as many wavelengths need be meas-ured as there are components in the mixture; in practice, it is often better to obtain the complete absorption curve of the sample and analyze it by adding or subtracting the absorption curves of the com-ponents. This provides a better check on errors of measurement, and may lead to the recognition and identification of unsuspected com-ponents of the mixture. With the present availability of recording spectrophotometers and computing equipment, the labor involved in the more elaborate procedures is not prohibitive.

Several computing devices have been developed especially for the purpose of analysis of absorption spectra. French et al. (1954) built a graphical computer in which heavily inked curves on moving tables are traced by photoelectric curve followers which continuously set potentiometers so that their output voltage is proportional to the curve height. The tables either will move at a constant rate or can be con-trolled by a variable voltage. Curves can be added, subtracted, multi-plied, divided, or otherwise operated upon, and complex curves can be fitted by algebraic combinations of simpler curves.

Noble et al. (1960) constructed an analog computer which generates several distribution functions with known parameters and presents their sum on an oscilloscope. The oscilloscope image can be projected on to an experimentally determined absorption spectrum and the parameters adjusted until the curves match. I t is much faster in operation than the computer of French et al., but involves making assumptions about the shapes of absorption curves that need not be made in use of the latter device.

A somewhat similar result to that of the computer of Noble et al. can be obtained with commercially available analog computers using func-tion generators that approximate experimental curves by a series of straight line segments. This is, of course, subject to the limitation that the curves should be sufficiently simple to be reasonably approximated by 20-25 line segments.

The alternative approach to the analysis of spectral data into its components is to digitize the output of the spectrophotometer and use a digital computer for the addition, subtraction, or other operation. How-ever, for many experimenters plotted curves provide a greater compre-hension of the spectra and of the effect of variation of components on the overall result than do numerical relations.

I t is sometimes difficult to recognize that an absorption spectrum is in fact composed of several overlapping bands. The derivative spectro-

92 MARY BELLE ALLEN

photometer, which plots the first derivative of absorbance with respect to wavelength against wavelength, was developed by French (1957) to cope with this problem. A diagram of this instrument is shown in Fig. 3. An extreme example of the ability of the derivative spectro-

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-ψ^ is plotted against X by a ratio recorder

FIG. 3. Diagram and principles of the derivative spectrophotometer (from French, 1957).

4. SPECTROSCOPY:SPECTROPHOTOMETRY 93

photometer to resolve overlapping absorption bands is shown in Fig. 4, in which the resolution of two bands with the same peak position, differ-ing only in band width, is shown.

§> 0.2 l·

E - 0 . 2 l ·

3 0 Wavelength

FIG. 4. Integral and derivative absorbance curves for two symmetrical absorption bands peaking at the same wavelength but of different band width. The deriva-tive curve clearly shows that the resultant band is formed of two overlapping components (from French, 1957).

Bailey (1961) has modified the Cary spectrophotometer to record the derivative of absorbance by attaching a generator that produces a voltage proportional to the rate of pen travel. An accessory of this type could probably be made commercially available if sufficient interest were shown in it.

94 MARY BELLE ALLEN

The most extensive use of derivative spectrophotometry has been in the work of French (1958, 1959) and his colleagues on the structure of the red absorption band of chlorophyll in the living cell. In contrast to chlorophyll a in solution, the spectrum of chlorophyll a in the living cell has been shown to be composed of several overlapping absorption bands. An example of this is shown in Fig. 5. It will be noted that the

i i i i i—|—τ—i i i i—i i i i i—ΓΊ—I—r- r - |—i I I i i I—r~r

I I I i L_J I i i L J i i L J L_J i i » l i i i ι I ι ι ι L ll 6 0 0 650 7 0 0 750

Wave length mji

FIG. 5. Integral and derivative absorbance curves for the red band of chloro-phyll in the crysomonad Ochromonas danica. The complexity of this band in the living organism, while visible in the integral curve made with a high-resolution spectrophotometer, is emphasized by the derivative curve (from Allen et al, 1960).

complexity of the absorption band can be detected in the absorbance curve, but is more prominently displayed in the curve of the derivative of absorbance.

4. Other Special Methods—Differential Spectroscopy and Rapid Reactions

Whenever a change in absorption is to be measured, the sensitivity of detection of the change can be greatly increased and difficulties due to scattering and opacity of samples minimized by the use of differential (not to be confused with derivative) spectrophotometry. In this method,

i i i i i—|—τ—i i i i—i i i i i—ΓΊ—I—r- r - |—i I I i i I—r~

.,-, ,\~-AbsorPtlon

\ ,,

Ochromonas danicatypical spectra of young cells

------


as its name implies, the difference of absorbance between a control and the reacted sample is measured.

Although difference spectra can be measured with any double-beam spectrophotometer, some problems studied with this technique have de-manded special instruments that can detect absorbance differences of as little as 10-4 OD units, and have a fast response and high stability. The principles and practice of construction of such spectrophotometric equipment have been extensively described by Chance and his associates (Chance 1940, 1951, 1954; Yang and Legallais, 1954). Two instruments devised by this group are illustrated in Figs. 6 and 7. The original papers

monochrom -ator

set to λ Α

exit slit

monochromator

set to XA

tungsten Isrnp

vibrating

photomuk'plier

half silvered mirror

Fid. 6. Schematic diagram of a time-sharing system for rapid measurement of absorbance changes occurring at two different wavelengths. Light reaching the photomultiplier from the vibrating mirror is modulated into a square wave. By appropriate demodulation the portions of this wave due to mixtures of XA and XB

are rejected and signals obtained that are proportional to the absorbances at XA and XB (from Chance, 1951).

should be consulted for further details. In photobiological work it is often of interest to measure the effect

of light on absorption spectra. An instrument designed by Duysens (1952) for this purpose is illustrated in Fig. 8. Kok (1957a, 1957b, 1959) has elaborated on this technique and has designed instruments with a series of rotating slotted disks to give light and dark periods of different duration and to separate in time the measuring and the actinic

96 MARY BELLE ALLEN

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i t

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FIG. 7. Optical system (above) and schematic diagram (below) of a split-beam spectrophotometer for measurement of difference spectra. Light from the mono-chromator is split into two beams by a chopping mirror and the ratio of the intensities of the two beams is measured. Results are recorded on a linear wave-length scale at a maximum rate of 6 m/i/sec. The noise level corresponds to a change of optical density of 10"4 at 400 ταμ with a spectral interval of 3 m/£. The overall accuracy on standard solutions is about 2% (from Yang and Legallais, 1954).

FIG. 8. Schematic diagram of apparatus used to measure change-of-absorption spectra. The straight filament of the tungsten lamp, Li, is imaged, by means of a lens, on the entrance slit of the monochromator. This slit is divided by a horizontal strip, which splits up the light into two beams bi and bi. The sector disk modulates both beams with the same frequency: the phases of the beams, however, differ by w radians. Since the phototube is connected to an amplifier provided with a phase-sensitive galvanometer, the beams cause opposite deflections. They are so adjusted as to compensate each other approximately. A change of the intensity of beam bi, owing to a change in absorption of the suspension, causes a displacement of the galvanometer image. This displacement is a measure of the change in absorption. The sensitivity of the apparatus is proportional to the intensity of the beams. The change in absorption of the bacterial suspension is brought about by irradiation with strong light from the tungsten lamp La (from Duysens, 1952).

97

98 MARY BELLE ALLEN

beam so that light from the latter does not affect the measurements. Kok's publications should be consulted for descriptions of this equipment.

The light-induced absorbance changes in algae and purple bacteria, typical examples of which are shown in Fig. 9, have proved valuable

0.4 h

0.3 h

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Spectrum of the change of absorption '(enlarged)-

*t>—o—~-o

0.02

H 0.01

H 0.01

900 850 800 750 * Wavelength in rryi

(a)

FIG. 9. Difference spectra of illuminated vs. darkened photosynthetic bacteria: (a) Rhodospirillum rubrum in the region of bacteriochlorophyll absorption (from Duysens, 1952) ; (b) the same bacterium in the region of cytochrome absorption (from Chance, 1957).

Optical density increment (cm )

o ό o

Optical density increment (cm""')

66 AHxaKOxoHJOHxoads : Adoosonxoads *f

FIG

. 9.

(b)

100 MARY BELLE ALLEN

tools for studying the mechanisms of photosynthesis. Comparison of the difference spectra with the absorption spectra of known pigments in other cellular constituents can indicate which pigments are involved in the reactions, while measurement of the time course of the changes can show the sequence of the reactions.

Since many absorbance changes in biological materials are reflections of the presence of transitory intermediates, methods for measuring rapid reactions are often required. One such method is to have the reactants flow rapidly through the observation vessel. This procedure, originally used by Hartridge and Roughton (1923), has been extensively used by Chance and his associates (Chance 1940, 1951, 1954; Chance et ai., 1961). Operation of such a rapid-flow device is illustrated in Fig. 10. If the volumes of the two reactants are greatly different, special prob-lems arise, which can be overcome by the use of properly designed mixing chambers (cf. above references for details of their design). Such accelerated flow methods can measure reactions from 1.5 to 30 msec. For somewhat longer times (750 msec) a stopped flow method is used, in which the reactants are rapidly mixed as in the accelerated flow method, but measurements are made in a stationary cuvette after mixing has occurred.

The American Optical Company manufactures a rapid scanning spectrophotometer which produces absorption spectra in the visible region (400-700 m/x) on the screen of a cathode ray tube 60 times a second. This is a single-beam instrument, whose performance is conse-quently dependent on the stability of the light source. Beinert and Sands (1961) have used this instrument in a study of the transient inter-mediates in the oxidation-reduction reactions of flavins and flavo-proteins. Motion pictures of the oscilloscope screen provide a graphical recording of the formation and disappearance of these evanescent molecules.

For measurement of light-induced absorbance changes taking place in as little as 10~5 sec, Witt et al. (1959) developed a variation of the light-flash spectroscopy used by Norrish and Porter (1949) for studying transient intermediates in photochemical reactions. In Witt's technique of light-flash photometry, absorbance changes as small as 0.1% and the time course of these changes from 10~5 sec to several minutes can be measured. Absorbance changes at two wavelengths can be compared, or absorbance and fluorescence measured simultaneously. The effect of illumination periods from 10~5 sec to several seconds and with different frequency can be studied. A diagram of the apparatus is shown in Fig. 11. The capacities of this equipment for investigation of complex photobiological processes are illustrated by its use in the study of

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FIG. 11. Diagram of Witt's light flash photometry apparatus. The photo-biologically active material is placed in the cuvette. The reaction is carried out with pulses of short duration from the discharge lamp, B, or of longer duration from the continuous lamp and rotating sector, D. The monochromatic measuring light


photosynthesis (Witt and Müller, 1959; Witt et al, 1960; Müller and Witt, 1961; Witt et al, 1961). Five types of short-lived absorption changes differing in duration and in wavelength dependence were ob-served in Chlorella. These could be correlated with excited states of chlorophyll, with cytochrome oxidation, and with the reduction of an unknown material that might be plastoquinone. The original papers should be consulted for details of this work.

5. Measurement of Action Spectra An action spectrum is a measure of the effectiveness of different

wavelengths of light in carrying out a photobiological process. Com-parison of action and absorption spectra may lead to an identification of the pigments sensitizing the photobiological process. Since the reac-tions that may be measured are numerous and diverse, a discussion of the measurement of action spectra is best centered around the problems that are common to all, namely, sources of light and measurement of light. Equipment for action spectrum measurements must usually be constructed by the experimenter, so that some knowledge of, and in-genuity in the application of, optics, mechanics, and electronics is essential for work in this field.

The principal types of light sources commonly used are listed in Table I. Tungsten filament lamps are probably the most convenient sources for the visible, near ultraviolet, and near infrared. For most purposes, lamps with short compact vertical filaments, either ribbons or closely coiled, are most suitable. Various types of projection bulbs and automobile headlight lamps meet these requirements. Many of the lamps operate at high amperage and low voltage, and hence require a suitable transformer. The lamp envelope darkens with use due to depo-sition of tungsten on the walls, so that the luminous output of the lamp decreases. A tungsten lamp containing iodine vapor, which combines with the tungsten and prevents its deposition, has recently been de-veloped by the General Electric Company.

Xenon, zirconium, and high-pressure mercury arcs produce a con-

passes through the cuvette onto the photomultiplier PM-1. Part of the measuring light can be deflected onto the photomultiplier PM-2 by means of a half-silvered mirror. The photocurrents from these two beams are equalized by the compen-sation circuit. The outputs of both photomultipHers pass through a differential amplifier and an electronic shutter, and are combined on an oscilloscope screen. Changes in intensity due to changes in absorbance in the cuvette thus appear on the oscilloscope screen. Effects of the flash and of fluorescence are removed by appropriate filters. With photomultiplier PM-3 either changes in absorption at a second wavelength or the fluorescence of the system can be measured.

MARY BELLE ALLEN

TABLE I SUMMARY OF LIGHT SOURCES

Continuous Sources Visible

Tungsten filament lamps with straight compact coils or ribbon filaments Pointolite lamps Zirconium arc Xenon arc Carbon arcs, plain and cored (high intensity) High-pressure mercury arc

Ultraviolet Hydrogen lamps, high voltage, low voltage, or electrodeless High-pressure mercury arc Xenon, krypton, or argon discharges Zirconium lamp

Infrared Nernst glower Globar

Line Sources Iron arc (used as wavelength standard in visible and ultraviolet) Low-pressure mercury arc Osram lamps (metal vapor discharge lamps, emitting spectra of sodium, potassium,

rubidium, cesium, helium, neon, cadmium, mercury, thallium, or zinc) Other metal-vapor or gas-discharge lamps Copper spark

tinuum of high intensity. Different models vary greatly in luminous output and spectral distribution. It should not be forgotten that these sources also have emission lines superimposed on the continuum. Carbon arcs, especially those with cored carbons used in theatrical projection, give high intensities of light, but tend to wander and vary in intensity. The continuum is overlaid by bands around 5000 Â and by the cyanogen bands in the violet and near ultraviolet. The emission in the ultraviolet is low.

The various metal vapor and gas discharge lamps are useful as calibration sources and, sometimes, for irradiation, if the wavelengths emitted are suitable.

Monochromatic light is necessary for the measurement of action spectra. This is obtained from the continuous sources either by a mono-chromator or by narrow band interference filters. Much older work on action spectra was carried out with broad-band glass or gelatin filters or with colored solutions. The response obtained with such broad spectral bands is usually difficult to interpret, and narrow bands should be used if at all possible.

104


Monochromators may be either of prism or grating type. The grating instruments are more convenient to use, having a linear wavelength scale. However, they may have more stray light than prism instru-ments. Gratings also tend to lose light in unwanted orders of diffraction, although this can be overcome for selected spectral regions by the use of suitably blazed gratings. When light of highest spectral purity is required, a double monochromator should be used.

It is often difficult to obtain enough light through a monochromator to carry out the desired photobiological reaction. Narrow band inter-ference filters, which are formed by deposition of successive layers of metallic or dielectric materials on glass, transmit more light. The art of production of these filters has developed greatly in recent years, and it is now possible to obtain them with almost any band width (from 1Â to 3000 Â) and any wavelength desired in the visible and near infrared. Transmission in the band ranges from 30 to 80-90%. The production of stable filters for the ultraviolet is still something of a problem, but one that is expected to be solved soon.

For proper results interference filters must be used with a parallel beam of light. Use of a nonparallel beam will shift the wavelength trans-mitted by the filter, or may even result in the band being split into a doublet. Interference filters also transmit light at wavelengths far removed from the nominal transmission band, so that they must be used with appropriate trimming filters. Some manufacturers supply these as integral parts of the filter, others do not. Trimming is espe-cially important when measurements of the light energy passing the filter are to be made. The filter must transmit no energy other than that in its nominal band out to 14,000 Â. A water cell will take care of longer wavelength radiation.

Measurement of the light incident on the photobiological experiment is a problem that has no easy solution. Three types of detectors can be used: (1) nonselective detectors, of which the most common is the thermopile, (2) spectrally selective detectors, such as photocells and photomultipliers, and (3) actinometers, in which photochemical reac-tions of known quantum yield are used to measure the integrated amount of energy received. Photocells and photomultipliers are the most sensi-tive light detectors and the easiest to use. However, when they are to be used with light of different wavelengths, as in an action spectrum, they must be calibrated against a spectrally nonselective detector. The stability of response of the photoelectric devices is somewhat uncertain, so that the calibration should be checked at frequent intervals.

Thermopiles, although less sensitive, respond equally to all radiant energy reaching them. They are calibrated against a standard lamp, which is a low-voltage carbon filament lamp obtainable from the


National Bureau of Standards. A number of precautions must be taken in calibrating the thermopile and in using it to measure the experimental light to be sure that it is responding only to the radiant energy which it is wished to measure and not to other sources of radiation in the room (including the experimentor!). Instructions for calibration of thermopiles with standard lamps have been published (Stair and John-ston, 1954), and are supplied with the lamps. In experimental measure-ments it is especially important to be sure that no stray infrared radiation reaches the thermopile.

Chemical actinometers, which may in some cases be useful, are not primary detectors and must be calibrated against a thermopile. They have advantages in (1) irregularly shaped experimental vessels, and (2) those fortunate situations in which an actinometer reaction can be found which has the same action spectrum as the reaction under investi-gation. The decomposition of oxalic acid catalyzed by uranyl ion has been extensively used in the ultraviolet (Leighton and Forbes, 1930; Bowen, 1946), whereas reactions of chlorophyll derivatives (Gaffron, 1927; Livingston and Pariser, 1948; Warburg and Schocken, 1949) have been used as actinometers for chlorophyll-sensitized reactions.

The foregoing brief account of the problems of action spectrum measurement is only intended to indicate the types of devices used and to point out some of their pitfalls. In addition to the treatises on absorp-tion spectroscopy mentioned in Section 1, the following references will be useful to anyone planning to make measurements of this type: Strong (1938) (old, but still very useful) ; Harrison et al (1948) ; Can-non (1960).

A few principles which are common to all measurements of response to light may also be outlined. Since most, if not all, photobiological reactions are linked to "dark" enzymatic reactions, the reactions are light-dependent only over a limited range of light intensities. As the light is increased, a point is reached at which the rate of the dark reactions limits the overall process, and the reaction is said to be light saturated. Although there are sometimes variations in the saturation rate with the wavelength of light (cf. McLeod, 1961), action spectra are usually meaningful only when the light intensity is such that light limits the reaction. It is further necessary either to establish the linearity of the light response or to remove the necessity for linearity by measuring the amount of light of each wavelength necessary to give a constant response.

Another basic requirement is that all parts of the sample receive equal intensities of light, i.e., a thin layer of lightly pigmented material should be used. Thick samples usually lead to nonlinear light responses.

4. SPECTROSCOPYISPECTROPHOTOMETRY 107

6. Interpretation of Action Spectra It is a fundamental law of photochemistry that light must be ab-

sorbed in order to have an effect. The effectiveness with which different wavelengths cause a photochemical or photobiological response is thus proportional to the absorption spectrum of the active pigment. An ac-tion spectrum can thus be used to identify the light absorbing pigment. For example, the action spectrum for photoreactivation of Streptomyces griseus exposed to ultraviolet light can be fitted to the absorption spectrum of a porphyrin (Keiner, 1951). Other examples are given in this volume, Chapters 7, 8, 10, and 11, and in Volume II, Chapters 13, 14, 17, and 18.

However, the results obtained are not always as readily interprétable. The active pigment may occur in small quantity, marked by larger quantities of inactive pigments, so that no identifiable bands correspond-ing to it occur in the absorption spectrum. For example, the action spectrum for phototaxis of the dinoflagellate Prorocentrum shows a peak at 570 m/A, although the absorption curve of the organism has no special features at this wavelength (Halldal, 1958). In the important case of the photomorphogenic effects of light on plant development, action spectra that revealed a hitherto unknown pigment led to the isolation of the phytochrome system (see Hendricks, 1960, for a review).

Other complications occur in multipigment systems in which light energy absorbed by one pigment can be transferred to another which is the actual catalyst of the photoreaction. The best examples of this are the pigment systems of photosynthetic organisms. These systems contain one or more chlorophylls, carotenoids, and, in some cases, biliprotein pigments. Light absorbed by all of these is effective in photosynthesis. For green algae and higher plants the action spectrum of photosynthesis rather closely resembles the absorption spectrum of the living material, but falls below it in the region of carotenoid absorption in the blue and on the long wavelength side of the primary absorption band of chlorophyll in the red (Emerson and Lewis, 1943). In red and blue-green algae, which contain biliproteins, the action spectrum follows the absorp-tion spectrum in the region of biliprotein absorption, but falls below it in the chlorophyll absorption bands (Haxo and Blinks, 1950). It is not, however, concluded from these results that all pigments of the cell catalyze the photosynthetic reactions, or that chlorophyll in the red and blue-green algae does not. This is because it is possible to show that the other pigments of the cell transfer their absorbed energy to a chlorophyll complex. If the action spectrum for excitation of chlorophyll fluorescence is measured, it is found that it does not follow the absorp-


tion spectrum of chlorophyll, but includes light absorbed by carotenoids and biliproteins (Duysens, 1952). This means that light absorbed by these pigments can raise chlorophyll to an excited state, and hence that energy is transferred to it.

The explanation of the apparent photosynthetic ineffectiveness of chlorophyll in some organisms has come from action spectrum measure-ments in the presence of various wavelengths of background light (Emerson et al, 1957). It was found that appropriate wavelengths of supplementary light could restore the chlorophyll to photosynthetic effectiveness. The action spectrum for increasing the ability of chloro-phyll a to catalyze photosynthesis was found to correspond to the absorption spectrum of chlorophyll b, a carotenoid, or a biliprotein. The reviews in Allen (1960) and McElroy and Glass (1960) should be consulted for the details of this reaction, which provides an excellent example of the use of action spectra of various types in understanding a complex photochemical system.

REFERENCES

Allen, M. B., ed. (1960). "Comparative Biochemistry of Photoreactive Systems." Academic Press, New York.

Allen, M. B., French, C. S., and Brown, J. R. (1960). In "Comparative Biochemistry of Photoreactive Systems" (M. B. Allen, ed.), pp. 33-52. Academic Press, New York.

Amesz, J., Duysens, L. N. M., and Brandt, D. C. (1961). J. Theoret. Biol. 1, 59-74. Bailey, G. (1961). Personal communication to J. C. Murchio. Barer, R. (1955). Science 121, 709-712. Beinert, H., and Sands, R. H. (1961). In "Free Radicals in Biological Systems"

(M. S. Blois, Jr., et al., eds.), pp. 17-52. Academic Press, New York. Bowen, E. J. (1946). "The Chemical Aspects of Light," 2nd ed. Oxford Univ. Press,

London and New York. Cannon, C. G. (1960). "Electronics for Spectroscopists." Interscience, New York. Chance, B. (1940). J. Franklin Inst. 294, 155. Chance, B. (1951). Rev. Sei. Instr. 22, 619-638. Chance, B. (1954). Discussions Faraday Soc. 17, 120. Chance, B. (1957). In "Research in Photosynthesis" (H. Gaffron, ed.), pp. 184-188.

Interscience, New York. Chance, B., Bicking, L., and Legallais, V. (1961). In "Free Radicals in Biological

Systems" (M. S. Blois, Jr., et al, eds.), pp. 101-111. Academic Press, New York. Charney, E., and Brackett, F. S. (1961). Arch. Biochem. Biophys. 92, 1-12. Clark, C. (1955). In "Physical Techniques in Biological Research" (G. Oster and

A. W. Pollister, eds.), Vol. 1, pp. 206-325. Academic Press, New York. Duysens, L. N. M. (1952). "Transfer of Excitation Energy in Photosynthesis."

Dissertation, Utrecht. Duysens, L. N. M. (1956). Biochim. et Biophys. Acta 19, 1-12. Emerson, R., and Lewis, C. M. (1943). Am. J. Botany 30, 165-178. Emerson, R., Chalmers, R., and Cederstrand, C. (1957). Proc. Natl. Acad. Set.

U. S. 43, 133-143.


French, C. S. (1957). Proc. of IJSA. Instrumentation and Control Symposium pp. 83-94.

French, C. S. (1958). Proc. 19th Ann. Biol. Colloq. Oregon State Coll. pp. 52-64. French, C. S. (1959). In "Photoperiodism and Related Phenomena in Plants and

Animals" (R. B. Withrow, ed.), pp. 15-39. Publ. No. 55, Am. Assoc. Adv. Sei., Washington, D. C.

French, C. S., Towner, G. H., Bellis, D. R., Cook, R. M., Fair, W. R., and Holt, W. W. (1954). Rev. Sei. Instr. 25, 765-775.

Gaffron, H. (1927). Ber. 60, 755. Gibson, K. S. (1949). Natl. Bur. Standards (U. S.) Cire. 484. Halldal, P. (1958). Physiol. Plantarum 11, 118-153. Harrison, G. R., Lord, R. C , and Loofbourow, J. R. (1948). "Practical Spectros-

copy." Prentice-Hall, Englewood Cliffs, New Jersey. Hartridge, H., and Roughton, F. J. W. (1923). Proc. Roy. Soc. (London) A104,

376. Haxo, F. T., and Blinks, L. R. (1950). J. Gen. Physiol. 33, 389-422. Hendricks, S. B. (1960). In "Comparative Biochemistry of Photoreactive Systems"

(M. B. Allen, ed.), pp. 303-322. Academic Press, New York. Hiskey, C. F. (1955). In "Physical Techniques in Biological Research" (G. Oster

and A. W. Pollister, eds.), pp. 74-130. Academic Press, New York. Jaycock, M. J., and Parfitt, G. D. (1962). Nature 194, 77. Keiner, A. (1951). Sei. American 184, 22. Kok, B. (1957a). Nature 179, 583-584. Kok, B. (1957b). Acta Botan. Neerl. 6, 316-336. Latimer, P. (1959). Pfont Physiol. 34, 193-199. Latimer, P., and Rabinowitch, E. (1956). J. Chem. Phys. 24, 480. Latimer, P., and Rabinowitch, E. (1957). In "Research in Photosynthesis" (H.

Gaffron, ed.), pp. 100-107. Interscience, New York. Latimer, P., and Rabinowitch, E. (1959). Arch. Biochem. Biophys. 84, 428-441. Leighton, W. G., and Forbes, G. S. (1930). / . Am. Chem. Soc. 52, 3139. Livingston, R., and Pariser, R. (1948). / . Am. Chem. Soc. 70, 1510. Lundegardh, H. (1951). Nature 167, 71. Lundegardh, H. (1960). Biochim. et Biophys. Acta 41, 245-251. McElroy, W., and Glass, B., eds. (I960). "Light and Life." Johns Hopkins Press,

Baltimore, Maryland. McLeod, G. (1961). Plant Physiol. 36, 114-117. Mie, C. (1908). Ann. Physik [4] 25, 377. Müller, A., and Witt, H. (1961). Nature 189, 944-945. Murchio, J., and Allen, M. B. (1962). Photobiology 1, 259-266. Noble, F. W., Hayes, J. E., Jr., and Eden, M. (1960). In "Medical Electronics"

(C. N. Smyth, ed.). Thomas, Springfield, Illinois. Norrish, R. G. W., and Porter, G. (1949). Nature 164, 658. Oster, G. (1948). Chem. Revs. 43, 319. Oster, G. (1949). Rec. trav. chim. 68, 1123. Rabideau, G. S., French, C. S., and Holt, A. S. (1946). Am. J. Botany 33, 769-777. Scott, J. F. (1955). In "Physical Techniques in Biological Research" (G. Oster and

A. W. Pollister, eds.), pp. 131-205. Academic Press, New York. Shibata, K., Benson, A. A., and Calvin, M. (1954). Biochim. et Biophys. Acta 15,

461-470. Söderborg, B. (1913). Ann. Physik [4] 41, 381-402.


Stair, R., and Johnston, R. G. (1954). / . Research Natl. Bur. Standards 53, 211-215. Strong, J. (1938). "Procedures in Experimental Physics." Prentice-Hall, Englewood

Cliffs, New Jersey. Warburg, O., and Schocken, V. (1499). Arch. Biochem. 21, 263. West, W. (1960). In "Physical Methods of Organic Chemistry" (A. Weissberger,

ed.), Part III, pp. 1799-1958. Interscience, New York. Witt, H., and Müller, A. (1959). Z. physik. Chem. (Frankfurt) [N.S.] 21, 1-23. Witt, H., Moraw, R., and Müller, A. (1959). Z. physik. Chem. (Frankfurt) [N.S.]

20, 193-205. Witt, H., Moraw, R., Müller, A., Rumberg, B., and Zieger, G. (1960). Z. physik.

Chem. (Frankfurt) [N.S.] 23, 133-138. Witt, H., Müller, A., and Rumberg, B. (1961). Nature 191, 194-195; 192, 967-969. Wood, R. W. (1934). "Physical Optics," 3rd ed. Macmillan, New York. Yang, C , and Legallais, V. (1954). Rev. Sei. Instr. 25, 801-807.

Chapter 5

THE PHOTOCHEMICAL REACTIONS OF PHOTOSYNTHESIS

F. R. Whatley and M. Losada1

Department of Cell Physiology, University of California Berkeley, California

1. Introduction Those organisms which live by the conversion of light into chemical

energy are called "photosynthetic." There are two groups of such organisms—the (green) plants and the photosynthetic bacteria. These two groups are mainly distinguished on the basis of their ability to use water as the photoreductant in photosynthesis (green plants) or their need for a photoreductant at a much greater reducing potential than water (photosynthetic bacteria).

These chlorophyll-containing cells carry out several partial processes during the overall process which we call photosynthesis. (1) Chlorophyll is used to absorb light. (2) The absorbed light is converted into a form of chemical energy, which the living cell can use for biochemical work. (3) The chemical energy is stored in a form which is available to the cell for subsequent biochemical negotiations not themselves directly dependent on light energy. It should be emphasized that the chemicals containing the stored energy are the ultimate energy sources for all organisms which are unable to use light energy directly.

The first of these processes, the absorption of light by chloroplasts and the physical aspects of the light reaction, is treated in Chapter 6 of this volume. The related question of the function of the accessory pigments in plants is discussed in Chapter 7.

It is our purpose in this chapter to discuss principally the second process, the biochemical mechanisms whereby light energy is converted into chemical energy in both plants and bacteria. We shall also discuss briefly some aspects of the third process, the mechanisms of energy storage.

1 Present address: Seccion de Bioquimica, Centro de Investigaciones Biologicas, Calle Velazquez 138, Madrid, Spain.

I l l

112 F. R. WHATLEY AND M. LOSADA

Changing Concepts in Photosynthesis

After the discovery of photosynthesis in green plants during the last quarter of the eighteenth century (largely due to the efforts of Priestley and Ingenhousz) considerable emphasis was placed on the identification of the first products of the process. Oxygen was accepted to be one of the primary products of photosynthesis and it was thought to arise by the splitting of the carbon dioxide molecule by light. The other primary product was earlier identified as starch, which was thought to result from the "hydration" of the carbon remaining when carbon dioxide was photodecomposed. Photosynthesis was thus defined by the simple equation:

light C02 + H20 ► (CH20) + 02 (1)

chlorophyll

Further experimental work on the mechanism whereby the assimilation occurred focused attention on a number of carbon compounds which might precede starch as the primary product of photosynthesis. Physio-logical experiments suggested that monosaccharides and disaccharides might be formed in advance of starch, and much discussion took place about the nature of the first sugar formed. A speculative suggestion by Bayer proposed formaldehyde as a precursor to the sugars, but this suggestion received no experimental support. More recently the work of Calvin and his associates (see review of Bassham and Calvin, 1957) has shown clearly that the three-carbon compound phosphoglyeerie acid should be regarded as the first stable product of photosynthesis with respect to carbon assimilation. The mechanism whereby this compound is further metabolized in photosynthetic organisms via the reductive pentose cycle and the reversal of some segments of the glycolytic path-way has been worked out in considerable detail.

The assimilation of C02 was once thought to be an exclusive feature of photosynthetic cells. However, the ability of many organisms to assimilate carbon dioxide in the dark has now been well documented. It has been shown that several autotrophic bacteria possess the same enzymatic apparatus for carbon dioxide fixation in the dark that is found in photosynthetic carbon dioxide fixation in green plants. The discussion about which carbon compound is the first to be formed when carbon dioxide is fixed would thus seem to be less pertinent to the question of the first product of photosynthesis than it seemed initially.

A number of photosynthetic bacteria are known which, under ana-erobic conditions, use carbon dioxide in the light if they are provided with a suitable reducing compound, such as hydrogen sulfide. These or-

5. PHOTOCHEMICAL REACTIONS OF PHOTOSYNTHESIS 113

ganisms do not evolve oxygen. After a careful comparative study of a number of photosynthetic bacteria van Niel (see review, 1941) sug-gested that light does not photodecompose carbon dioxide itself, a reac-tion which would have involved oxygen evolution, but that light is responsible for the formation of a reductant for carbon dioxide. Van Niel emphasized the nature of the photosynthetic process as an oxida-tion-reduction reaction, in which the reductant was produced in the light from water (green plants) or from a substance like hydrogen sul-fide (photosynthetic bacteria). The current investigations on the pri-mary reactions of photosynthesis, under review in this chapter, pick up the trail at this point. According to the mechanism for carbon dioxide fixation currently proposed (the reductive pentose cycle of Fig. 1; see p. 115) the only requirement for the continued assimilation of carbon dioxide is a supply of adenosine triphosphate, together with reduced phosphopyridine nucleotide. The essence of photosynthesis now appears to lie in the way in which these two compounds are formed as a result of the photochemical reactions. An excellent historical review of the changing concepts of photosynthesis has been written by Arnon (1961a).

2. The Chloroplast as the Photosynthetic Unit Photosynthetic cells carry out the chemical energy changes of respira-

tion and cellular metabolism at the same time that they perform the conversion of light into chemical energy during photosynthesis. Since some of the cofactors, enzymes, and intermediates involved in these two types of energy conversion may be identical, there results an inter-mixing of the components and it becomes difficult to distinguish with certainty the photosynthetic from the respiratory pathway. One way to avoid this difficulty is to isolate an organelle from the photosynthetic cell which can carry out the overall photosynthetic process, but which is free from the complication of respiration and associated metabolic processes. Chloroplasts isolated from other cytoplasmic particles do not respire; moreover, they contain the photochemically active chlorophyll. This makes them potentially suitable for the study of photosynthesis in a* simplified system. The experiments of Hill which began in 1937 (see review, 1951) showed that isolated chloroplasts could carry out the photochemical evolution of oxygen when given an artificial electron acceptor such as ferric oxalate, according to the equation

2Fe + + + + H 2 0 -» 2Fe + + + 2H+ + 1/2 0 2 (2)

This demonstrated that at least a part of the photosynthetic apparatus could be isolated with the chloroplasts. The isolated chloroplasts were not then shown to be able to assimilate carbon dioxide, and the con-


sensus of the active investigators in this field was that the cooperation of other cytoplasmic components, especially of soluble enzymes, was needed for the complete process of photosynthesis to occur.

By altering the methods of isolating the chloroplasts and testing their photochemical activities it was shown by Arnon et al. (1954) that whole chloroplasts can assimilate carbon dioxide. The reduction of carbon dioxide to carbohydrate was carried out at room temperature by isolated chloroplasts, unaided by other cellular particles or enzyme systems, with no energy supply except visible light. Molecular oxygen was evolved and the products of carbon dioxide assimilation by chloro-plasts were found to be the same as those observed in photosynthesis in whole cells by Benson (1951) and by Bassham et al. (1954).

In this way direct experimental evidence was obtained that chloro-plasts represent the cytoplasmic bodies in which the complete photo-synthetic apparatus is localized. Since the isolated chloroplasts did not respire, extracellular photosynthesis by chloroplasts could be studied independently of at least some of the cellular activities which cannot be kept separated from photosynthesis in the intact cell. Most of the experimental data presented in this article were obtained by using iso-lated chloroplasts from green plants or the equivalent chromatophores from photosynthetic bacteria.

Carbon Dioxide Assimilation by Isolated Chloroplasts

Using radioactive C02 to follow the progress of the reaction, C02 assimilation was found to proceed at a constant rate for up to 1 hour in whole chloroplasts. The major insoluble product was starch. Oxygen evolution in an amount equivalent to the C02 taken up was observed. The soluble radioactive products included phosphate esters of fructose, glucose, ribulose, sedoheptulose, dihydroxyacetone, and glyceric acid, glycine, malic and aspartic acids (see Allen et al., 1955).

Experiments on extracellular photosynthesis by isolated chloroplasts have been further extended. Not only spinach (the plant on which the earliest experiments were carried out) but also sugar beet, Swiss chard, sunflower, pokeweed {Phytolacca americana) and New Zealand spinach (Tetragonia expansa) yielded chloroplasts which could all carry out the same assimilation of C02 to carbohydrate (Whatley et al.} 1960). This list will undoubtedly be extended in the future. Our immediate inten-tion is simply to indicate that experiments on spinach chloroplasts have a general validity. The assimilation of C02 to the level of carbohydrate by isolated spinach chloroplasts has been confirmed by investigators in several other laboratories. In addition, Gibbs and Cynkin (1958) found that the C14-labeling of starch synthesized by isolated chloroplasts was


the same as that in starch formed during photosynthesis by intact cells.

The C02 assimilation has been investigated not only with whole chloroplasts but also with disrupted chloroplasts (Whatley et al, 1956). Both whole and broken chloroplasts gave a similar pattern of products of C02 fixation. On extraction with dilute salt solution whole chloro-plasts yielded a non-green "chloroplast extract" containing the water-soluble enzymes needed for C02 fixation in the dark. All of the individ-ual enzyme systems required for the conversion of C02 to carbohydrate, as envisaged in the reductive pentose cycle, have been identified in this chloroplast extract by Losada et al (1960a) and Trebst et al (1960).

A general scheme for C02 assimilation by isolated chloroplasts is briefly summarized in Fig. 1. The validity of the scheme shown in Fig. 1 was supported by a physical separation of light and dark reactions of

Triose phosphate

1 Hexose u ^phosphate STARCH

FIG. 1. Diagram of the reductive carbohydrate cycle in chloroplasts. The cycle consists of three phases. In the carboxylative phase (I), ribulose-5-phosphate (Ru-5-P) is phosphorylated to ribulose diphosphate (RuDP), which then accepts a molecule of C02 and is cleaved to two molecules of phosphoglyceric acid (PGA). In the reductive phase (II) PGA is reduced and converted to hexose phosphate. In the regenerative phase (III) hexose phosphate is converted into storage carbo-hydrates (starch) and into the pentose monophosphate needed for the carboxylative phase. All the reactions of the cycle occur in the dark. The reactions of the car-boxylative and reductive phases are driven by ATP and TPNH2 formed in the light (Arnon, 1961b).

photosynthesis in chloroplasts (Trebst et aï., 1958). The light phase was carried out first by a complete chloroplast system in the absence of C02, and resulted in an evolution of oxygen, accompanied by an ac-cumulation of substrate amounts of TPNH2 and ATP. These products of the light phase are circled in Fig. 1. The green portion of the chloro-


plasts was then discarded and when C02 was next supplied to the re-maining nongreen portion of the chloroplasts (equivalent to the "chloro-plast extract" mentioned above) it was converted to sugar phosphate in the dark. The light and dark phases, when carried out separately in sequence, yielded essentially the same final photosynthetic products as the continuously illuminated chloroplast system. These products in-cluded hexose and pentose monophosphates and diphosphate, phospho-glyceric acid, and dihydroxyacetone phosphate together with a little phosphoenolpyruvate and malate.

The same products of C02 assimilation by chlorophyll-free chloro-plast extracts, including the phosphorylated sugars, were also obtained in a totally dark chemosynthesis, where TPNH2 and ATP were not supplied by a prior photochemical reaction but were prepared chemically or enzymatically (TPNH2), or derived from animal material (ATP). This complete dark chemosynthesis by enzymatic components derived solely from chloroplasts is in harmony with the experiments of Racker (1955) who used a multienzyme system composed of enzymes from rabbit muscle, yeast, and spinach leaves. Moreover, experiments from the laboratories of Calvin, Horecker, and Ochoa had led to the con-clusion that C02 assimilation proceeded by way of a reductive pentose cycle driven by ATP and TPNH2 (see review by Vishniac, Horecker, and Ochoa, 1957). The experiments of Trebst et al. are in agreement with this conclusion.

The results on isolated chloroplasts have thus underlined the es-sence of photosynthesis in green plants, namely, the energy conversion process which results in the storage of light energy in the "energy-rich" compounds ATP and TPNH2. The carboxylation reaction leading to the formation of phosphoglyceric acid requires ATP, and the reduction of phosphoglyceric acid to the level of carbohydrate requires both ATP and TPNH2. The distinction between photosynthetic and nonphotosyn-thetic cells lies in the manner in which ATP and reduced pyridine nucleotides are formed. Nonphotosynthetic cells form these compounds at the expense of energy released by dark reactions, whereas photo-synthetic cells form them at the expense of light energy.

3. Photosynthetic Phosphorylation It was shown in several different laboratories that the TPNH2

needed could be made by illuminated chloroplasts, with a simultaneous evolution of oxygen. However, the source of the ATP in photosynthesis was not clear. In early models of ATP formation in photosynthesis it was proposed that the reduction of pyridine nucleotide was carried out by illuminated chloroplasts and that the resulting reduced pyridine


nucleotide was reoxidized with molecular oxygen by mitochondria (Vish-niac and Ochoa, 1952). The coupled chloroplast-mitochondrial system only differed from the conventional oxidative phosphorylation system in the source of the reduced pyridine nucleotide. In the former system the pyridine nucleotide was reduced by light, and in the latter system it was reduced by a respiratory substrate. In both cases the phosphoryla-tion reactions which lead to the synthesis of ATP depended on enzymes located in mitochondria.

The chlorophyll-containing cells in the most specialized photosyn-thetic tissue (palisade parenchyma of the mesophyll of the leaf) possess very few mitochondria (James and Das, 1957), although mitochondria occur in considerable numbers in other nongreen plant cells. It was therefore difficult to see how oxidative phosphorylation by mitochondria could produce enough ATP in photosynthetic tissues, in which the rate of photosynthesis greatly exceeds the rate of respiration.

In 1954 isolated chloroplasts were found to synthesize ATP in the light without the aid of mitochondria (Arnon et al., 1954). When con-ditions were arranged so that assimilation of exogenous C02 was pre-vented, isolated chloroplasts used light energy to esterify inorganic phosphate according to the overall reaction

light nP + nADP ► nATP (3)2

At least two fundamental differences were apparent which demonstrated that this light-induced ATP formation was not identical with oxidative phosphorylation by mitochondria. ATP formation in the illuminated chloroplasts occurred: (1) without the net consumption of molecular oxygen and (2) without the addition of a chemical substrate to supply the free energy for the synthesis of pyrophosphate bonds. The light-induced formation of ATP by chloroplasts was therefore named photo-synthetic phosphorylation, to distinguish it from oxidative phosphoryla-tion and from anaerobic phosphorylation occurring at the substrate level, as in glycolysis. In both of the latter processes ATP formation occurs at the expense of energy liberated by the oxidation of chemical substrates, whereas the only "substrate" consumed in photosynthetic phosphorylation is light.

Although photosynthetic phosphorylation when first discovered de-pended on the presence of air it proceeded without a net consumption of oxygen, and it became apparent that the oxygen was needed only as

*The symbol "n" is used to indicate that the number of molecules of ATP formed from ADP and inorganic phosphate in each complete reaction sequence is not known with certainty.


a catalyst. Further investigation of the process in spinach chloroplasts resulted in the identification of FMN (flavine mononucleotide) (What-ley et al., 1955) and vitamin K3 (Arnon et al, 1955) as catalysts for photosynthetic phosphorylation. At optimal concentrations of either FMN or vitamin K3, photosynthetic phosphorylation became inde-pendent of external oxygen, and proceeded rapidly in an atmosphere of nitrogen or argon. Without added FMN or vitamin K, or on adding very low, "microcatalytic," concentrations of the cofactors, photosyn-thetic phosphorylation remained dependent on oxygen, although no net oxygen consumption was observed. These observations are in agreement with the results of Wessels (1958), Jagendorf and Avron (1959), and Nakamoto et al. (1959), who found that photosynthetic phosphoryla-tion with suboptimal amounts of cofactors is oxygen-dependent, but be-comes oxygen-independent at higher concentration of the cofactors.

Later experiments in our laboratory laid special stress on the ana-erobic photosynthetic phosphorylation with optimal catalytic concen-trations of FMN and vitamin K, on the premise that this type of phos-phorylation is more fundamental to photosynthesis in general than the oxygen-catalyzed type, because it would also apply to bacterial photo-synthesis in which oxygen is not involved.

Soon after the discovery of photosynthetic phosphorylation in iso-lated chloroplasts, Frenkel (1954, 1956) reported a light-dependent phosphorylation in the photosynthetic bacterium Rhodospinllum rwb-rum which turned out to be similar to that in the chloroplasts. Other investigators subsequently demonstrated photosynthetic phosphorylation in cell-free preparations of the obligately anaerobic photosynthetic bac-teria Chromatium and Chlorobium. It thus became clear that green plants and photosynthetic bacteria share a common anaerobic mech-anism for a light-induced phosphorylation which does not depend on external substrates or on oxygen consumption.

Despite certain differences the energy conversion process itself ap-peared to be basically independent of oxygen. The importance of a suitable redox potential for bacterial photophosphorylation may require the addition to the reaction medium of an optimal amount of a reducing compound, such as DPNH2, succinate, or ascorbate (Frenkel, 1954, 1956; Horio and Kamen, 1962) ; these compounds, however, are not con-sumed during the reaction, but appear simply to affect the redox level of the particles. Mg++ ion is needed for the phosphorylation and ADP acts as the phosphate acceptor.

Nozaki et al. (1962) found that freshly isolated chromatophores from Rhodospirillum rubrum, which had been isolated under anaerobic conditions, did not require the addition of a cofactor, and could sustain


a high rate of phosphorylation alone. On aging of the particles the rate of cyclic photophosphorylation decreased, but was restored to the origi-nal rate by the addition of ascorbate or other reductant. This restored photophosphorylation was antimycin-sensitive, like the original. The original rate of photophosphorylation was also restored to aged particles by the addition of phenazine methosulfate, but in this case the reaction was not antimycin-sensitive. Nozaki et al. (1962) are inclined to regard the cofactor-stimulated phosphorylations by aged particles as "arti-ficial" and the ascorbate-stimulated system as "physiological."

4. The Electron-Flow Mechanism of Photosynthetic Phosphorylation

The demonstration of anaerobic photosynthetic phosphorylation pro-vided direct experimental evidence for the idea that the conversion of light into chemical energy is independent of the classical manifestations of photosynthesis in green plants: C02 reduction and oxygen evolution. The sole product of the anaerobic photosynthetic phosphorylation is ATP, and the most important fact which must be explained is that a high-energy pyrophosphate bond is formed at the expense of absorbed light energy. Although earlier proposals for a mechanism of ATP pro-duction were based on a photolysis of water, i.e., the formation in the light of reduced ([H]) and oxidized ([OH]) moieties from water, fol-lowed by a recombination of these moieties via a series of electron car-riers to provide the energy for ATP formation (see for example Arnon et al, 1956), later experiments led to different interpretations. There seemed no fundamental reason to connect ATP formation either with a photolysis of water or with C0 2 reduction.

The simplest hypothesis to account for ATP formation was to as-sume that it is coupled with a release of free energy when an electron drops from a higher to a lower energy level, such as occurs during the oxidation-reduction reactions of the electron transport in mitochondria or during glycolysis. But since photosynthetic phosphorylation needs no added substrate (electron donor) and consumes no oxygen (electron acceptor) the chloroplast or chromatophore must be able to generate both the donor and the acceptor by using light energy.

I t was proposed by Arnon (1959, 1961b) that the phosphorylating particle (chloroplast or chromatophore) operates as a closed catalytic system. Arnon suggested that during the primary photochemical act, one component of the closed system, chlorophyll, bound to protein, becomes excited when it absorbs a photon and expels one of its electrons that has been raised to a higher energy level. The excited chlorophyll thus


becomes the electron donor. When it loses its electron the chlorophyll protein complex assumes an "oxidized" state (i.e., becomes deficient in electrons) and in this way also becomes the electron acceptor in photo-synthetic phosphorylation.

The expelled electron returns in a stepwise manner via an electron-transport chain within the chloroplast to the oxidized chlorophyll com-plex which, on accepting the electron returns to its normal ground state. On its return path (downhill) the expelled electron releases free energy as it passes through several electron carriers. The electron carriers are considered to be the cofactors, vitamin K and FMN (or related physio-logical equivalents), and the cytochromes. These intermediate electron carriers are coupled with enzyme systems catalyzing the phosphorylation process during which electron energy is converted into the pyrophosphate bond energy of ATP. A diagrammatic representation of this concept is given in Fig. 2.

©- ^ • C o f actor

V* r - P

LIGHT ~p-

ADP

ADP * ©

FIG. 2. Scheme for anaerobic cyclic photophosphorylation catalyzed by vitamin K3 or FMN. (Araon, 1961b.)

Certain nonphysiological cofactors such as phenazine methosulfate have been found to catalyze photosynthetic phosphorylation in both chromatophores and chloroplasts, presumably acting by providing arti-ficial shortcuts from the physiological electron pathway between the excited chlorophyll complex and cytochrome (Fig. 3).

At low light intensity the overall rate of cyclic photophosphoryla-tion is limited by the electron flux. Under these conditions the rate of photophosphorylation obtained using chloroplasts with vitamin K3 or FMN as catalysts was about twice that with phenazine methosulfate, suggesting (although by no means proving) that with the physiological cofactors more phosphorylating sites may be operating (see Arnon 1961b, Figs. 9 and 10).


(ir! ) PMS

1 qi Kf) '^"V cy>

LIGHT ~p-ÂSP Κ ^ )

FIG. 3. Scheme for anaerobic cyclic photophosphorylation catalyzed by PMS. (Arnon, 1961b.)

The stepwise interaction of an electron, which has become activated by light, with the intermediate electron acceptors in the photosynthetic particle constitutes the energy conversion process in photosynthetic phos-phorylation. On account of the cyclic path traveled by the activated electrons this type of phosphorylation was termed cyclic photophos-phorylation.

In cyclic photophosphorylation the electrons flow from chlorophyll to a cofactor, from the cofactor to cytochrome, and from cytochrome back to chlorophyll. During the cyclic flow of electrons the physiological electron carriers present in the photosynthetic particles undergo oxida-tion-reductions that are coupled to phosphorylation reactions leading to the production of ATP. The proposed mechanism of cyclic photophos-phorylation may be divided into three phases: (1) the light-induced generation of an endogenous electron donor and an endogenous electron acceptor, (2) electron transport from the donor to the acceptor via a photosynthetic electron transport chain, and (3) phosphorylation reac-tions coupled to electron transport. Phases (2) and (3) are analogous to, and in some respects identical with, their counterparts in oxidative phosphorylation, whereas phase (1) is peculiar to photosynthetic phos-phorylation. As was stated earlier this type of cyclic photophosphoryla-tion, whose overall experimentally observed characteristics are ade-quately represented by the formulation of Eq. (3), is carried out by isolated chloroplasts or chromatophores. In the two sections which fol-low (Sections 4.1 and 4.2) we shall show how the electron-flow theory of cyclic photophosphorylation proposed by Arnon was also extended to the formation of reduced pyridine nucleotide by plants and photosyn-thetic bacteria.


4.1 Noncyclic Photophosphorylation in Bacteria

As was already stated, C02 assimilation during photosynthesis re-quires not only ATP but also reduced pyridine nucleotide. The forma-tion of reduced pyridine nucleotide by photosynthetic bacteria may be accomplished in one of several ways. If hydrogen is present as an elec-tron donor the direct reduction of pyridine nucleotide in the dark could provide the reduced pyridine nucleotide required for C02 reduction, as was shown for Chromatium by Ogata et al. (1959). The function of light in bacteria supplied with hydrogen gas would then be restricted to the formation of ATP by cyclic photophosphorylation, a concept dis-cussed by Losada et ai. (1960b). However, many electron donors be-sides hydrogen can be used by photosynthetic bacteria. For example, succinate which does not have a sufficiently low redox potential to re-duce pyridine nucleotide directly, will support C02 fixation by several photosynthetic bacteria in the light. An extra energy supply is there-fore needed for the reduction of pyridine nucleotide by succinate. In photosynthetic bacteria the extra energy is supplied by light, and there is evidence to suggest that this energy is supplied by way of an electron-flow mechanism. For example, cytochromes in photosynthetic bacteria become oxidized when the cells are illuminated (Section 5.5) and the oxidized cytochromes may be reduced by electrons donated by succinate or by thiosulfate (Nozaki et al., 1959). It may be noted here that thio-sulfate can act only as an electron donor and not as a "hydrogen" donor.

LIGHT FIG. 4. Scheme for noncyclic electron flow in Chromatium. (Arnon et al., 1961b.)

5. PHOTOCHEMICAL BEACTIONS OF PHOTOSYNTHESIS 123

Thus, electrons of moderate reducing potential, which are more oxi-dizing than pyridine nucleotide, can be transferred via cytochromes to chlorophyll, and then, on receiving a quantum of light energy, be raised to a reducing potential equivalent to that of hydrogen gas. The elec-trons in this case do not cycle, as they do in cyclic phosphorylation. In-stead they are ultimately accepted by an external electron acceptor. Here the electron pathway becomes noncyclic, and continued electron flow depends on a continuous supply of electrons from an external elec-tron donor to an external acceptor of which three have been found in bacteria. These are nitrogen, protons (H+), and pyridine nucleotide (Arnon et al., 1961b). Evidence is on hand, and will be discussed sub-sequently, that the transfer of electrons along this pathway is accom-panied by ATP formation (Nozaki et al., 1961). A diagrammatic repre-sentation of noncyclic electron flow in bacteria is given in Fig. 4.

4.2 Noncyclic Photophosphorylation in Plants

In green plants water (OH") is the normal electron donor for the reduction of triphosphopyridine nucleotide (only a few algal species can adapt to the use of hydrogen gas for the direct reduction of TPN). The use of OH" requires a large input of light energy in order to raise electrons from a potential of +0.81 volt to a potential equivalent to that of molecular hydrogen (—0.42 volt) or TPN (—0.324 volt). In the light, isolated chloroplasts can reduce TPN with water as the donor, and incidentally evolve oxygen, provided that the enzyme system needed for the transfer of the activated electron from chlorophyll to TPN is present in sufficient amounts.

In 1958 Arnon et al. made the very unexpected finding that the photoreduction of TPN, accompanied by oxygen evolution, was coupled with ATP formation when the chloroplasts were provided with ADP and inorganic phosphate. The stoichiometry of this reaction is shown in Eq. (4).

TPN + P + ADP + H20 -► TPNH2 + ATP + 1/2 02 (4)

Ferricyanide and a number of dyes can replace TPN in Eq. (4), and the reduction of these nonphysiological compounds is also accompanied by ATP formation. The electron flow involved in this reaction re-sembles the electron flow in the Hill reaction. The Hill reaction itself [Eq. (2)] may be regarded as a nonphysiological variation of non-cyclic photophosphorylation in which the phosphorylating steps have become uncoupled and in which an artificial electron acceptor replaces TPN.

The reduction of TPN by chloroplasts may be viewed as being


analogous to the noncyclic electron flow in bacteria, differing from it only in those aspects that reflect the special enzymatic properties of the chloroplasts. Unlike the photosynthetic bacteria, chloroplasts contain neither nitrogen-fixing enzymes nor hydrogenase. As a consequence the electron acceptor B (Fig. 7; see p. 125) of the noncyclic electron-flow mechanism in chloroplasts is coupled via TPNH2 only to C02 reduction and not, under physiological conditions, to photofixation of nitrogen or photoproduction of hydrogen gas.

However, the most characteristic difference between the noncyclic electron flow in chloroplasts and bacteria is in the electron-donor sys-tem. Chloroplasts which have lost the ability to photoevolve oxygen, either through aging or by poisoning, are nevertheless able to photo-reduce TPN with electrons supplied via 2,6-dichlorophenolindophenol from ascorbate (Vernon and Zaugg, 1960). We have found recently that noncyclic photophosphorylation in chloroplasts [Eq. (4)] can be ex-perimentally separated into two distinct photochemical reactions: (1) a photooxidation of water (OH-) that yields oxygen [Eq. (5)] and (2) a noncyclic photophosphorylation of the bacterial type, i.e. a photore-duction of triphosphopyridine nucleotide coupled with the formation of adenosine triphosphate [Eq. (6)] (Losada et αΖ., 1961). The two re-actions were separated experimentally using reduced (A-) and oxidized (A) indophenol dyes as summarized in the following equations:

light 20H- + 2A > 1/2 02 + 2A- + H20 (5) 2A- + 2TPN + 2H+ + ADP + P -> TPNH2 + 2A + ATP (6)

Sum: TPN + ADP + P + 20H~ + 2H+ -> ATP + TPNH2 + 1/2 02 + H20

[The summary equation should be compared with Eq. (4) above.] The bacterial type of noncyclic electron-flow mechanism that is envisaged as operating in chloroplasts when the participation of water as the elec-tron donor is blocked is shown diagrammatically in Fig. 5. The electron

FIG. 5. Scheme for noncyclic photophosphorylation of the bacterial type in chloroplasts. (Losada et al., 1961.)


donor A- represents a reduced indophenol dye and the electron acceptor B represents TPN or its equivalent. We envisage the formation of ATP as occurring during the electron transport between cytochromes and chlorophyll.

The photooxidation of water is represented in Fig. 6. We regard the

| p| ent|C+)«jy—OH"< H20

* [OH] LIGHT ^ o 2

FIG. 6. Scheme for photooxidation of water by chloroplasts. (Losada et al., 1961.)

photooxidation of water by chloroplasts as an auxiliary reaction to sup-ply electrons at an intermediate reducing potential for a second photo-chemical reaction, during which a phosphorylation coupled with a re-duction of pyridine nucleotide actually occurs. Figure 7 shows the com-

LIGHT

1 pigment | φ Q ü r0H"* H20

♦ [OH] LIGHT ^ o 2

FIG. 7. Scheme for noncyclic photophosphorylation in chloroplasts. (Losada et al., 1961.)

bined scheme for noncyclic photophosphorylation in green plants that we now envisage. The intermediate A is both the electron acceptor for the first and the electron donor for the second light reaction. We have used indophenol dye as an experimental reagent to distinguish be-


tween the two photochemical reactions. The natural intermediates with which the dye has interacted have not been fully identified although it is known that they include plastoquinone (Section 5.12) and also prob-ably cytochromes.

5. Evidence for the Electron-Flow Mechanism in Photosynthetic Phosphorylation

There are several lines of evidence which support the foregoing schemes for cyclic and noncyclic photophosphorylation. We shall dis-cuss the occurrence in chloroplasts and chromatophores of the com-pounds implicated in the electron-flow mechanisms, and shall consider evidence that the various postulated intermediates are able to react in the manner outlined above.

5.1 Some Constituents of Chloroplasts

Chloroplasts are organelles 1-8 μ in diameter which possess a lamel-lar structure. These cell constituents contain all of the chlorophyll which occurs in the cell and it is believed that this chlorophyll is localized in the lamellae. In green plants two types of chlorophyll, chlorophyll a and b, are found. In the blue-green and red algae only chlorophyll a is present and it is accompanied by the phycobilins, phycoerythrin and phycocyanin. Chlorophyll b and the phycobilins function as accessory pigments, as discussed in Chapters 6 and 7. A number of carotenoids, principally carotene and xanthophylls are always associated with the chlorophyll, although the nature of their participation in the photo-synthetic process is not clear. It is thought that the carotenoids may have a protective antioxidant function. It is considered that the high lipid content is of great importance in determining the orientation of the chlorophyll molecules within the lamellar structure. The lipids con-centrated in the chloroplast contain only a small amount of neutral triglycérides. They are characterized by the possession of galactolipids (e.g., /?-galactopyranosyl diglyceride), the plant sulfolipid (sulfodeoxy-glycosyl diglyceride), and the phosphatidyl glycerols (Benson, 1961).

The chloroplasts are remarkable for their high protein content (up to 50% of the dry weight). About half of the protein in whole chloroplasts is readily water-soluble. Aqueous extracts of chloroplasts contain a number of catalytically active proteins, including the enzymes needed for the operation of the reductive pentose cycle, as well as catalysts, such as ferredoxin, involved in electron flow. Ferredoxin is under active investigation at this time. In aqueous extracts of chloroplasts Losada


et al. (1960a) identified phosphoribulokinase, carboxydismutase, phos-phoglycerate kinase, triose phosphate dehydrogenase, triose phosphate isomerase, aldolase, fructose diphosphatase, and the enzymes involved in the regeneration of ribose-5-phosphate from fructose-6-phosphate (in-cluding transaldolase and transketolase).

Special cytochrome components have been discovered in photosyn-thetic cells of green plants and several algae and appear to be concen-trated in the chloroplasts. Cytochrome f was isolated, as a heme protein of molecular weight 110,000, from photosynthetic tissues and shown to be present in chloroplasts (Hill and Scarisbrick, 1951; Davenport and Hill, 1952; Davenport, 1952; and Hill, 1954). Cytochrome f has an un-usually oxidizing redox potential (E'0, pH 7 = +0.365 volt) and when isolated from the chloroplasts occurs in the reduced form, characterized by a very sharp «-band at 554.5 ηΐμ. In addition to cytochrome f, Hill (1954) detected another cytochrome of the b-group, which he called cytochrome b6; it is autooxidizable, has an E'0 pH 7 = —0.06 volt, and has a sharply defined absorption band at 563 τημ. Katoh (1959a) has described the isolation and properties of a cytochrome of the c-type from various algae of the families Rhodophyceae, Phaeophyceae, Chloro-phyceae, and Cyanophyceae. The cytochromes from these widely differ-ent algae were practically identical with respect to their redox potentials and absorption spectra, with E'0 pH 7 = +0.30 to 0.34 volt, and an «-band at 553 τημ. They appear to be the algal "equivalents" of cyto-chrome f (itself a cytochrome of the c-type). In leaves the molar ratio cytochrome f: chlorophyll is about 1:400 (Hill and Scarisbrick, 1951).

Another iron-containing protein called ferredoxin, (see Section 5.6) which is not a heme compound, has been recently identified in chloro-plasts. It is a component of the enzyme system involved in the reduction of TPN by chloroplasts, which was previously described as photosyn-thetic pyridine nucleotide reductase by San Pietro and Lang (1958). As will be shown in Section 5.6 it functions as an electron carrier in con-junction with a flavin enzyme. It has a molecular weight of about 14,000 and a redox potential E'o, pH 7.5 = —0.43 volt, and is the most reducing electron carrier known in chloroplasts, as has been shown by Tagawa and Arnon (1962). It occurs normally in the oxidized form and is present in the ratio ferredoxin: chlorophyll = 1:400.

Chloroplasts also contain a number of characteristic quinones. Dam et al. (1948) showed that the naphthoquinone, vitamin K (as determined by biological assay), is contained in chloroplasts. Lichtenthaler (1962) was able to demonstrate the presence of vitamin K in green leaves of a number of different plants by chromatography and chemical tests, and

128 F. R. WHATLEY AND M. LOS ADA

Kegel and Crane (1962) found that vitamin Kt could be extracted from spinach chloroplasts and chemically identified. It occurs in the ratio of vitamin K: chlorophyll = 1 : approximately 30-80.

In addition to naphthoquinone Crane (1959) found that a substituted benzoquinone, which was first isolated from alfalfa by Kofler (1946), was characteristically localized in chloroplasts of higher plants. The compound was named plastoquinone by Crane. It is also known by such names as Kofler's quinone and Q-254. Plastoquinone was found to be dimethoxybenzoquinone with a 9-isoprenoid side chain. It is chemically related to coenzyme Q (ubiquinone) which is absent from the chloro-plast, although present in the mitochondria in the leaf (Crane, 1959). The ratio plastoquinone: chlorophyll is 1:10, or even higher (Crane, 1961).

A constituent which occurs in rather variable quantities in chloro-plasts is ascorbic acid, which Molisch early associated with the ability of chloroplasts (in $itu) to reduce silver compounds. The concentration of ascorbic acid undergoes a considerable seasonal variation. Two other important constituents which have been found in small amounts in chloroplasts in our laboratory are flavin nucleotides (Ohta and Losada, 1959) and TPN (Rosenberg, 1955). A number of metals (copper, iron, manganese, and zinc) also appear to be present in chloroplasts. Of these, copper and iron are concentrated in the chloroplasts, whereas the manganese and zinc are distributed throughout the whole cell. (See, for example, Whatley et at, 1951.)

5.2 Some Constituents of Chromatophores

Chromatophores are particles about 1000 Â in diameter which can be isolated from a number of photosynthetic bacteria by sonication followed by differential centrifugation. There is some doubt whether the chromatophores isolated in this way represent structures which actually occur in the intact bacteria or are formed as an artifact from a cyto-phamic membrane during sonication (Tuttle and Gest, 1959; Cohen-Bazire and Kunisawa, 1963). The chromatophores contain all the photosynthetic pigments of the bacterial cells. They contain bacterio-chlorophyll, which resembles chlorophyll a in plants. However, bac-teriochlorophyll is not accompanied by an accessory pigment like the chlorophyll b or phycobilin of plants. Chromatophores contain caro-tenoids in large amounts, and phospholipoprotein. Newton and Newton (1957) found that a protein could be isolated from the chromatophores of Chromatium which contained glycerol, ethanolamine, and phosphorus in equimolar amounts, associated with bacteriochlorophyll, carotenoids, and cytochromes in the ratio 10:5:1. Large amounts of nonheme iron


were present. Pyridine nucleotides and flavins were also present in small amounts (Newton and Newton, 1957).

Special cytochromes have been discovered in the photosynthetic bacteria, and it appears that they are closely associated with the photo-synthetic activities of these organisms. Elsden et al. (1953) discovered and later Horio and Kamen (1961) purified a special cytochrome c component from Rhodospinllum rubrum, which they called cytochrome c2. Although in many ways similar in properties to mammalian cyto-chrome c, it was not oxidized appreciably in air in the presence of the cytochrome oxidase system; its redox potential is close to that of cytochrome f. (E'0 pH 7 = +0.33 volt). A very similar cytochrome was crystallized from Rhodopseudomonas palustns by Morita (1960).

Cytochrome c2 is found in photosynthetic bacteria which are obligate anaerobes, such as Chromatium and Chlorobiunij as well as in species which are able to use oxygen in the dark.

There is also evidence that cytochrome b components are present in photosynthetic bacteria. Different from these, but having a redox potential of approximately 0 volts, is the "Rhodospinllum heme protein" (RHP) investigated by Horio and Kamen (1961). I t contains two heme groups per molecule, and is thought by Kamen and his associates to participate in the electron-flow pathway of both Rhodospinllum and Chromatium.

5.3 The Electronic Nature of the Primary Light Reaction

Arnold and Clayton (1960) have presented evidence to support their belief that the first step in photosynthesis is the separation of an electron and a hole in a chlorophyll semiconductor system, i.e., that the first step is purely electronic in nature. Using dried films of isolated chroma-tophores they found that, upon illumination, the chlorophyll in the chromatophores undergoes a reversible shift towards shorter wavelengths in every absorption band, and that this shift is almost the same from 300°K to 1°K. At the lowest temperature no chemical reaction can take place, so that the spectral changes must accompany a physical change in the system. If dried chromatophores were illuminated there was a sudden change in the dielectric constant of the system, which suggested that electrons and holes had become spatially separated. The system returned to its original state as soon as the light was turned off.

When intact bacteria were illuminated, the chlorophyll peaks were unchanged since, the authors conclude, the flow of electrons in the undamaged cells is continuous and uninterrupted. This contrasts with isolated chromatophores, which showed the spectral changes. The


separation of electrons from holes in the chromatophores thus appears to be the primary photochemical reaction and Arnold and Clayton concluded that this separation precedes the first oxidation-reductions which involve cytochromes.

Chance and Nishimura (1960) have studied the effect of low temper-ature on the oxidation of cytochrome c2 in intact Chromatiwn cells. These investigators observed that at 300°K a rapid oxidation of reduced cytochrome c2 occurred when the light was turned on and the rate of this change was measured. When the light was turned off the oxidized cytochrome became reduced by dark chemical steps. At 80°K the oxidation of reduced cytochrome c was again observed to occur rapidly on illumination. In a subsequent dark period no reduction of cytochrome c occurred, since chemical changes do not go on at liquid nitrogen temperatures. Chance and Nishimura concluded that illumination of the Chromatium cells initiates a temperature-insensitive electron-transfer reaction between bacteriochlorophyll and a closely associated cyto-chrome c2.

5.4 Participation of Cytochromes in Photosynthesis by Chloroplasts

In his electron-flow theory Arnon (1959) suggested that cytochromes participate in photosynthesis through oxidation by the photochemically produced oxidant (Chl+). This was partly based on the occurrence of cytochrome f in chloroplasts and on the experiments of Lundegardh and Duysens (see below) on its photooxidation. No cytochrome f oxidase has ever been detected in plants (cytochrome f is not oxidized by cyto-chrome c oxidase) and thus cytochrome f does not react with oxygen (Hill and Scarisbrick, 1951; Lundegardh, 1962). However, in illuminated leaves of the "golden" variety of several species of plants, Hill (1954) had made spectroscopic observations from which he inferred that the cytochrome-f component was oxidized and the cytochrome be reduced on illumination. James and Leech (1958) report observations on isolated chloroplasts which appear to support this idea.

Other more extensive experiments with suspensions of Chlorella showed a difference spectrum (absorption spectrum in the light minus that in the dark) with a minimum at 420 ηΐμ, which was interpreted as revealing the oxidation of cytochrome on illuminating Chlorella and its reduction in the dark (Lundegardh, 1954; Duysens, 1954a). Similar experiments were also carried out with leaf extracts by Lundegardh (1954, 1962). A clear-cut demonstration for a photooxidation of cyto-chrome f was obtained by Duysens (1955) with the red alga, Por-phyridium cruentum. In this case the «-band of cytochrome f was not


completely masked by the chlorophylls, and changes around 550 mu were clearly seen. Similar changes were also observed by Chance and Sager (1957) with a Chlamydomonas mutant low in chlorophyll and carotenoids, although they made reservations about interpreting their results as indicating the participation of cytochrome f in photosynthetic electron flow. Chance and Sager emphasized the importance of further experiments on particles isolated from cells to distinguish between cyto-chrome participation in the pathways of photosynthesis and respiration.

Cell-free extracts of Porphyra tenera were found to catalyze the cyanide-insensitive photooxidation of reduced cytochrome f (Katoh, 1959b). The algal plastids which carried out this oxidation had lost their phycobilin pigment and were unable to evolve oxygen. This implies that the phycobilins are not involved in the cytochrome photooxidation, and that cytochrome f is not involved directly in the evolution of oxygen.

The experiments of Katoh recall those of Nieman and Vennesland (1957; Nieman et al., 1959), which showed that reduced cytochrome c is oxidized on illumination (but not in the dark) by isolated chloro-plasts treated with digitonin. This activity was termed "cytochrome c photooxidase." The photooxidation was not inhibited by 10^ M cyanide (different from the dark cytochrome oxidase) but was sensitive to heat, suggesting that an enzymatic component was involved, and differentiating the reaction from the numerous photooxidations catalyzed by chloro-phyll solutions. Many of the typical inhibitors of photosynthesis did not affect the photooxidation of cytochrome c, but several mercury compounds were inhibitory (Bishop et al., 1959).

Our own experiments on the photooxidation of cytochrome c by digitonin-treated chloroplasts show that although added cytochrome c can be photooxidized with oxygen as the terminal acceptor it can equally well be photooxidized by TPN, provided the necessary inter-mediate carriers are present (Horton and Whatley, 1962). We may con-clude that, in these experiments with digitonin-treated chloroplasts, we are studying experimental manifestations of some activities of the terminal portion of the electron-flow pathway.

The evidence listed above shows that cytochromes are indeed charac-teristic components of green plants, and there are strong indications that the cytochrome-f components become oxidized in the light and reduced again in the dark. However, as pointed out by Hill and Bonner (1961), there has been no unequivocal demonstration of this in the 550-mju, region with normal green cells or chloroplasts. The evidence for cytochrome participation in photosynthesis in bacteria is more direct.

132 F. R. WHATLEY AND M. LOS AD A

5.5 Participation of Cytochromes in Photosynthesis by Bacteria

A "cytochrome c photooxidase" activity, like the one described in Section 5.4 for digitonin-treated chloroplasts, was found in extracts of several species of photosynthetic bacteria (Vernon and Kamen, 1953; Kamen and Vernon, 1954). In the earlier experiments these in-vestigators showed that the system was destroyed by heating at 65 °C for 10 min, although later they drew attention to its relative heat stability by comparison with the dark cytochrome c oxidase. They emphasized the fact that the oxygen requirement for the photooxidase is artificial, since the photosynthetic bacteria do not take up oxygen in the light (even if some do in the dark), and we now consider that they were investigating the terminal portion of the electron-flow pathway (Fig. 4).

An oxygen-independent photooxidation of endogenous cytochrome was observed by Duysens (1954b) in Rhodospirillum rubrum suspensions in the presence of substrate. At low light intensities spectral changes indicated that a cytochrome component became oxidized rapidly (1 sec) ; in the dark it became reduced rapidly (1 sec). Similar results were obtained by Chance and Smith (1955). A reversible light-induced oxida-tion of cytochrome c2 in cell-free preparations of Chromatium has been measured by Nozaki, Ogata, and Arnon (1959). Spectral observa-tions were also extended to extracts of Rhodospirillum rubrum capable of cyclic photophosphorylation (Smith and Ramirez, 1958; Smith and Baltscheffsky, 1959). Cytochrome c2 in the extracts was oxidized on illumination under conditions where phosphorylation was taking place. The spectral changes were intensified when an inhibitor which pre-vents the reduction of the oxidized cytochrome was present. In the absence of a phosphate acceptor (ADP) no cytochrome c2 was oxidized. A role for this bacterial cytochrome in photosynthesis is thus clearly indicated, and these experiments support the proposal that the forma-tion of ATP occurs at the site of cytochrome oxidation in the scheme for cyclic photophosphorylation in bacteria (Fig. 2). There is as yet no direct evidence to justify a similar site for ATP formation in green plant photophosphorylation, although by analogy it must be considered as the most probable.

5.6 Pyridine Nucleotide Reduction and Ferredoxin

The photoreduction of pyridine nucleotides by chloroplasts was first shown in coupled systems, in which the reduced pyridine nucleotide did not accumulate. For example, the photoreduction of DPN was demonstrated by the reduction of pyruvate to lactate whéh the chloro-

5. PHOTOCHEMICAL REACTIONS OP PHOTOSYNTHESIS 133

plasts were incubated in the light with pyruvate and lactic dehydro-genase from rabbit muscle together with a catalytic amount of DPN. Under strictly anaerobic conditions the photochemical reduction of pyridine nucleotides was accompanied by the evolution of oxygen (see Vishniac and Ochoa, 1951; Arnon, 1951; Hendley and Conn, 1953). However, in 1956, San Pietro and Lang showed that in the presence of large amounts of chloroplasts DPN (and to a lesser extent TPN) could become reduced in the light and be accumulated as reduced pyridine nucleotides, without the need for a coupled pulling reaction. The chloro-plasts were found to contain a soluble protein factor, which Arnon et al. (1957) found preferentially to reduce TPN with the evolution of a stoichiometric amount of oxygen, and which they termed the "TPN-reducing factor." San Pietro and Lang (1958) subsequently purified this factor (by which treatment it became TPN-specific) and named it photo-synthetic pyridine nucleotide reductase, since it appeared to catalyze TPN-reduction only in the presence of illuminated chloroplasts.

It was shown (Davenport, 1959) that photosynthetic pyridine nucleo-tide reductase is identical with a protein studied by Davenport et al. (1952), purified by Davenport and Hill (1960) and called the met-hemoglobin-reducing factor. Hill (1951) believed that the methemo-globin-reducing factor was, in fact, the primary electron acceptor in photosynthesis, in spite of the fact that its true substrate was not known to him for many years. Recently Tagawa and Arnon (1962) found that the TPN-reducing factor is closely related to the ferredoxin isolated by Mortensen et al. (1962) from bacteria and shown by them to act as an electron carrier between hydrogenase and various electron donors and acceptors. The ferredoxin from spinach is an iron-containing protein which contains neither heme nor flavin prosthetic groups. On the basis of the iron content (0.815%) and a molecular weight of approximately 14,000, there appear to be two Fe atoms per molecule. The spinach ferredoxin was found to be the most reducing electron carrier found so far in cellular metabolism and has a redox potential, E'0 pH 7.5 — —0.430 volt. This was determined by allowing it to react with hydrogen gas under the influence of a bacterial hydrogenase. It appears that only one electron is accepted by each molecule of oxidized ferre-doxin on becoming reduced. The spectra of the oxidized and reduced forms are shown in Fig. 8.

An analysis for ferredoxin in leaves and chloroplasts of spinach showed 1 mole of ferredoxin per 400 moles chlorophyll, which is reminiscent of the ratio cytochrome f: chlorophyll found by Hill and by Lundegardh.

Reduced ferredoxin has a high affinity for oxygen (it was not re-

134 F. R. WHATLEY AND M. LOS ADA

250 350 450 550 wavelength (πΐμ)

FIG. 8. Oxidized and reduced spectra of spinach ferredoxin. (Tagawa and Arnon, 1962.)

duced by hydrosulfite in an open tube, only under nitrogen) ; it is also reoxidized very rapidly by TPN, but only on the addition of a flavin enzyme present in broken chloroplasts and readily extracted from chloroplasts by acetone treatment. On partial purification the TPN-reducing enzyme was found in a flavin-containing fraction (Tagawa and Arnon, 1962). Spinach ferredoxin is not only reduced by hydrogen gas (with hydrogenase) but also by illuminated, washed chloroplast fragments under anaerobic conditions. Spinach ferredoxin thus acts as an electron carrier between either hydrogen in the presence of hydrogenase or illuminated chloroplasts on the one hand, and TPN in the presence of the flavoprotein enzyme on the other.

5.7 Photoproduction of Hydrogen

Photoproduction of molecular hydrogen was first observed by Gaffron and Rubin (1942) in the green alga Scenedesmus and by Gest and Kamen (1949) in photosynthetic bacteria. The evolution of hy-drogen was thought to derive either from the decomposition of a dicarboxylic acid or from the photolysis of water into [H] and [OH].


Gest and Kamen suggested that [H] is eventually liberated, with the aid of hydrogenase, as molecular hydrogen, where the [OH] is reduced back to water by reacting (in the case of algae) with endogenous hydrogen donors and (in the case of photosynthetic bacteria) with exogenous hydrogen donors.

Photosynthetic bacteria evolved hydrogen in the light only in the presence of organic acids which served as hydrogen donors. However, from the point of view of the electron-flow theory the photolysis of water does not occur, and the photoproduction of hydrogen could be viewed as a reduction of protons by a hydrogenase with the aid of electrons from excited chlorophyll molecules. The electrons come to the excited chlorophyll via cytochromes from the external electron donors and are raised to the reducing potential of molecular hydrogen during the primary photochemical act. If this were so, then, contrary to negative evidence in the past, photoproduction of hydrogen should occur not only at the expense of organic hydrogen donors but also at the expense of suitable inorganic electron donors, and these electron donors should be capable of reducing the bacterial cytochromes that are oxi-dized by light in the course of photosynthesis. The experiments of Arnon et al. (1961b) showed that in the light, but not in the dark, hydrogen gas was evolved by Chromatium cells in the presence of thio-sulfate. The photoproduction of hydrogen was inhibited by molecular nitrogen and ammonium ions. Thiosulfate was also able to reduce oxidized Chromatium cytochromes (Nozaki et al., 1959). Thus, electrons donated from an inorganic source can be used to bring about the evolu-tion of hydrogen by the electron-flow pathway involving cytochromes, illuminated chlorophyll, and hydrogenase (Fig. 4).

I t has recently been found that hydrogen photoproduction can also be experimentally carried out by illuminated chloroplasts, if they are supplemented with a suitable bacterial hydrogenase system (Arnon et al., 1961c; Mitsui and Arnon, 1962; Paneque and Arnon, 1962). If oxygen production is prevented, so that electrons cannot be donated into the electron-flow chain by water, either ascorbate or cysteine may still donate electrons which can then be raised to the level of molecular hydrogen by the primary photochemical act. The photoproduction of hydrogen gas is accompanied by the formation of adenosine triphosphate.

5.8 Effect of Chloride

The role of chloride in photosynthesis was discovered by Warburg in 1949 when he found that chloride, which could be replaced by bromide but not by other anions, was essential for oxygen evolution by isolated


chloroplasts. This observation was confirmed by Arnon and Whatley (1949) but since chlorine had not been shown to be an essential element of green plants, they did not wish to accept Warburg's conclusion that chloride is a coenzyme of photosynthesis. Later Broyer et al. (1954) and Martin and Lavollay (1958) proved that chloride is an essential micronutrient for green plants. Bové et al. (1959) then confirmed Warburg's conclusion that chloride is essential for those photosynthetic reactions in which oxygen is liberated. Chloride was not required for the anaerobic cyclic photophosphorylation carried out by both bacterial particles and chloroplasts. In the absence of chloride chloroplasts behave like bacterial chromatophores, i.e., they are able to carry out the anaerobic cyclic photophosphorylation but are unable to evolve oxygen. Losada et al. (1961) have recently shown that chloride is in fact involved in the auxiliary reaction of chloroplasts, the photooxidation of water. On the basis of these experiments with chloride, oxygen evolution appears to be an additional secondary feature of photosynthesis in green plants which is not essential to the primary conversion of light energy into the energy of the pyrophosphate bonds.

5.9 Effect of Ferricyanide

On the basis of the mechanism for cyclic photophosphorylation proposed above, the electrons ejected by light from chlorophyll a return to the chlorophyll by a cyclic pathway. If this is so, then cyclic photophosphorylation will be abolished if the electrons are prevented from completing the cycle because of capture by some external electron acceptor. Ferricyanide is such an electron acceptor, and has a great affinity for trapping electrons. Furthermore, since ferricyanide supports a vigorous noncyclic photophosphorylation, it is obviously not toxic to the phosphorylation reactions themselves. But ferricyanide can con-tinue to accept electrons in the noncyclic photophosphorylation system only when chloride is present to enable water to donate the necessary electrons. If ferricyanide is added to a system carrying out cyclic photo-phosphorylation in the absence of chloride the withdrawal of electrons from the cyclic electron pathway should result in an inhibition of the phosphorylation.

This prediction has been experimentally verified by Bové et al. (1959), as shown in Table I. The addition of ferricyanide abolished cyclic photophosphorylation both in chloroplasts and chromatophores. Ferrocyanide was not inhibitory. The reduction of ferricyanide by the addition of ascorbate before or during illumination fully restored the cyclic photophosphorylation. When chloride was added to the ferri-cyanide-inhibited system, OH~ could now donate electrons in the


TABLE I INFLUENCE OF FEBRICYANIDE (IN THE ABSENCE OF CHLORIDE) ON

CYCLIC PHOTOPHOSPHORYLATION BY SPINACH CHLOROPLASTS AND BACTERIAL CHROMATOPHORES (Chromatium)a

(Bové et al, 1959)

Treatment Chloroplasts Chromatophores

Control 9.2 4.9 Ferricyanide, 1 μΐη 0.5 0.4 Ferricyanide, 2 μπι 0.5 0.5 Ferricyanide, 3 Aim 0.5 0.4 Ferricyanide, 5 /xm, reduced by ascorbate6 7.2 6.2 Ferrocyanide, 5 /an 9.4 5.4

° As micromoles of phosphate esterified in 30 min. 6 Sodium ascorbate (5 μπι) was tipped in from a sidearm 15 min after the beginning

of the experiment, and illumination (35,000 lux) was then continued for 30 min.

chloroplast system to cause the reduction of the ferricyanide, and as soon as all the ferricyanide was reduced, cyclic photophosphorylation was restored in the chloroplasts.

Since water cannot be used to donate electrons for the reduction of ferricyanide in chromatophores, the addition of chloride to the bac-terial system does not bring about the reversal of the ferricyanide in-hibition. The conclusion that the inhibition by ferricyanide resulted from the capture of electrons which would otherwise have passed along the cyclic electron-transport pathway was strengthened by the observation that the inhibition was produced by very low concentrations of ferri-cyanide. This would be expected if the quantity of ferricyanide required to capture electrons from the cyclic system need only be sufficient to leave all the catalytic components of the system in the oxidized form.

5.10 Evidence for Two Light Reactions in Noncyclic Photophosphorylation

I t was stated earlier (Section 4.5) that noncyclic photophosphoryla-tion in chloroplasts has been biochemically separated into two distinct photochemical reactions: (1) a photooxidation of water (OH-) that yields oxygen [Eq. (5)] and (2) a noncyclic photophosphorylation of the bacterial type, i.e., a photoreduction of triphosphopyridine nucleotide that is coupled with the formation of adenosine triphosphate [Eq. (6)] . The two reactions were separated by using (A-) and oxidized (A) indophenol dyes as shown in the equations. Evidence in support of these equations is given in Tables I I and I I I .

Table I I shows that when catalytic amounts of 2,6-dichlorophenol-indophenol replaced water (OH-) as the electron donor (treatment 3), illuminated chloroplasts formed adenosine triphosphate and reduced


TABLE II NONCYCUC PHOTOPHOSPHOBYLATION OF THE BACTERIAL TYPE BY CHLOROPLASTS

(Losada et al.y 1961)

Effective Oxygen TPN ATP electron evolved reduced formed

Addenda donor (^atoms) (/xmoles) (jumoles)

1. None Water 3.0 3.4 2.4 2. Chlorophenyl dimethyl urea (CMU) None 0 0.5 0.2 3. Dye, ascorbate, CMU Reduced dye 0 3.2 3.4

(ascorbate)

TABLE III PHOTOOXIDATION OF WATER

(Losada et αΖ., 1961)

Adenosine Effective Oxygen triphosphate electron produced formed

Treatment acceptor 0*atoms) Gmnoles)

1. Ferricyanide Ferricyanide 3.3 3.5 2. Ferricyanide + 0.2 jumoles Oxidized dye 3.5 0.8

trichlorophenolindophenol (Ferricyanide)

triphosphopyridine nucleotide without evolving oxygen. The participation of water as an electron donor in these reactions was blocked by the addition of p-chlorophenyl dimethylurea to the reaction mixture while at the same time omitting chloride. When the participation of water as an electron donor was not blocked (Table II, treatment 1), the reduction of triphosphopyridine nucleotide and the coupled phosphorylation were accompanied by oxygen evolution. When the participation of water was blocked but no substitute electron donor was supplied (Table II, treat-ment 2) no significant reaction occurred at all. Treatment 1 (Table III) shows a type of electron flow in which the reduction of ferricyanide was accompanied by oxygen evolution and ATP formation. Treatment 2 (Table III) shows a type of electron flow in which the dye was the effective electron acceptor; a catalytic amount of the dye was kept oxidized by a chemical reaction with ferricyanide. In this case ATP formation was suppressed, although oxygen evolution continued. Treat-ment 2 thus demonstrated the occurrence of the photooxidation of water. The electrons were diverted from participation in the cytochrome chain through capture by the indophenol dye when it was added. Thus, al-though there was a reduction of ferricyanide accompanied by oxygen evolution, the electrons failed to pass along the phosphorylating portion


of the electron-flow pathway. In this case we have what appears to be a "classical" Hill reaction.

Additional support for the existence of these two reactions, as dis-tinguished by use of the dye, came from the experiments of Arnon et al. (1961a) on the effectiveness of monochromatic light in catalyzing the two partial reactions. In the red region of the spectrum the photooxidation of water was found to be catalyzed best at about 644 ηΐμ, and to decrease at longer wavelengths in contrast to the photoreduction of TPN (by ascorbate) which showed a minimum with light at 661 m/* and which increased sharply at shorter and longer wavelengths. These experi-ments strongly indicated the participation of two different pigment systems in the two partial reactions. I t is suggested by these data that the photooxidation of water is catalyzed by the accessory pigment, chlorophyll b, and the subsequent photoreduction of TPN by chloro-phyll a.

There are other lines of evidence to suggest the occurrence of two light reactions in the photosynthesis of plants. The experiments of Emerson, Blinks, Myers and French, Govindjee and Rabinowitch, Hoch and Kok, Witt, Allen, and others all lead to the conclusion that light absorbed in the far red is ineffective unless accompanied by "substrate quantities" of light at other wavelengths. These results are discussed in detail in Chapters 6 and 7. The experiments of Myers and French (1959) are of special interest here. These authors concluded from their results that a relatively stable chemical intermediate was formed by one light reaction and consumed by another. Similarly, the clear experiments of Duysens et al. (1961) showed that the cytochrome of Porphyra tenera was predominantly oxidized when illumination was with red light (ab-sorbed by chlorophyll) but became reduced on illumination with green light (absorbed by the accessory pigment phycobilin). Although the biochemical separation of the two light reactions in chloroplast systems by Losada et al. does not rest on the above results on whole cells, or on the increase in photosynthetic efficiency observed in whole cells by many workers using light of different wavelengths, it is not inconsistent with these observations. Mention should also be made of the very interesting paper by Hill and Bendall (1960) proposing a scheme for two light reactions in green plants and bacteria, to which they were lead by the known characteristics of several of the cytochromes believed to be intermediates. Our own evidence would suggest that two light reactions occur only in green plants.

5.11 Participation of Oxygen

Cyclic photophosphorylation can be experimentally demonstrated in chloroplasts and chromatophores to be an anaerobic process. If water is

140 F. B. WHATLEY AND M. LOSADA

used as the electron donor in noncyclic photophosphorylation by chloro-plasts, oxygen is excreted as a by-product. If another donor is substituted [cf. Eq. (6)] TPN is still photoreduced and coupled ATP formation results but no oxygen is evolved. A special case of noncyclic photo-phosphorylation occurs when oxygen acts as the terminal electron accep-tor and becomes reduced to hydrogen peroxide or water. This happens, for instance, in a cell-free system containing microcatalytic amounts of FMN, and gives rise to an oxygen-dependent pseudocyclic photophos-phorylation characterized by an oxygen exchange. If experimental con-ditions are arranged so that the hydrogen peroxide (the initial reduction product of oxygen) is not decomposed into water, an oxygen uptake results. Arnon et al. (1961a) showed that the same ATP formation took place in illuminated chloroplasts whether the photoactivated electrons were accepted (1) by TPN, resulting in net oxygen evolution, (2) by molecular oxygen (water as the product) resulting in oxygen exchange, or (3) by oxygen (hydrogen peroxide as the product) resulting in net oxygen uptake.

The replacement of TPN by oxygen results in the loss of bio-chemically useful energy, which would otherwise have gone to form a strong reductant, TPNH2. By contrast, the role of oxygen as the terminal electron acceptor in respiration, which results in ATP formation, is physiologically useful.

5.12 Participation of Plastoquinone

The ability to carry out the Hill reaction is lost when lyophilized chloroplasts are extracted with petroleum ether. Bishop (1959) found that plastoquinone would restore the Hill reaction with indophenol dyes as electron acceptors. We have now investigated the role of plastoquinone in the photoreduction of TPN by water, which is the physiological counterpart of the Hill reaction. Since the noncyclic electron flow in chloroplasts has been shown to be composed of two partial reactions: (1) the photooxidation of water leading to oxygen evolution and (2) the subsequent photoreduction of TPN, usually coupled with ATP formation, it has been possible to identify the site of action of plasto-quinone with more certainty. Plastoquinone was found to be needed only for the photooxidation of water and not for the subsequent reduction of TPN (Fig. 9). Plastoquinone may very well be the initial electron acceptor of the auxiliary light reaction catalyzed by the accessory pigment (Arnon et al, 1962).

Krogmann (1961) demonstrated that plastoquinone was needed for the cyclic phosphorylation catalyzed by phenazine methosulfate. These observations were extended (Arnon et al, 1962) to include the anaerobic cyclic photophosphorylations catalyzed by FMN and vitamin K3 which


were also found to be plastoquinone-dependent. Although no definite evidence is available to show the site of action of plastoquinone in the cyclic phosphorylation, the simplest hypothesis is to suppose that it operates at the same site where it has been shown to function in noncyclic electron flow.

5.13 Role of Manganese

A specific influence of manganese directly on photosynthesis in the green alga Ankistrodesmus was shown by Pirson et al. (1952). This organism can easily be adapted to hydrogen metabolism, under which condition it photoreduces C02 at the expense of hydrogen gas. If the organism was grown under manganese deficiency it lost its ability to photosynthesize, but the ability to photoreduce was retained (Kessler, 1957a). When normal cells adapted to hydrogen were illuminated at increasing light intensities reversion to oxygen evolution soon occurred, but with manganese-deficient cells the reversion did not occur until a very high light intensity was supplied. In the manganese deficient cells a vigorous hydrogen uptake could occur under high light intensity. Kes-sler concluded that the role of manganese was mainly, if not exclusively, concerned with oxygen evolution, which is the only partial process of photosynthesis not needed in photoreduction. The addition of manganese to deficient cultures of a number of algae (but not to isolated chloro-plasts) quickly restored their capacities for photosynthesis and the Hill reaction.

5.14 Participation of Carbon Dioxide

Carbon dioxide is required in catalytic amounts for the operation of the Hill reaction (Warburg et al., 1959). This was confirmed and ex-tended to a demonstration of a C02 requirement in the photochemical reduction of ferricyanide, TPN, flavin mononucleotide, and indophenol dyes (Stern and Vennesland, 1960; Stern, 1961). Warburg has suggested that carbon dioxide participates in the evolution of oxygen directly, and postulates that its activity in this system represents the major pathway for the entry of carbon into the plant. This interesting idea of à mechanism to explain the effect of C02 still requires considerable experimentation before it can be accepted. I t appears to be contradictory to much of the biochemical evidence available at the present time.

5.15 Inhibitors of Photosynthesis

A certain amount of information on the mechanism of photophos-phorylation has come from the use of inhibitors. We shall very briefly mention a few results. I t has been shown that several treatments prevent the photooxidation of water. These include a deficiency of chloride (see


Section 5.8), a deficiency of manganese (see Section 5.13), and the addition of phenylurethane, substituted dimethylureas, hydroxylamine, and o-phenanthroline. The site of action of all these treatments has been shown to be concentrated at the "oxygen-evolving end" of the electron-flow chain. The anaerobic cyclic photophosphorylations catalyzed by various cofactors are not affected by these inhibitors—only those special photophosphorylations experimentally arranged to go through oxygen are affected. Moreover, the bacterial type of noncyclic photo-phosphorylation (e.g., ATP formation coupled with the photoreduction of TPN by ascorbate) is not affected.

Other inhibitors uncouple the phosphorylation steps from the electron flow, as can be seen clearly from experiments with noncyclic photo-phosphorylation. Low concentrations of NH4

+ ion uncouple the phos-phorylation (Krogmann et al., 1959), as does atabrine and the anti-biotics gramicidin and valinomycin (Baltscheffsky, 1960). The use of valinomycin led to the interesting conclusion that there are two phos-phorylation sites in chromatophores of Rhodospinllum rubrum, since only a 50% inhibition was obtained even with high concentrations of valinomycin. The addition of arsenate in the presence of ADP and Mg++

also uncouples the phosphorylation, presumably by way of forming an unstable intermediate in place of the normal phosphate intermediate (Krogmann et al., 1959). Other treatments which effectively uncouple phosphorylation are to lower the salt concentration of the medium, to lower the concentration of the chloroplasts at a slightly acid pH, or to freeze the chloroplasts.

Other substances have been found to inhibit the electron flow and to prevent the operation of cyclic photophosphorylation. A number of quinoline-iV-oxide derivatives have been found to inhibit cyclic phos-phorylation, but about 100 times as much of them was needed to inhibit the chloroplasts as to inhibit chromatophores (Baltscheffsky, 1959). The site of action of the quinoline derivative was by-passed by phenazine methosulfate. Antimycin A and oligomycin also inhibited the chroma-tophore phosphorylation. Dinitrophenol, which is a strong inhibitor of oxidative phosphorylation at low concentrations, does not inhibit photo-synthetic phosphorylation in chloroplasts or chromatophores at these concentrations. Inhibition by p-chloromercuribenzoate is by an un-coupling of the phosphorylation, except in those cases where the reduc-tion of TPN is involved. It then apparently acts on the TPN-reducing system (ferredoxin or enzyme), which makes it impossible to test for uncoupling. 5.10 Existence of Light and Dark Phases in Photophosphorylation

In Section 4.5 the existence of light and dark phases in C02 fixation


was described. The light phase was considered to involve the steps leading to ATP and TPNH2 formation (cyclic and noncyclic phos-phorylation). Experiments have now been done which demonstrate the existence in the photophosphorylation itself of light and dark phases. Two types of experiments have been carried out: the first used the flashing-light technique with chromatophores from Rhadospinllum rubrum (Nishimura, 1962), and in the second the effect of temperature at low light intensities with chloroplasts was studied (Hall and Arnon, 1962).

In the experiments of Nishimura, the first rapid photochemical steps (induced by a brief high-intensity light flash) occurred only on illumination and was not affected by the presence of the cofactor, phenazine methosulfate. The second dark process took place both dur-ing the flash and between flashes. I t was possible to distinguish two steps in the dark process by the use of inhibitors: (1) the electron-transfer process, which was inhibited by 2-n-heptyl-4-hydroxyquinoline-iV-oxide and slowed by decreasing the temperature from 26° to 15°C and (2) the esterification of phosphate accompanying the electron trans-port. Nishimura (1962) considers that, after the photoactivated elec-tronic states of chlorophyll, "the first chemical process which takes place in photosynthesis is probably the light-induced oxidation of cyto-chrome. The rapidity of the process suggests that the oxidation of cytochrome takes place during the short illumination. And the rest of the photosynthetic reaction can proceed thermochemically." Nishimura believes that the first reduced substance, which he did not identify, but which we believe is probably ferredoxin (Section 5.6), reduces the oxi-dized cytochrome by electron transport along an oxidation-reduction chain coupled with phosphorylation. This formulation is in agreement with the electron-flow scheme shown in Fig. 2.

Variants of cyclic and noncyclic photophosphorylation in isolated chloroplasts were investigated by Hall and Arnon (1962) over a range of temperatures from —10° to -|-150C. These experiments show an ap-preciable light-induced ATP formation below 0°C which under certain conditions is independent of temperature within the range —10° to -f-15°C. Hall and Arnon found that at low light intensity (4000 lux) the rate of cyclic photophosphorylation catalyzed by phenazine metho-sulfate in the presence of a large amount of chloroplast material (con-taining 2 mg chlorophyll) was unaltered between —10°C to 15°C, which shows that the reaction was limited by the temperature-insensitive light reaction, whereas at a higher light intensity (40,000 lux) the phos-phorylation became temperature-dependent indicating that the reac-tion was now limited by a temperature-dependent dark reaction. This means that in the PMS system the thermochemical reactions of


ATP formation (occurring in the dark) keep pace with the low electron flux produced by a low light intensity, but not with the high electron flux resulting from a high light intensity. Evidence was also presented to show that even at the low light intensity where the PMS-catalyzed phosphorylation was temperature-independent, the phosphorylation in the cyclic system catalyzed by vitamin K3 or FMN was temperature-dependent and limited by a dark reaction in the electron-transport sys-tem. Similarly, noncyclic phosphorylation with TPN or ferricyanide proved to be temperature-dependent even at low light intensity, and was also limited by a component, of the electron-transport system. The fact that the cyclic phosphorylations with PMS and vitamin K3 behave differently in these experiments provides supporting evidence for the two electron-flow schemes in Figs. 2 and 3, which indicate that PMS by-passes a rate-limiting dark step present in the vitamin-K catalyzed system.

In addition to these experiments of Nishimura, and of Hall and Arnon, it may be appropriate to mention that the photoreduction of TPN itself (which we regard as a phase of photosynthetic phosphoryla-tion) can also be experimentally shown to comprise a light and a dark phase. It is known (Tagawa and Arnon, 1962) that the reduction of TPN by chloroplasts requires the presence of ferredoxin. It has now been demonstrated (Whatley et al, 1963) that illuminated chloroplasts under strictly anaerobic conditions can reduce "substrate amounts" (0.3 gniole) of added ferredoxin, which may be readily observed from the spectral changes accompanying the reduction (see Fig. 8). After the reduction of the ferredoxin was complete the light was turned off and the reduced ferredoxin was shown to be stable. The subsequent addition of TPN caused a rapid reoxidation of the ferredoxin in the dark and an accompanying reduction of TPN, which was catalyzed by an enzyme present in the chloroplast fragments. On further exposure to light the ferredoxin was seen to become reduced again but it became fully re-oxidized in the dark by the TPN, progressive reduction of which was to be seen as an increased absorption centered around 340 m/x. When all the added TPN had been reduced, the ferredoxin became reduced and remained in the reduced state in the dark. In this way the photoreduction of ferredoxin by chloroplasts was distinguished from its subsequent dark oxidation by TPN.

It may be of value at this point to present a summary of our present knowledge on the mechanism of noncyclic photophosphorylation in chloroplasts. Figure 9 shows a scheme in which the redox potentials of the various intermediates are taken into account. The dark arrows represent thermochemical reactions proceeding in the dark. The open


TPN

LIGHT

E'0,PH7

cytb6

FIG. 9. Scheme for noncyclic photophosphorylation in terms of redox poten-tials. (After Whatley et al, 1963.)

arrows represent the intake of light energy by the chlorophyll, and the elevation of an electron to a more reducing potential where it is accepted by an appropriate electron acceptor [plastoquinone (Q) or ferredoxin (FD)] . The phosphorylation is shown to accompany the dark reactions in the cytochrome chain. The point of entry of electrons from ascorbate is indicated as subsequent to the site where plastoquinone operates.

6. Some Examples of Photosynthesis We wish to emphasize again that the essence of the photosynthetic

process is the conversion of light energy into well-defined forms of chemical energy, by the reactions known as cyclic and noncyclic photo-phosphorylation, and not the subsequent utilization of these "energy-rich" compounds (PNH2 and ATP) for the synthesis of other cellular substances. The dark reactions involved in these secondary transforma-tions (e.g., assimilation of C02 into sugar, of sugar into starch, of amino acids into proteins, or of acetate into lipids) are not peculiar to the photosynthetic organisms. However, it may perhaps be desirable to classify the different types of photosynthesis according to the com-pounds being assimilated or produced at the expense of the chemical energy stored in the first stable products of the photochemical reactions.


In the photosynthetic organisms a large number of synthetic reactions, driven by the energy of light, can take place simultaneously, even if one or other of them is predominant under certain conditions. (A similar diversity of simultaneous reactions occurs in nonphotosynthetic organ-isms.) Let us consider some well-established examples of endergonic reactions driven by light, all of which must be considered examples of photosynthesis.

6.1 Reactions Driven by ATP

6.1.1. PHOTOASSIMILATION OF GLUCOSE

In 1959, Maclachlan and Porter documented an example of what, from our point of view, can be considered one of the most simple cases of photosynthesis. These investigators reported that tobacco leaf disks synthesized starch and sucrose, when given glucose solutions and kept in light under anaerobic conditions. They concluded that the phosphorylation of glucose, an essential step in such a synthesis, was brought about by light-induced reactions which proceeded anaerobically (ATP formation by cyclic photophosphorylation).

6.1.2. PHOTOASSIMILATION OF ORGANIC ACIDS

Stanier et al. (1959) showed that the main function of organic substrates in photosynthesis by Rhodospirillum rubrum is to serve as readily assimilable sources of carbon. The assimilated carbon is stored intracellularly in the form of two principal reserve materials: poly-ß-hydroxybutyric acid and polysaccharide. Substrates which can be di-rectly converted to pyruvate, with an accompanying generation of reducing power, such as succinate, yield mostly polysaccharide.

An uncomplicated example of the direct photoassimilation of an organic substrate is the conversion of /?-hydroxy-butyrate to poly-/?-hydroxybutyrate by Rhodospinllum rubrum. Merrick and Doudoroff (1961) have recently shown with cell-free preparations of Rhodospirillum rubrum that the immediate substrate for polymer synthesis is ß-hydroxybutyryl-CoA. Therefore in the photoassimilation of jß-hydroxy-butyric acid the only role which must be attributed to light is ATP synthesis by cyclic photophosphorylation, the only mechanism available to the cell for making ATP under anaerobic conditions. This photo-synthesis can therefore be more correctly represented by the coupled reactions:

liiçht nADP + nP > nATP (3)

CoA nATP + nC4He08 > (C4He02)„ + nADP + nP + nH20 (7)


Chromatium can be grown photosynthetically in the absence of C02. Losada et al. (1960b) have given evidence for an acetate-dependent photosynthetic cycle, in which the main role of light is the formation of ATP by cyclic photophosphorylation. The photosynthetic cycle for the assimilation of acetate was different from the pentose reductive cycle for C02 assimilation. When Chromatium was grown with C02 and H2, the role of light for the assimilation of C02 was again only in the formation of ATP since the reduced pyridine nucleotide required for the reduction of C02 may be formed in the dark by a reaction catalyzed by hydrogenase.

In related experiments, Fuller et al. (1961) showed that Chromatium can grow in the light on malate, pyruvate, acetate, glutamate, suc-cinate, aspartate, citrate, and glucose in the absence of added C02. These workers also showed that when acetate or malate are the only carbon substrates for growth, the ribulose diphosphate-carboxylating enzyme is suppressed, confirming that in these cases the pentose reductive cycle did not operate. Finally Pringsheim and Wiessner (1960) concluded that sev-eral green algae (Chlamdobotrys, Euglena, Chlorogonium, and Chlorella) can grow anaerobically in the absence of carbon dioxide when acetate and light are provided. In this case also light energy is required only to supply ATP by cyclic photophosphorylation.

6.1.3. PHOTOACTIVATION OF ORGANIC COMPOUNDS

Amino acid-activating enzymes in isolated chloroplasts from spinach leaves have been found (Bové and Raacke, 1959; Marcus, 1959) and it has been demonstrated that ATP produced photosynthetically can be utilized for the activation. Other ATP-requiring enzymes, e.g. the acetate-activating enzyme, and glutamine synthetase, have been shown to be present in spinach chloroplasts.

6.2 Reactions Driven by Photochemical Reductant

6.2.1. PHOTOPRODUCTION OF H2 AND PHOTOREDUCTION OF N2

The light-dependent evolution of hydrogen in photosynthetic bacteria and green algae which was discussed earlier may be regarded as a par-ticular case of noncyclic photophosphorylation, and as such might be construed as a type of photosynthesis (although it might also be regarded as a way to get rid of electrons not wanted for reducing various metabolites).

In photosynthetic bacteria, N2 photofixation may be viewed as another example of noncyclic electron flow. I t is certainly a very important example of a photosynthesis (Arnon et al., 1961b).


The photochemical reduction of elementary nitrogen in the blue-green alga Anabaena cyUndrica has been reported (Fogg and Than-Tun, 1958). The production of "extra oxygen" during the assimilation of nitrogen gas indicates that electrons from water are utilized in the reduction of both nitrogen and C02. When Anabaena assimilated nitrogen gas at light intensities saturating for photosynthesis, more 0 2 was liberated than in similar preparations in which nitrogen was supplied in the reduced form, e.g. as glycine or an ammonium salt.

6.2.2 PHOTOREDUCTION OF NITRATE

C. B. van Niel et al. (1953) reported that at high light intensity sus-pensions of Chlorella pyrenoidosa, supplied with nonlimiting concentra-tions of C02, produce oxygen at a greater rate when N03~ is simul-taneously present. In that case the photosynthetic quotient, C0 2 /0 2 , is considerably lower than in the absence of N03~, even though the rate of C02 assimilation is not reduced. From these results van Niel et al. con-cluded that the photochemical N03" reduction, discovered by Warburg and Negelein (1920), can best be interpreted as a process in which nitrate acts directly as an alternate and additional hydrogen acceptor in photosynthesis.

Illuminated grana in the presence of TPN, and purified nitrate reduc-tase will also reduce nitrate (Evans and Nason, 1953; Jagendorf, 1956) which indicates that the photochemical reaction first produces the TPNH2 required for nitrate reduction.

In addition, Kessler (1957b) showed the rapid light-induced reduction of nitrite by the green alga Ankistrodesmus braunii in a nitrogen atmos-phere when C02 was excluded. Manganese was required for this reaction.

6.3 Reactions Driven by ATP and TPNH2: Photoassimilation of C02

As already discussed (Section 2) the conversion of C02 into carbo-hydrate requires both ATP and TPNH2. In both green plants and photo-synthetic bacteria light has to supply the energy for the synthesis of ATP. Reduced pyridine nucleotide has to be formed by a light-driven reaction in green plants where the electron donor is OH", but not neces-sarily in photosynthetic bacteria, where it is produced either by a dark or light reaction according to the electron donor used, e.g. hydrogen gas, thiosulfate, or succinate. I t must be emphasized, however, that the reduc-tive pentose cycle leading to C02 fixation is undoubtedly the most im-portant type of photosynthesis in plants, and is responsible for the storage of the major part of the chemical energy captured in ATP and TPNH2 during the conversion of light into chemical energy by photo-synthetic phosphorylation. The first stable chemical compounds formed


by the photochemical reactions are present only in catalytic amounts in the living cell, and an appreciable energy storage only results from the use of these initially formed substances to drive the C0 2 fixation.

7. Comparison of Photosynthesis and Chemosynthesis As we stated earlier, organisms which grow by converting light into

chemical energy are considered to be "photosynthetic." After the initial conversion of light into chemical energy in the form of ATP and TPNH2

the subsequent energy transformation leading to synthesis of new mate-rials are "chemosynthetic"—that is, the energy source for these subse-quent reactions is chemical. In their later energy transformations the photosynthetic organisms are behaving like those organisms, such as animals and bacteria, which depend entirely on chemical energy and are unable to use light energy themselves.

Now we are accustomed to thinking of organisms which can grow on a simple inorganic medium as "autotrophic." The green plant, Chlorella, is autotrophic; so is the sulfur bacterium, Thiobaaillus, which grows by fixing C0 2 on a sulfur-containing medium. But these two organisms have a very different energy metabolism. Chlorella obtains its energy by converting light into chemical energy, which is then available for meta-bolic use. I t makes ATP at the expense of light energy. To obtain "hydrogens" from water at the reducing potential of TPNH2 suitable for the reduction of C0 2 (when aided by ATP) it must make two separate inputs of light energy raising the electron from the water to a potential of approximately 0.0 volt with the first, and then to a potential of approximately —0.4 volt with the second light reaction. The "substrate," water, provides no useful chemical potential. Thiobacillus obtains its energy by oxidizing, say, thiosulfate to sulfate; electrons from thiosul-fate are carried along an oxidation chain to oxygen, the terminal acceptor, and ATP is produced by coupled phosphorylation. The chemical energy of the substrate is then made available as the metabolically useful ATP. Reduced PN is also made available from the chemical energy of the substrate. By this conversion of the chemical energy of the substrate into the energy of ATP and PNH2 Thiobacillus provides itself with the compounds needed to drive the C02-fixation cycle. Now let us consider the energy conversions in the photosynthetic bacterium, Chrom-atium, which is also autotrophic. This bacterium grows on an inorganic medium containing, say, hydrogen gas or thiosulfate, and uses C02 as its carbon source. Chromatium grows only in the absence of oxygen, but it must be provided with light energy to enable it to fix C02. I t obtains its ATP by converting light energy into chemical energy, and generates its PNH2 either directly in the dark (as when hydrogen gas is available to


it) or in the light by means of the noncyclic electron-flow mechanism (as with thiosulfate). When the PNH2 comes from hydrogen gas it is appar-ent that the substrate needed by Chromatium may provide chemical energy for the fixation of C02 directly. However, when the substrate is thiosulfate, which cannot reduce the pyridine nucleotide directly, one in-put of light energy is needed for each electron transferred from the sub-strate to pyridine nucleotide via the bacterial type of noncyclic electron flow. The additional input of light energy needed for the photooxidation of water by green plants is not required by Chromatium and we may con-clude that the substrate for bacterial photosynthesis provides an amount of energy which is at least equivalent to the energy contained in the intermediate A (Fig. 7) of the electron-transport mechanism (i.e. is equivalent to a redox potential of approximately 0.0 volt).

The basic difference among these three autotrophic organisms resides in the way in which they get the energy needed for their metabolism.

TABLE IV NOMENCLATURE OF ORGANISMS BASED ON THEIR ENERGY SOURCES

Class of organism Energy source Light reactions Examples

1. Photoergonic Light Two light reactions; Green plants water as reductant

2. Photochemoergonic Light in coop- One light reaction; Photosynthetic eration with reductant more bacteria reductant reduced than water

3. Chemoergonic Chemical No light reaction Nonphotosynthetic bacteria, animals, fungi

Group 3 might be subdivided as follows :

(I) energy by Fermentation: Energy obtained from reactions of organic compounds; (oxidoreductions with organic substances as both elec-tron donor and electron acceptor). Examples: (1) glucose —» 2CO2 + 2 ethanol

(2) 2 pyruvate —► acetate + CO2 + lactate

(II) energy by Respiration: Energy obtained by oxidoreductions when the electron acceptor is inorganic. The donor may be inorganic (e.g., H2S, H2, NH8, ferrous iron) as in some bacteria, or it may be organic (carbohydrate, lipid, protein) as in most living organisms. Examples of acceptors in this group are: (1) 02 —» H20 "Aerobic" organisms (2) S04" —> H2S Sulfate-reducing bacteria (3) NOT -> N2, N20 Dentrifying bacteria (4) C02 —► CH4 Methane bacteria


With these examples in mind we should like to propose a nomenclature of organisms based on their energy sources, which resembles the nomen-clature of bacteria proposed in 1946 by Lwoff et al., but which goes beyond it in some respects. The proposed nomenclature is shown in Table IV. The terms photoergonic, chemoergonic, and photochemoergonic used to describe the various classes of organisms were first introduced in an article by Arnon and Losada (1963).

The subdivision of the photosynthetic bacteria into those able to use (1) inorganic or (2) organic electron donors appears to be unnecessary in a nomenclature based on energy sources; the important point in the photosynthetic bacteria is that all the substances needed for bacterial photosynthesis provide a part of the overall energy requirement directly as chemical energy. Similarly when we consider the chemoergonic organ-isms it is not particularly significant whether the electron-donor sub-stances are organic or inorganic—they all have the same function. What is more important is perhaps whether these substances donate their electrons to an organic electron acceptor, as in fermentation (when most of the chemical energy available in the organic substrates is not re-leased) or to an inorganic electron acceptor, as in respiration (when almost all of the chemical energy available in the organic substrates is released). Although oxygen is quantitatively the most important electron acceptor it functions in essentially the same way as nitrate and sulfate in those organisms which employ the latter as terminal electron acceptors. Thus we may speak of nitrate and sulfate respiration, and the distinction between aerobic and anaerobic respiration becomes secondary.

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Chapter 6

PHYSICAL ASPECTS OF THE LIGHT REACTION IN PHOTOSYNTHESIS

Roderick K. Clayton

Biology Division, Oak Ridge National Laboratory,1

Oak Ridge, Tennessee

1. Introduction The central physical problem of photosynthesis is concerned with

the manner in which light energy is absorbed by chlorophyll, transmitted to a photochemical site, and converted to chemical energy. Recent years have seen a great proliferation of mechanisms, some conceptual and some demonstrated, which could be important for these primary events. The problem is to learn what does take place. It is therefore essential to consider what restrictions are imposed by existing knowledge, much of which is biochemical.

To this end we shall first examine some biochemical aspects of photosynthesis, as related to photochemical systems and reaction centers. It will then be possible to formulate the biophysical problems with some clarity. The next step will be to survey the physical and chemical properties of molecules and molecular aggregates of chlorophyll and to see which of these properties are exhibited in vivo. The hypotheses that can be entertained will then be self-evident; a brief evaluation of these will bring this chapter to a close.

2. The Biophysical Problem Delineated

2.1 Biochemical Outlines

Photosynthesis in green plants and algae can be defined in terms of three consecutive processes:

1. The energy of light quanta affords a separation of oxidizing and reducing entities; chlorophyll mediates this primary photochemical process.

1 Operated by Union Carbide Corporation for the United States Atomic Energy Commission.

155

156 RODERICK K. CLAYTON

2. The primary oxidizing and reducing entities provide starting points for a variety of electron-transfer reactions. As a result, chemical bond energy is stored, high-potential reducing substances are generated, and oxygen is released from water. The most popular (but by no means proven) point of view is that reducing power is stored as reduced pyri-dine nucleotide, and energy as ATP.

3. The energy and reducing power thus formed are used in the conversion of C02 to sugar and other cell constituents.

The behavior of the photosynthetic bacteria deviates from that of green plants and algae in several respects. The primary photocatalyst [Bacteriochlorophyll (BChl) in the purple bacteria and Chlorobium-Chl in the green sulfur bacteria] differs from the chlorophyll (Chi) of green plants and algae, absorbing mainly in the near infrared and near ultraviolet rather than in the red and blue-violet regions of the spectrum (Table I ) . The bacteria cannot liberate 0 2 from H 2 0 ; instead, they

TABLE I APPROXIMATE WAVELENGTHS OF THE PRINCIPAL ABSORPTION MAXIMA OF

CHLOROPHYLL AND BACTERIOCHLOROPHYLL IN ETHER SOLUTION AND in Vivo

In ether In vivo

Chi a 430, 660 niju ~ 4 3 5 , 670-680 BChl 360, 770 375, 800,850, 870-890

may release the oxidation products of substrates that are essential for their photosynthetic growth. Suitable substrates (depending on the species of organism) are H2, H2S, thiosulfate, and a great variety of organic compounds, such as acids and alcohols. The need to manufacture a high-potential reductant may be weakened or eliminated if a highly reduced substrate is fed to the bacteria; the essential function of bac-terial photosynthesis then becomes simply the formation of ATP at the expense of light energy (Stanier, 1961). The essential difference between photosynthesis in green plants and bacteria may be simply that the latter lack an enzyme for releasing 0 2 from H 2 0. The requirement is about 8 quanta per C02 for bacteria as well as for plants (see Gaffron, 1962). Alternatively, the bacteria may lack an entire photochemical system that the green plants possess (Section 2.3), but we shall see that in this view the quantum requirement for bacterial photosynthesis is hard to understand.

I t was van Niel (1935, 1949) who laid the foundations of this general picture of photosynthesis, by showing how the primary photo-chemical events could rationally be separated from subsequent "dark"

6. LIGHT REACTION IN PHOTOSYNTHESIS 157

reactions (oxidations, reductions, syntheses, transfers, etc.). His formula-tion (ca. 1940) is outlined in Fig. 1. The oxidizing and reducing entities

h . ^ i U H20

M- \ ^■»SUGARS, ETC.

FIG. 1. A representation of van Niel's formulation of photosynthesis (see van Niel 1935, 1941, 1949).

generated photochemically are denoted [OH] and [H] respectively. They are regarded as products of the photolysis of water, but they are not necessarily OH radicals and H atoms. [H] provides the necessary re-ducing power, and [OH] is disposed of either through conversion to 0 2 (in green plants and algae) or through oxidation of a substrate (in bacteria). In this relatively primitive representation the mechanics of energy conversion, storage, and utilization are omitted from considera-tion. Also the identities of enzymes operating between [H] and C02, and between [OH] and 0 2 or H2A, are left unspecified.

More recent formulations, drawing upon advances in the identifica-tion of electron transport processes, seem to present a more sophisticated appearance, but the essential validity of van NiePs picture has survived. By way of comparison, a scheme for bacterial photosynthesis, repre-senting recent suggestions by Arnon and others (Arnon et al., 1961; Losada et al., 1961) is outlined in Fig. 2. This picture incorporates such specific electron carriers as cytochrome and DPN. The storage of energy is in the pyrophosphate bond of ATP and in the energy level (reduction potential) of DPNH. The most striking departure in Fig. 2 is that the formalism of water splitting has been abandoned in favor of an electron flow circuit passing through BChl. An electron, given some of the energy of a light quantum, is driven from BChl to an acceptor such as DPN+; the "hole" in the BChl-system is filled by another electron from cyto-chrome. A similar but more elaborate scheme for photosynthesis in green plants and algae will be described later.

S~ SUBSTRATE

^ » OXIDIZED SUBSTRATE

RODERICK K. CLAYTON

ENERGETIC ELECTRON FROM BChl

h i /

A

BChl

\

ί

ANABOLIC REACTIONS

Cyt. <-

ADP + Pj

e- FROM SUBSTRATE

FIG. 2. An electron-flow diagram for bacterial photosynthesis, representing proposals of Arnon et al. (1961) and Losada et al. (1961).

Figure 1 implies that water is involved in the primary photo-chemistry; in Fig. 2 the BChl· is supposed to react chemically (losing and gaining electrons) as well as physically. These implications could be taken literally or they could be regarded as accidents of the type of formalism used. The point is that light energy and Chi are essential to a separation of oxidant and reductant ([OH] and [H], or holes and electrons). Any scheme preserving this feature can be adapted to new information on the detailed reactions of Chi and water.

In plants, and to some extent in bacteria, the remainder of the bio-chemical pattern consists in the conversion of C02 to sugar via the pentose cycle, drawing on the supply of ATP and reductants generated photochemically. This process, elucidated chiefly by Calvin (1962) and collaborators, is not peculiar to photosynthesis. Chemoautotrophic bacteria can perform the reactions of the pentose cycle (Suzuki and Werkman, 1958).

Although reduced pyridine nucleotide has been implicated (Vishniac and Ochoa, 1953; San Pietro and Lang, 1958; Frenkel, 1961) as the most important stable reductant generated in photosynthesis, this point is by no means proven. In the meantime Franck (1961) retains the position that a molecule such as phosphoglyceric acid is reduced directly (e.g., to phosphoglyceraldehyde) in a 2-quantum, two-step photochemical reaction.

158


2.2 The Photosynthetic Unit

We shall now consider briefly the architecture of a "photosynthetic unit": a set of several hundred Chi molecules that act cooperatively in harvesting the energy of light quanta and channeling this energy to a single reaction center.

Emerson and Arnold (1932b) measured the amount of 0 2 that was released in response to a single flash of light (/-Ί0"5 sec) in Chlorella. In these experiments the maximum quantum efficiency was about one 0 2 molecule evolved per 8 quanta absorbed. If every Chi molecule (or perhaps every set of eight Chi molecules, in view of the quantum efficiency) has associated with it a complete set of the enzymes needed for photosynthesis, one can expect that a sufficiently intense flash (which excites every Chi molecule) will cause one 0 2 molecule to be evolved for every eight Chi molecules present. Actually the maximum yield per flash was found to be one 0 2 per about 2400 Chi molecules. In spite of this low stoichiometric yield, the quantum efficiency was high (e.g., 8 quanta/02) as long as light saturation was not exceeded. In normal green plant photosynthesis one C02 molecule is reduced for every 0 2 molecule evolved. The conclusion was inescapable that a set of 2400 Chi molecules serves a single machine for reducing C0 2 and evolving 02, and that 8 quanta absorbed anywhere in the set can make this machine operate once, to reduce a single C0 2 molecule and produce one 0 2 molecule.

Another argument, leading to the same conclusion, was drawn by Gaffron and Wohl (1936), who computed that, in a suspension of Chlorella exposed to saturating illumination, each Chi molecule absorbs a light quantum every 8 min or so. At this rate, an hour would elapse before a single Chi molecule could absorb the 8 quanta needed to re-duce one C02 molecule. Nevertheless, the full rate of C0 2 reduction (and of 0 2 evolution) is established soon after illumination is begun, even in thoroughly dark-adapted cells to which dim light is applied. Clearly the quanta absorbed by many Chi molecules must be channeled to a common reaction center in order for the reduction of C02 to begin without delay.

A unit of about 2400 Chi molecules was established on the basis that 0 2 and reduced C02 (requiring 8 quanta per molecule) are the first stable products of photosynthesis. I t appears now that photo-synthesis begins with partial reactions leading to the formation of stable intermediates; reduction of C02 then draws upon the supply of these intermediates. The experiments of Emerson and Arnold might then be interpreted as follows: A unit of 300 Chi molecules serves a reaction center where a single electron-transfer act occurs with a quan-


turn requirement of unity. The reaction products (formed in a single light flash) of eight of these units provide the wherewithal for the reduction of one C02 molecule. Thus one C02 is reduced for every 2400 Chi molecules present, but the basic unit contains 300 molecules. If the unit reaction requires 2 quanta, then the unit contains about 600 Chi molecules, and so forth.

The only available evidence with regard to photosynthetic bacteria (W. Arnold, quoted by van Niel, 1941) indicates that 400 BChl mole-cules cooperate in the reduction of one molecule of C02. The efficiency of bacterial photosynthesis is about 8 quanta/C02 (Larsen et al, 1952) so the size of the unit on the basis of a 1-quantum reaction is 50 BChl molecules.

The existence of the photosynthetic unit is shown in several other ways: (1) When chloroplasts are broken into progressively smaller fragments, their photochemical activity (02 evolution) declines abruptly as the number of Chi molecules per fragment becomes less than about 100 (Thomas et al, 1953). (2) The herbicide 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), present in Scenedesmus at a concentration of one molecule per 200 molecules of Chi, suppresses photosynthetic 0 2

evolution completely (Bishop, 1958). (3) The concentration of light-reacting carriers such as the cytochromes is appropriate to the hypo-thetical size of the unit: about 0.3% of the Chi concentration in plants (Kok, 1961), and about 3% of the BChl concentration in photosynthetic bacteria (Chance and Nishimura, 1960).

I t can be taken as proved, then, that several hundred Chi molecules cooperate in harvesting light quanta for photosynthesis. A few quanta (1 to 8), absorbed anywhere in the unit, lead to the formation of one molecule of a photosynthetic product. If these quanta are absorbed within a short time, <0.04 sec according to Emerson and Arnold (1932a), the system is saturated; additional quanta absorbed during this time are ineffective. After about 0.04 sec, during which time the photo-product is utilized or otherwise removed, the unit is free to perform its function again. A corollary of these considerations is that the Chi unit possesses a reaction center at which the photo-product is made available to the cell, one molecule at a time.

The primary reactions carried out by the photosynthetic unit, and the mechanisms involved in these reactions, will be the main subject of this chapter. There is little doubt that the reaction center is part of the Chi unit, and that it involves one or more Chi molecules in a specialized environment. Light quanta are absorbed anywhere in the unit, and their energy is channeled to the reaction center. This channeling could occur through the intermolecular transfer of excitation energy and perhaps also


through the conduction of excited electrons or holes (electron vacancies). Upon reaching the reaction center, the excitation energy (or charge) promotes a reaction that yields a stable product.

The unit could be a definite set of Chi molecules plus a reaction center, functionally isolated from other units in the cell. Alternatively, it could be a statistical entity; a large aggregate of Chi molecules studded here and there with reaction centers. Morphological investiga-tions have some value with regard to these possibilities. The chloroplasts of green plants (see Rabinowitch, 1959) are seen in the electron micro-scope to contain "grana," cylindrical bodies about 1 X 1 μ that resemble stacks of disks (about 10 disks per granum). The disks are connected to those of adjacent grana by lamellar membranes; it appears that the lamellae, present throughout the chloroplast, are thickened locally to form the grana. The lamellar surface is grainy, as if covered with spher-ical macromolecular clusters. Each cluster, of diameter 70 to 100 Â, could accommodate a few hundred Chi molecules. In photosynthetic bacteria the BChl is contained in spherical subcellular particles termed chromatophores. The chromatophores of Chromatium are about 300 Â in diameter and contain about 600 molecules of BChl apiece (Bergeron, 1959). Chromatophores from Rhodospirillum rubrum and Rhodopseudo-monas sphéroïdes are somewhat larger. Thus the smallest photosynthetic structures that can be resolved in the electron microscope (the spherical cluster on the lamellar surface in chloroplasts, and the chromatophore in photosynthetic bacteria) are of such a size as to contain one or a small number of photosynthetic units.

2.3 The Cooperation of Two Distinct Photochemical Systems in Photosynthesis

A single quantum of red light does not provide enough energy to break an O—H bond in water and stabilize oxidizing and reducing entities at the levels of 0 2 and sugar, respectively. Mechanisms should therefore be sought by means of which the energy of 2 quanta could be summed in a photochemical act. Against this background, the "two-light" effects discovered by Emerson (Emerson et al., 1957; Emerson and Rabinowitch, 1960) promised at once to be of special interest. These effects, observed in green plants and algae, are: (1) the quantum efficiency of photosynthesis declines sharply at wavelengths greater than 680 πΐμ, in a region that is well within the red absorption band of Chi a and (2) the efficiency of this far-red light is enhanced by an admix-ture of shorter-wave light. I t appears that light has two distinct actions in photosynthesis; one of these is missing in far-red light. In terms of the summation of 2 quanta, one can imagine that quantum I can be of


any wavelength, but quantum II must be of wavelength less than 680 m/*.

The meaning of the Emerson red-drop and enhancement phenomena has been brought into a new focus by several recent observations. Myers and French (1960) observed that enhancement occurs even when the two qualities of light are separated by dark intervals of several seconds. This shows that the cooperation of the two light effects involves a rela-tively stable intermediate. Duysens et al. (1961) reported that in algae a cytochrome (probably Cyt f, in the light of other investigations) is oxi-dized by far-red light and then becomes reduced if shorter-wave light is superimposed. Kok (1959, 1961; also Kok and Gott, 1960; Kok and Hoch, 1961) has described a green plant pigment with absorption bands at 430 and 700 ιημ that is bleached (oxidized) in far-red light and restored (reduced) by shorter-wave light. This pigment, termed P700, is prob-ably a modification of Chi a; it occurs in plants in association with roughly equimolar amounts of Cyt f. Titrations with ferricyanide indi-cate that P700 is a one-electron transferring agent with a potential of about 430 mv; the potential of Cyt f is 365 mv. There is about one molecule of P700 for every 400 Chi a molecules in typical plants. Witt et al. (1961) have also observed the light-induced oxidation of such a pigment; the kinetics indicate that it is first oxidized and then receives electrons from cytochrome. The extensive experiments of Witt and collaborators (Witt and Moraw, 1959; Witt and Müller, 1959; Klingen-berg et al., 1962) also suggest that a quinone (potential about zero mv) is reduced in illuminated plants, whereas Hill and Bendall (1960) have implicated Cyt b6 in an electron transfer sequence, adjacent to Cyt f. Taken together, the observations of these and other investigators (e.g., Kautsky et al, 1960; Losada et al., 1961) have been construed to support the following picture: The Emerson phenomena arise from the interaction of two distinct photochemical systems that must operate sequentially to promote photosynthesis. The two systems are coupled by a set of electron carriers (quinone, cytochromes, and P700). One system (System I, activated by far-red light) oxidizes these carriers and the other (System II, activated by shorter wavelengths) reduces them.

In the context of this picture there is abundant evidence, based on action spectra and on the effects of inhibitors, that System II is con-cerned with 02 evolution and System I with the formation of reductant at the level of TPNH (see Duysens et al., 1961). A working hypothesis, reached more or less simultaneously2 by Duysens, Kok, Witt, Hill and

2 The development of this hypothesis should be attributed largely to Emerson's discovery of the enhancement phenomenon and to the incisive experiments of Duysens, Kok, and Witt and collaborators.


Bendall, Kautsky, and their collaborators, is represented in Fig. 3. Light quanta absorbed by System II raise electrons from a potential of about +800 mv (the 02 electrode potential3) to zero mv. These electrons "fall"

TO H+TPN+, ETC.

- 4 0 0 mv ■ L •e-

Omv e~—

+400 mv-

nuff SYSTEM

Π (SHORT Xîè

~\ QUINONE?! Cyt.b? Cyt.f P700 C

ADP + Pi

ATP

_L

SYSTEM I

(FAR RED)é

e-

+800 mv H*0 -*—>(

FIG. 3. The cooperation of two photochemical systems in photosynthesis ("series formulation"). This scheme is drawn from hypotheses of Duysens et al. (1961), Kautsky et al. (1960), Hill and Bendall (1960), and Losada et al. (1961).

to a potential of +400 mv through a system of carriers including cyto-chrome and P700. Electrons are raised from +400 mv to —400 mv (the potential of TPNH) at the expense of quanta absorbed by System I. In Fig. 3 the formation of ATP is coupled with the flow of electrons from 0 to +400 mv, but other sites of phosphorylation (e.g., a coupling with an electron pathway from —400 to 0 mv) have not been excluded. The sketchiest part of this scheme is, of course, the mechanism by which 02 is evolved. Arnon (1959) has proposed that OH" ions are oxidized to 02 while H+ ions are reduced to the H in TPNH. In this way the 2 quanta absorbed by Systems I and II cooperate in breaking the O—H bond: removal of H+ (System I) increases the chemical potential of OH" (operated on by System II) and vice versa. There is as yet no

8 The reaction 4 OH" -> 2H20 + 02 + 4e~ has, at 25°C and pH 7, a potential ΕΌ = 815 mv.


evidence for the existence of enzymes catalyzing such speculative partial reactions as OH" -> OH + e~.

In terms of Fig. 3, the photosynthetic bacteria can be regarded as organisms possessing System I only (cf. Fig. 2). In these bacteria, sub-strates provide electrons at a potential of about 0 mv, replacing the electrons delivered by System II . H2-adapted algae which carry on a "bacterial" photosynthesis using H2 as the electron-donor substrate (Gaffron, 1944), can be thought of in the same way. In strong light these algae revert to "green plant" photosynthesis, but in the presence of DCMU (which inhibits 0 2 evolution and presumably blocks the oper-ation of System II) this reversion does not occur (Bishop, 1958).

Three implications of Fig. 3 are subject to criticism and have provided the basis for a vigorous attack by Gaffron (1962). First, the type of water splitting proposed by Arnon involves difficulties when examined carefully. The reactions that deplete H+ and OH" ions (e.g., the TPN-reducing and 02-evolving reactions) must occur together both spatially and temporally. If they do not, there will be local or transitory changes in pH of such magnitude as to limit both reactions severely.

Second, the characterization of photosynthetic bacteria and H2-adapted algae as organisms using only System I is contrary to what is known about quantum efficiencies. If green plant photosynthesis re-quires 8 quanta per molecule of C02 reduced (Gaffron, 1960), then the bacteria and the H2-adapted algae should need only 4 quanta per C02, provided they are given substrates (such as H2) at an oxidation level of 0 mv. Actually, the H2-linked photoreduction of C02 in algae requires about 8 quanta per C0 2 (Rieke, 1949), and so does the reduction of C02

in bacterial photosynthesis (Larsen et al., 1952). Furthermore the quan-tum requirement in the bacteria does not depend on whether the substrate is strongly or only moderately reducing. Gaffron (1962) argues that bacterial photosynthesis, as well as green plant photosynthesis, involves the primary splitting of water by a mechanism involving 2 quanta. This produces, in the bacteria, an oxidant that is almost at the level of 02. The essential difference between photosynthetic bacteria and green plants is that the latter possess a special manganese-containing enzyme that mediates the terminal stage of 0 2 evolution.

A third implication of Fig. 3 is that 0 2 evolution is strictly a function of System II , except insofar as System I depletes H+ ions to keep pace with the depletion of OH- ions. A more complicated interaction of the two systems is indicated by the observations of French and Fork (1961) on 0 2 evolution and consumption in response to far-red and shorter-wave light. According to their data both systems cooperate in evolving 02, and the far-red system promotes a reaction that consumes 02.


Instead of the "series" formulation shown in Fig. 3, Gaffron (1962; also verbal communication) has proposed a "parallel" formulation in which a hypothetical manganese-containing enzyme (written MnEO in its reduced form and MnE0 2 in its oxidized form) mediates the evolution of 02. In this scheme, shown in Fig. 4, Y and Z might be regarded as

(Chi!) Y(OH)2 + MnEO—>Y + MnE02+ H20

S ft > f DCMU

(Chin) Z(OH) 2 + MnE02->Z + MnEO + H20+02

FIG. 4. Two-pigment cooperation ("parallel formulation") as suggested by Gaffron (1962).

cytochromes. They are oxidized in the light, by the far-red and shorter-wave systems (Chlï and Chln respectively), to the forms Y(OH)2 and Z(OH)2. These are the oxidizing entities equivalent to [OH] in van Niel's formulation; the appearance and fate of the reducing entities (van NiePs [H]) are not shown in Fig. 4. Energy can be transferred from Chln to Chli, but not in the opposite direction. This is a natural assumption since the "far-red" Chli uses lower energy quanta than does Chln. A little thought will show that such striking observations as that of Duysens et al. (1961), on the oxidation and reduction of a cytochrome in two qualities of light, are accommodated by the scheme of Fig. 4 as well as by that of Fig. 3. Gaffron has advanced the scheme of Fig. 4 by way of example, as a possible alternative to that of Fig. 3. Models embracing some features of both schemes can probably be constructed.

I t should be emphasized that in Fig. 3 the cooperation of two light quanta, to split water and stabilize 0 2 and reducing power, is such that Systems I and II each carry out a 1-quantum "partial process." In Gaffron's view there are 2-quantum processes, leading to water splitting, within each system separately (see Fig. 4). From the latter point of view the occurrence of 2-quantum processes in bacterial photosynthesis is not incompatible with the reported absence of "two-light" (enhance-ment) effects in the bacteria.

The pigments involved in Systems I and II can be discussed in the same way whether Fig. 3 or Fig. 4 is preferred. The operation of System I is sensitized by a nonfluorescent form of Chi absorbing maximally at wavelengths greater than 670 ηΐμ (Rabinowitch and Govindjee, 1961; Duysens, 1952; Kok and Hoch, 1961; French, 1961). The pigmentations


of different plants give different impressions as to the abundance and the wavelength of maximum absorption of this "far-red Chi a." The overall impression, gained from absorption spectra and action spectra for photosynthesis and fluorescence, is that far-red Chi a absorbs mainly in the region 680-695 ϊημ and is more abundant than such trace pigments as P700. Light energy absorbed by carotenoids is also active in the far-red system (see Kok and Hoch, 1961), presumably through energy transfer to far-red Chi a. System II appears to be sensitized by Chi b (absorption maximum at about 650 ηΐμ), by a fluorescent form of Chi a absorbing at 670 m/x, (Chi a670), and by phycobilins that transfer ex-citation energy to Chi b or to Chi a670 (Rabinowitch and Govindjee, 1961; Duysens, 1951). The reactions of Systems I and II have been termed the far-red and the accessory-pigment reaction. Chi ae7o and far-red Chi a may differ in their degree of aggregation; the most spe-cific suggestion in this respect is that the former is a monomer and the latter is aggregated (Brody and Brody, 1961a). The absorption spectra of these pigments are such that it is easy to activate System I selectively (by using 700 τημ light), but it is doubtful that System II can be activated without much concomitant activation of System I.

An entirely different interpretation of the two-light effects in green plant photosynthesis has recently been offered by Franck and Rosenberg (1963). In this formulation there is only one pigment system: one kind of Chi unit with its reaction center. The far-red and shorter-wave effects arise from two kinds of excited states in Chi, the strongly polarized ηπ* state and the less polarized ππ* state. "P700" is simply the ri?!-* state of active-center Chi associated with cytochrome. These states will be described fully in Section 3.1 ; for the present it will suffice to say that in its ηπ* excited state, Chi should be more reactive than in its 7Γ7Γ* state. Far-red light causes ηπ* excitation of the reaction-center Chi; shorter wavelengths arouse predominantly ππ* excitation. Both of these modes of excitation can promote the photochemical reac-tions of photosynthesis, but the η,π* excitation can also bring about a photo-oxidation in which Chi is oxidized and 02 reduced. This puts the Chi temporarily out of commission; in this way far-red light inhibits photosynthesis. Shorter-wave light, by flooding the system with ππ* excitation, suppresses the nm* Chl-oxygen reaction and allows the oxi-dized Chi to recover. Thus, for recovery from the photooxidation reac-tion, shorter-wave light is about as effective as darkness. This formulation can be made to accommodate most of the information on two-light effects, including the observations of Duysens, Kok, and Witt. It also accounts for the inhibiting effect of 685-700 πΐμ light observed in Anacystis (Emerson and Rabinowitch, 1960). To be sure, this hypothesis


is speculative and susceptible to experimental tests that have not yet been applied. In any case it adds to the variety of ways in which the two-light effects can be interpreted, and shows that the increasingly popular formulation of Fig. 3 is not the only one to be taken seriously.

Whatever interpretation proves to be correct, the mechanism will involve photosynthetic units and their reaction centers. Evidence for these will now be considered in more detail.

2.4 Evidence for Photosynthetic Reaction Centers

The concept of a photosynthetic unit implies the existence of a reaction center serving that unit. The possible existence of two distinct photochemical systems in the photosynthesis of green plants and algae raises new questions. Does the concept of the unit apply to each system separately or to the whole? Is each system a morphological entity, spatially separated from the other and possessing its own reaction center?

A reaction center for the far-red system might be built around the Chl-like pigment absorbing at 700 to 705 τημ (Kok's P700). Corre-spondingly the photosynthetic bacteria possess a special component of BChl, denoted BChl2, that may serve as a photochemical reaction center (Clayton, 1962a). Evidence relating to these possibilities will now be considered.

It was mentioned in Section 2.3 that P700 is associated with an equimolar quantity of Cyt f, and that far-red light causes it to become oxidized and in turn to accept electrons from the Cyt f. Although there is only one molecule of P700 for every 400 molecules of Chi a, fewer than 5 quanta (absorbed by Chi a) are needed for the oxidation of one P700 molecule (Kok and Gott, 1960). A convincing argument can be drawn, then, that light energy absorbed by Chi a is channeled efficiently to a reaction center containing P700 and Cyt f. At this center electrons are transferred from P700 to an unidentified acceptor and perhaps ultimately to TPN. P700 is then restored by taking electrons from Cyt f. P700 is probably the same as the 700 to 705 m/x-absorbing pigments described by Witt et al (1961), by Butler (1961), and by Allen (1961), the exact location of the absorption maximum depending on the species of plant or alga. Butler (1961) has suggested that this pigment is simply Chi a in a unique structural environment: a close coupling with cyto-chrome shifts its absorption band to 700-705 τημ and endows it with the properties of a photochemical reaction center. It will be shown in Section 5.3 that the arrangement of P700 molecules in chloroplasts is highly oriented.

In photosynthetic bacteria there is a specific BChl component, BChl2,


that comprises about 3% of the total BChl (Clayton, 1962b). Light absorbed by BChl causes a reversible alteration of BChl2, characterized mainly by bleaching of its long-wave absorption band (at 870-890 ηΐμ) (Clayton, 1962b; Duysens et al, 1956). The change is similar to that caused by chemical oxidation (515 mv potential) (Goedheer, I960), and requires about 3 quanta (or fewer) per molecule (Clayton, 1962c). In Chromatium there is a cytochrome (Cyt C423.5) that is oxidized in the light; BChl2 and Cyt c423.5 are present in approximately equimolar amounts. At liquid nitrogen temperature the light reaction of BChl2 occurs reversibly and Cyt c423.5 is oxidized irreversibly. The kinetics of the reactions of BChl2 and Cyt c423.5 at room temperature indicate that BChl2 is oxidized and then accepts electrons from Cyt c423.5 (cf. Arnold and Clayton, 1960; Clayton, 1962a-d; Chance and Nishimura, 1960; and J. M. Olson and Chance, 1960). It appears, then, that BChl2 in photo-synthetic bacteria is the counterpart of P700 in green plants: a modified chlorophyll that forms a reaction center in conjunction with cyto-chrome, and that mediates the photochemical transfer of electrons from cytochrome to an unidentified acceptor. The Chi of the green bacteria is of still another type (or types) termed Chlorobium Chi. J. M. Olson (verbal communication) has obtained preliminary evidence that a trace of BChl is present in these bacteria and could be a part of a reaction center.

The light reactions of P700 and BChl2 have been identified as oxida-tions because the light-induced changes in absorption spectrum resemble those caused by chemical oxidation. For P700 this means simply a bleaching around 700 τημ. The change in BChl2 is characterized by bleaching of the long-wave band and a slight blue-shift of an absorption band at 800 τημ. These changes could result either from the complete removal of a ground-state electron (oxidation) or from the promotion of such an electron to a long-lived excited state (excitation). Thus it cannot be taken as proved that the light-altered forms of P700 and BChl2 are identical to the chemically oxidized forms.

There is some evidence (e.g., Witt and Moraw, 1959) that a reduced form of Chi, having an absorption maximum at 520 m/x, is produced in illuminated plant tissues. No information has accrued, however, that would implicate reduced Chi as a component of a photosynthetic re-action center.

2.5 The Nature of the Biophysical Problem

The foregoing sections were meant to provide a framework in which the biophysical problems of photosynthesis could be formulated. We may now ask two questions. How does a photosynthetic unit, harvesting


light quanta, deliver an effect to a reaction center? What does the reaction center then do to produce separated oxidizing and reducing entities, and what are these entities? These questions draw our attention to certain kinds of events. Chi absorbs light quanta and enters an ex-cited state. Energy or charge migrates from the site of light absorption to a reaction center. The reaction center traps this energy or charge and generates relatively stable oxidants and reductants.

In attempting to understand these events we must consider various properties of Chi: the nature of its excited states, its photochemical reactions with other substances, and its physical interactions with Chi and other molecules. In particular, these properties should be studied as they occur in a highly condensed state, comparable to the state of aggregation in chloroplast lamellae or chromatophores. The properties of Chi can then be used in interpreting the behavior of photosynthetic tissues and in constructing theories for the primary reactions of photo-synthesis.

3. Excited States of Chlorophyll

3.1 Singlet States in Isolated Chlorophyll Molecules

Electronic states in organic molecules are determined by the electron orbitals of the component atoms (principally C, H, N, and 0) and by their interactions in the molecule (see McClure, 1960). The interactions of the atomic orbitals generate more distinct states in the molecule than existed in the component atoms. Thus, an atomic ground state of one kind (e.g., a 2p state of carbon) can yield, in a molecule, several distinct ground (normally occupied) and excited (normally unoccupied) states. These molecular states are generally far lower, on an energy scale, than the excited states of the component atoms. Transitions of electrons between molecular ground states and low-lying excited states involve energies in the range of visible light quanta ; they are, therefore, of prime importance in photochemistry.

The electrons occupying these states usually occur in pairs having oppositely directed electron spins, so that the net spin angular momen-tum is zero. In a singlet excitation this condition is conserved; the spin of the excited electron continues to neutralize that of its ground state partner. If this neutralization is imperfect the excitation has some triplet character; a pure triplet excitation would be one in which the spin of the excited electron has become reversed and is parallel to that of its partner. Triplet states are of lower energy than the corresponding singlet states. We shall return later to a consideration of singlet-triplet transi-tions, and confine ourselves at this point to singlet states.


In Chi and its relatives the ground states involved in optical transi-tions are probably of three kinds (Franck, 1958). Two of these are "n" states; i.e., states of electrons localized near single atoms and retaining their "atomic" character. The two potentially important n states are for electrons localized at N and 0 atoms, respectively. The third kind of important ground state is a π state, in which electrons are delocalized over the conjugated tetrapyrrole system (see Fig. 5). The optical

\s

PROTOCHLOROPHYLL

Γ 7 N N\ I H i — - / N > 1

CHLOROPHYLL

H i —

BACTERIOCHLOROPHYLL

FIG. 5. Conjugated systems (indicated by solid lines) in chlorophylls. See Rabinowitch (1956).

transitions accounting for the visible absorption bands of Chi and related molecules appear to be from these ground states to two or more π* (delocalized, excited) states. Thus a variety of η-»π* and π->π* (ηπ* and ππ*) transitions are implicated in the absorption spectrum of Chi.


The probability of a transition depends on the overlap between the wave functions of the ground and excited states involved in the transi-tion. In an ήπ* transition the electron passes from a localized state (at a single atom) to a delocalized state in a nearby conjugated system; here the wave functions have little overlap and the probabilities of absorption (n -» π*) and emission (π* —> n) are relatively small. Proba-bilities of 7Γ7Γ* transitions are generally more than ten times greater than those of η-π* transitions in the same molecule (Kasha, 1960a). Since the lifetime of an excited state is inversely proportional to the emission probability, the ηπ* states are intrinsically much longer lived than 7Γ7Γ* states. Various quenching effects will of course alter the life-times of these states (see later sections for information on internal conversion and intersystem crossing).

The detailed natures of n, π, and π* states are not well enough known to provide a complete theory of the absorption spectra of chlorophylls, but some correlations and assignations are plausible (see Rabinowitch, 1956, pp. 1793-1798). The major absorption bands (e.g., 430 and 660 τημ in Chi a) undoubtedly reflect ππ* transitions because they are so intense. These blue and red bands probably correspond to π* states in which the orbital angular momentum of the excited electron is antiparallel (blue) or parallel (red) to that of its ground state partner. The number of distinct π* states is increased further by asymmetry of the conjugated ring system. Thus in BChl the conjugated system is elongated (Fig. 5), and π* states can be differentiated according to whether the excited electron oscillates principally along or perpendicular to the long axis. This may account for the band at about 590 τημ that accompanies the 770 τημ band in BChl. By measuring the polarization of fluorescence emitted by BChl, using polarized exciting light, Goedheer (1957) has shown that the 590 and 770 πΐμ bands are related to two mutually perpendicular oscillators, both lying in the plane of the con-jugated system.

Evidence for absorption bands reflecting ηπ* transitions in Chi is as yet rather tenuous. Polar solvents, and traces of H20 in nonpolar solvents, can be expected to raise the energy gap of an η-π* transition in Chi and lower that of a ΤΓΤΓ* transition (Platt, 1956) .4 In dry benzene the absorption spectrum of Chi b shows a shoulder at 670 ιημ, next to the main band at 650 τημ (Livingston et al.y 1949). This shoulder, which vanishes when a trace of water is added to the benzene, might be due to an ηπ* transition (Becker and Kasha, 1955b). In wet benzene a shift from 670 to 650 τημ would conceal this minor absorption band.

4 Blue-shift of the wir* level in wet solvents is probably not a polarization field effect, but rather a specific effect of H20 on the n-electrons of the N atoms that bind the Mg atom (Franck, 1958).


The higher excited states of Chi and BChl are nonfluorescent; thus when Chi a absorbs blue (430 τημ) light, it emits red fluorescent light. The fluorescence signals a transition from the lowest excited state (corresponding to the red band) to ground. Information of this kind shows that when an electron enters a higher excited singlet level it is quickly converted, in radiationless transitions, to the lowest excited singlet state. Having reached the lowest excited singlet level through this process of internal conversion, the electron may then enter a meta-stable excited state, engage in a photochemical reaction, or return to the ground state. The important point is that light quanta generating higher singlet states are photochemically equivalent to those producing the lowest excited singlet state. An exception to this statement might be found in a condensed system (a Chi aggregate) if intermolecular energy transfer can occur via higher excited states more rapidly than internal conversion within a single molecule.

3.2 Triplet States and Intersystem Crossing

Paralleling the system of singlet excited states is a system of triplet states in which the spin of the excited electron is no longer antiparallel to that of its unexcited partner. The energy level of each triplet state is lower than that of the corresponding singlet state. This difference in energy levels is greater for ππ* than for ηπ* transitions because the spin coupling (between the excited electron and its partner) is closer in the former case.

The probability of an intersystem (singlet <-» triplet) transition is governed by quantum mechanical selection rules and by the ability of the external radiation field to interact with the electron spin. Radiative singlet-triplet transitions are usually about 106-fold less probable than the corresponding singlet-singlet transitions; the lifetimes of excited triplet states are correspondingly greater than those of singlet states.

The selection rules that allow or forbid electronic transitions involve the symmetry properties of the total wave function (positional and spin) before and after the transition. Any changes in electron spin must therefore be accompanied by changes in the symmetry of the positional wave function. Factors that disrupt the positional symmetry of a mole-cule will therefore facilitate a change of electron spin in a transition (see Rice and Teller, 1949).

Intersystem transitions involving light quanta are intrinsically improbable because the radiation field interacts with the electron spin only through its magnetic moment. This magnetic coupling is much weaker than the electric coupling through which a light quantum promotes an electronic transition. In some transitions (ηττ*, for example)


the coupling between spins of the excited electron and its partner is weakened and the spins interact mainly with other magnetic forces such as electron-orbital magnetic moments. These forces may realign the electron spins and thus encourage intersystem crossing. Radiationless in-tersystem crossing (e.g., between excited singlet and triplet states) may occur with high yield, especially if the initial state is long-lived and the change in energy level is small and negative.

Some practical consequences of these considerations can now be listed. (1) Excited triplet states, having lifetimes of the order of milliseconds (compared with about 10~9 sec for singlet states), may be important in trapping excitation energy and initiating photochemical events in photosynthesis. Their chemical reactivity may also depend on the fact that they are paramagnetic, and will attract other paramagnetic entities such as 02. (2) Triplet excitations are encouraged by factors that dis-rupt symmetry: collisions, molecular interactions, and fields associated with heavy nuclei. (3) Singlet-triplet conversion should be favored in highly polarized states (states such as ηπ*, in which a large electron displacement has occurred), because of the greater lifetimes of these states and because of the greater spin-orbital coupling. This expecta-tion may not be realized if other events, such as radiationless de-excita-tion, supervene. (4) A fortuitous matching of the energy levels of two different states (e.g., a singlet ττπ* and a triplet η-π*) may facilitate their interconversion.

Radiative de-excitations can be analyzed by measuring the emitted light: fluorescence from singlet states and the longer-lived phos-phorescence from triplet states. Of course, the absence of emitted light does not guarantee the nonexistence of an excited state, as many kinds of radiationless de-excitations (quenching processes) are possible. In Chi b, phosphorescence has been observed with maxima of intensity at 733 and 865 m/*,; these wavelengths probably correspond to the energy levels of τ&π* and ππ* triplet states, respectively. Chi a shows only a 755 m/A phosphorescence corresponding to the ηπ* triplet, but a ττπ* triplet around 870 τημ may be presumed to exist (Becker and Kasha, 1955a; Fernandez and Becker, 1959). Fluorescence of Chi a is maximal at 672 πιμ (C. S. French, quoted by Rabinowitch 1956, p. 1828) ; this fluorescence is from the 660 τημ ΤΓΤΓ* singlet level. Photosynthetic tissues exposed to light emit a long-lived luminescence having the same spec-trum as their fluorescence (Arnold and Davidson, 1954). This delayed light emission represents a return to the ground state, via the lowest excited singlet level, of electrons in long-lived states that are not necessarily triplets.

Molecules in triplet excited states can be observed spectrophoto-

174 RODEEICK K. CLAYTON

metrically, their absorption bands revealing transitions to higher triplet levels. Using a flash photometry technique applied to Chi in solution, Linschitz and Sarkanen (1958) have observed that a single flash of light converts as much as 90% of the Chi to a triplet state lasting several milliseconds (Evidence for triplet Chi in vivo is tenuous: see Section 5.4). The yield of triplet Chi a in dry benzene is about % that in benzene containing a trace of water (A. C. Pugh, quoted by Living-ston, 1960). At the same time, Chi a is fluorescent in wet benzene but not in dry benzene- As was indicated earlier, the lowest ηπ* level of Chi a probably stands below the lowest ππ* level in dry benzene, and above it in wet benzene. It may be that in wet benzene the singlet ππ* state is converted eflSciently to a triplet η-π* state of slightly lower energy and thence to a ιπτ* triplet state, whereas in dry benzene the lowest singlet (ηπ*) state is dissipated mainly through radiationless transitions to ground (see Livingston, 1960).

The simplest approach to understanding the behavior of Chi is through the use of a system such as Chi dissolved in benzene. Com-plexities are already abundant in this simple system, and the properties of Chi in vivo can be expected to be far more complicated. Nevertheless, the Chl-benzene system has yielded not only basic information but also some interesting ideas for the mechanism of photosynthesis.

3.3 Excited States of Interacting Chlorophyll Molecules. Energy Migration

Molecular interactions, by spoiling the symmetries of individual molecules, may facilitate entry into "forbidden" states such as triplets. The close association of unlike molecules can make heterogeneous charge-transfer states possible. Aside from these possibilities, there are two consequences of interaction in a homogeneous molecular aggregate that may be of first importance in photosynthesis. One is the intermolecular overlap of electron orbitale, leading to electric conductivity. The other is electric dipole (and multipole) interaction, leading to the migration of excitation energy.

With sufficient electron orbital overlapping, conductivity bands analogous to those in ionic crystals might occur. This fusion of orbitale may take place with the higher ("atomic") excited states of molecular crystals, but it appears to be absent in the molecular states that correspond to the visible absorption spectrum (Kasha, 1959). A degree of overlap sufficient to produce conductivity bands should alter the absorption spectrum drastically, and that is not observed. A limited conductivity facilitated by electron trapping does occur in molecular aggregates and will be discussed in the next section.

Energy migration through electric dipole interactions will be con-


sidered first for the simple case of two molecules. Two extremes cor-responding to very weak and very strong interaction will be examined. The first of these extremes is represented by a pair of molecules in a monomeric solution; the second by a tightly bound dimer.

Figure 6a portrays the sequence of excitation and energy transfer in

[^ + r^JH[^+ [^i^fc>[^t:

-EXC.\

-Θ-Θ Θ-Θ-GRQ

(QrQ2)

EXCITED (Qi · Q2)*

f t (ALLOWED)

(Qi -Q, ) *

t | | l (FORBIDDEN)

(Q, · Q2r

GROUND « V Q2)· -Ψ-Ψ,ΨΖ

FIG. 6. Diagrams showing excitation and energy transfer involving two mole-cules Qi and Q2. (a) Weak coupling; excitation localized in Qi or Q2. (b) Strong coupling; excitation delocalized over the dimer Qi*Q2. The vertical arrows indicate transition dipole moments in the molecules. The horizontal lines indicate energy levels of ground and excited states.

two molecules, Qi and Q2, for the case of very weak interaction (for a detailed treatment of this case see Förster, 1959). The excitation is first localized in Qi*; the arrow represents the transition dipole moment (dipole moment of Qi* minus that of Qi) imparted by the external radiation field. In the change from Qx* -f- Q2 to Qi + Q2*, the excitation of Q2 is promoted by the dipole field of Qi* rather than by an external electromagnetic field. This energy-transfer event is in competition with a simple radiative de-excitation (fluorescence) of Qi*. The probability of energy transfer is governed by the degree of coupling between the


transition dipoles of Qi and Q2. This localized picture is appropriate only when the coupling is relatively weak. Under these conditions the energy transfer rate is 109 transfers/sec or fewer. The transfer rate varies inversely as the sixth power of the intermolecular distance, and is predicted to be temperature-dependent. The unimolecular absorption spectrum is not altered appreciably by the interaction.

In the transfer act, Qi* usually returns to ground from the lowest vibrational level of its excited state (as it usually does in fluorescence). The concomitant excitation of Q2 involves the same energy change as the de-excitation of Qi. Thus the transfer probability (or rate) depends on the amount of overlap between the fluorescence spectrum of Qi and the absorption spectrum of Q2. For this reason (see Fig. 7) the slow

Q, AND Q2 ARE LIKE MOLECULES

ABSORPTION FLUORESCENCE ABSORPTION OF Q, , OF Q, ^ OF Q2

\

Qi AND Q2 ARE UNLIKE MOLECULES

FIG. 7. Diagrams showing the overlap of absorption and fluorescence bands for like and unlike pairs of molecules.

transfer described above can be more efficient between unlike molecules than between like molecules. The extension of the foregoing picture from two molecules to many molecules is obvious, as the events involve only two molecules at a time.

In the case of strong coupling (e.g·, in a dimer, Qi-Q2) it is not admissible to describe the excitation as being localized in one molecule or the other. A delocalized exciton theory developed by Frenckel (1931), Davydov (1948), Simpson and Peterson (1957), and others, and ex-

ABSORPTION FLUORESCENCE


tended by Kasha and collaborators (McRae and Kasha, 1958; Kasha et al., 1961) will be sketched.

Whereas the localized picture deals with excited states Qx* + Q2 and Qi + Q2*, the treatment for strong coupling involves a ground state (Q1/Q2) and an excited state (Qi'Q2)* that is split into two levels. The wave functions for the two localized conditions (Qi* + Q2 and Qi + Q2*) are ψι*^2 and ^ 2 * . Linear combinations of these yield the wave func-tions for the two levels of (Qi*Q2)*:

* = ^ ( ^ l V 2 Ψ M ^ ( 1 )

This is illustrated in Fig. 6b for a dimer whose transition dipole mom-ents5 are perpendicular to an axis through the center of each dipole. The negative sign in Eq. (1) is for the upper (antibonding) state in which the transition dipoles of the individual molecules are parallel. In this example the upper state is allowed and the lower state forbidden. This can be seen from a qualitative argument: The dimer is much smaller than the wavelength of the exciting light, so both molecules will be in the same small region of the radiation field. The phase of the electro-magnetic wave will be the same throughout this region, and so the transi-tion dipoles generated by the radiation field ought to be in phase with each other. If the transition dipoles are aligned with the dimer axis, the allowed configuration (-»->) is attractive and the forbidden one (-> «-) repulsive; in that case the lower of the two excited states is allowed. In an oblique arrangement, / \ and / \ , both configurations have "in-phase" components and both states are allowed. When this approach is extended to N coupled molecules, N excited states are generated through linear combinations of wave functions such as ^1^2 ...ψ8* — -ψΝ. Depending on the symmetry of the array, one can expect various situations as shown in Fig. 8 (see Kasha et al., 1961). In this figure, allowed states are shown by solid lines and forbidden states by dashed lines. Depending on the symmetry, all kinds of spectral mani-festations are predicted: blue-shift, red-shift, no shift, narrow or broad bands, band splitting, etc. Band splitting (Fig. 8b) has been seen in Chi dimers, the single maximum at 665 πΐμ being split into two at 648 and 682 m/A (Brody and Brody, 1961b).

In Fig. 8a, a triplet level is drawn between the top and bottom of

5 Although the excitation is treated as belonging to the dipole as a whole, it is sometimes helpful to visualize a quantum of excitation (an exciton) oscillating rapidly between the two members of the dipole. This notion will be introduced again when the extent of delocalization of an exciton is discussed in terms of the rate of its transfer from one molecule to another.


COMPLEX SYMMETRY OR DISORDERED CLOSE COUPLING

d

FIG. 8. Excited state levels in polymers, as compared with the monomer level, for aggregates having various geometries. The arrows show the alignment of transition dipole moments of molecules in the polymer. Various permutations, in which some of the arrows are reversed, give rise to various energy levels. Allowed levels are shown by solid lines; forbidden levels by dashed lines. See Kasha et al. (1961).

the set of singlet levels. In this example a subtle change in the structure of the aggregate [from a > cos"1 ( l / \ / 3 ) to a < cos-1 ( 1 / V 3 ) ] could cause an overwhelming change in the population of the triplet state. In Fig. 8d, a triplet state could be populated efficiently if it lay near the bottom of the exciton band.

All of these cases apply to models having strong coupling, or high delocalization of the excitation energy. Close proximity of two molecules (even to the extent that their electron orbitals overlap) does not guaran-tee strong dipole coupling; the dipole interaction will be zero if the


transition dipole moments are mutually perpendicular. Thus it is pos-sible to have marked spectral changes (due to electron orbital overlap) without strong dipole coupling and without extensive exciton migration. Conversely, the absence of a large spectral shift or broadening does not guarantee the absence of strong dipole coupling (see Fig. 8c), nor does it prove the absence of a high degree of order in an aggregate.

In the case of strong coupling (see Förster, 1960), the degree of delocalization of the excitation energy corresponds to a transfer rate ranging from 1012 to 1016 per second (cf. 109/sec for very weak coupling). No temperature dependence is predicted, and the transfer rate varies inversely as the third power of the intermolecular (dipole-dipole) separa-tion. The rate of transfer depends on the integrated area of the absorp-tion band rather than on the overlap between absorption and fluorescence bands.

Intermediate coupling, leading to transfer rates in the range 109 to 1012 per second, is transitional between the delocalized (strong coupling) picture and the localized (weak coupling) picture. The transfer rate is then equal to or somewhat less than the frequency of nuclear vibrations, and a complicated interplay between electronic and vibrational states is involved. For this transitional case, Förster (1960) predicts some temperature dependence, rather slight changes in absorption spectrum, and a transfer rate inversely proportional to the third power of the molecular separation.

The importance of exciton migration is obvious as a mechanism by which excitation energy in a photosynthetic unit can be delivered to a reaction center. Indeed, all current theories of photosynthesis invoke some combination of exciton migration and electron conduction or transfer.

The transfer of excitation energy in vitro has been established through numerous experiments on sensitized fluorescence (see Förster, 1959) :Λν + Α + Β-»Α* + Β - » Α + Β * - » Α + Β + hv'. Additional evi-dence (Goedheer, 1957) is found in the depolarization of fluorescence emitted by a collection of molecules exposed to polarized exciting light. In an ensemble of randomly oriented molecules, this depolarization will signify energy transfer if the molecules are not able to rotate during the lifetime of excitation. Also the quenching of fluorescence in a molecular aggregate may imply the transfer of energy to a site where radiationless de-excitation is especially rapid. Convincing evidence for energy transfer in vivo will be presented in Section 5.2.

The occurrence of energy transfer through electronic coupling of excited triplet states has been demonstrated by experiments showing sensitized phosphorescence (Terenin and Ermolaev, 1956).


4. Electron Transport in Molecular Crystals and in Solutions of Chlorophyll

4.1 Conduction, Charge Trapping, and Charge-Transfer States

The electron orbitale of ionic crystals overlap to such a degree that excited levels become conduction bands; an electron in a conduction band is not bound to any one atom and is free to move about in the crystal (see Kittel, 1953, for an introduction to this subject). In the unexcited crystal the ground state (the valence band) is filled and the conduction bands are empty; the crystal is nonconducting. Elevation of an electron to a conduction band permits the electron to move, and the vacancy (positive hole) created in the valence band can also move. If the crystal contains sites of electron affinity, the conduction electrons may become trapped while the holes continue to conduct charge (p-type crystal). Conversely the holes may be trapped, the electrons remaining free (n-type). Trapping centers are generally identified as flaws arising from atomic deletions, substitutions, or additions, or from breaks in the continuity of the crystal structure. A trapped electron can be restored to a conduction band by abstracting a small quantum of energy from its surroundings; it may then make a radiative transition to the ground state. This untrapping will be acceler-ated by heating, giving rise to thermoluminescence.

Excitation of electrons into conduction bands can arise from thermal agitation (semiconductivity) or through the absorption of light quanta (photoconductivity). The energy required for photoconduction is of course the energy of the effective light quanta. The energy gap for semiconduction can be obtained from an Arrhenius plot of the temper-ature dependence.

I t was mentioned in Section 3.3 that organic molecular crystals lack well-defined conduction bands corresponding to the lower excited states and ground states. Nevertheless, molecular crystals exhibit limited con-ductivity involving low-energy states, and the possible importance of this conduction for photosynthesis should be assessed.

The low mobility of charge carriers in organic semiconductors (about 10"4 to 10"15 of that in ionic crystals), together with spectral evidence for the absence of "good" conduction bands, indicates that the con-duction is an electron diffusion process that involves tunneling of the charge (quantum-mechanical penetration of energy barriers) from one molecule to the next (Garrett, 1960; Nelson, 1962; Tollin, 1960; Eley and Parfitt, 1955). Conduction in a molecular crystal begins, then, with a


separation of charge that does not involve an ionization band or continuum. The first step is a thermal or photo-excitation, followed by (or accompanied by) a transfer of the excited electron or the hole to a neighboring molecule. In an alternative description, an exciton is re-garded as a neutral "excitation particle" consisting of a closely coupled electron-hole pair that moves in the crystal lattice. Its dissociation pro-duces a conducting state in which the electron and the hole are separated. The essential problem in this field is to understand the nature of the low-energy conducting states and the mechanisms by which they are populated.

One aspect of the problem is that the ionization leading to conduction is effected at energies far below the molecular ionization potential. Thus in anthracene the ionization energy should be around 5 ev, but photo-conduction involves 3.2 ev quanta and the energy gap for semiconduction is only 1.9 ev (see Tollin, 1960). Obviously, mechanisms are at work that lower the ionization energy.

Traps with high affinity for electrons (or for holes) can lower the ionization energy by several electron volts. For example, the ionization potential of gaseous tri-p-tolylamine is 8 to 12 ev (Kasha, 1960b). When dissolved in a rigid solvent at 90°K, this substance is photo-ionized by 3 to 5 ev quanta. Electrons are captured by the solvent, leaving the tri-p-tolylamine in an oxidized state. Here the electron affinity of the solvent amounts to about 6 ev.

Where the electron trapping can be identified with certain molecular species (a donor and an acceptor molecule) it is possible to describe a charge transfer state (McGIynn, 1960): hv + D-A-»+D-A~. If this is to afford conductivity, the separated charges in the complex must be bound loosely enough to allow further separation under an external field: /iv + D - D - D - A - » D - D - + D - A - - > D + D D A - . Homogeneous charge-transfer states, in which D and A are identical, can also be conceived. For example, in the strongly polarized ηπ* state of Chi the π* electron might be transferred to a neighboring Chi molecule : hv + Chi · Chi -» Chi · (~Chl+) -» (Chi") · (Chl+). If the dissociation (second step) does not require too much energy it might be brought about by a thermal encounter or a strong electric field. Again it should be remarked that if this process were to occur efficiently, it would probably require a degree of electron-orbital overlap that would alter the absorption spectrum markedly.

There is some evidence (B. Rosenberg, 1958) that the entry into a conducting state involves a triplet excited state. The energy gap for semiconduction is often close to the lowest excited triplet level. Thus


in Chi the energy gap is 1.44 ev, corresponding to 860 τημ (Eley, 1962). The lowest (τπτ*) triplet level is at about 870 τημ.

In attempting to construct a model system that may have application to photosynthesis, Kearns et al. (1960) have uncovered an interesting case of conductivity in which phthalocyanine (PC) is the conductor and quinones, notably o-chloranil (CH), serve as electron acceptors. Dry films of PC, with or without a coating of CH, were examined in the dark and under light absorbed by PC. It was observed that the semi-and photoconductivity of PC increased by factors of 107 and 105, re-spectively, when CH was applied. Electrostatic measurements showed that electrons were transferred from PC to CH in the dark, and this transfer was increased reversibly by illumination. Electron spin reso-nance measurements showed a large signal in the dark that was decreased reversibly by light. These results were interpreted as follows:

Dark: PC + CH «± PC+ + CH" Light: PC* + CH- <=± PC+ + CH~

The charge carrier is PC+, or more accurately, the hole that is associated with PC and symbolized as PC+. The source of the ESR signal is CH", and the photoionization is facilitated mainly by the presence of CH" as an electron acceptor. The ESR measurement had special value in that the number of charge carriers could be determined; then from the current the mobility of the holes could be computed. The results were: The mobility is 10-4 cm2/volt sec, both for semi- and photoconductivity. The quantum efficiency for carrier production is about 100% in (PC + CH) and less than 10% in PC alone. The increased conductivity endowed by CH was ascribed mainly to the enhanced lifetime of the carriers, recombination of electrons and holes being prevented by the trapping action of CH and CH-.

Another system of this sort, a sandwich made of Chi and carotene, has been studied by Arnold and Maclay (1959). In this system electrons are moved from carotene to Chi in the light. Both components are photoconductors, but there was no evidence for an enormous change in conductivity when the two components were placed in contact with each other. The kinetics of light-induced polarization of the sandwich, and of photoconductivity, showed the participation of electron traps. In the photoconductivity of Chi, the charge carriers are holes (quoted by Livingston, 1960).

Arnold and collaborators have made extensive studies of photocon-ductivity, semiconductivity, delayed luminescence, and thermolumines-cence in chloroplasts and algae. These studies, made with living and dried plant materials, will be reported in Section 5.5.


4.2 Photochemistry of Chlorophyll in Solution

The study of the photochemistry of Chi in solution has established two important points. Chi can sensitize a variety of photochemical elec-tron transfer reactions. These reactions in all probability involve Chi in an excited triplet state.

Photochemical reactions involving Chi in organic solvents can be categorized as follows [see Livingston (1960) for a review of his own work and that of others, notably A. A. Krasnovsky] :

1. Photooxidations in which 0 2 is the ultimate electron acceptor. A wide range of substrates, including alcohols, hydrocarbons, and Chi it-self, can be oxidized. These reactions probably involve the Chi triplet magnetically coupled to 0 2 as a reactive oxidant.

2. Photoreduction of Chi, with ascorbic acid, H2S, or phenylhydrazine as electron donor. The final product is a pink, reduced Chi with an absorption maximum at 523 ηΐμ.

3. Electron transfer reactions in which ascorbic acid, H2S, and phenylhydrazine can serve as electron donors and reduced Chi is an intermediate. Suitable electron acceptors are azo dyes (such as methyl red), o-dinitrobenzene, riboflavin, DPN, and TPN.

4. Isomerization of poly-cis carotenes (Claes and Nakayama, 1959). The first of these four reaction-types can be deleterious (Griffiths

et ah, 1955) ; photooxidative killing and Chi destruction occur in plants that lack colored carotenoids.6 The second and third reactions may have importance for the mechanism of photosynthesis.

In general, the photoreductions involving Chi do not occur in non-polar solvents; they proceed well in alcohols or in pyridine containing some H 20. This is probably related to the fact (Livingston, 1960) that Chi forms 1:1 addition compounds with generalized bases such as alcohols and H 2 0. An exception is the photoreduction of Chi by phenyl-hydrazine, which occurs in toluene or ether (Bannister, 1959). I t was mentioned earlier that a trace of water shifts the ππ* and ηπ* levels of Chi in benzene, and changes dramatically the yields of fluorescence and triplet excitation (in dry benzene the triplet yield is reduced five-fold and fluorescence is abolished). Although the effect of H 2 0 and other polar molecules on the photochemistry of Chi is poorly understood, it is safe to say that Chi exposed to H 2 0 is photochemically reactive, while Chi in a dry nonpolar environment is relatively inert.

I t has not been rigorously proven, but it is extremely likely that the eThe influence of carotenoids on the photochemistry of Chi will be described

later in this section, and the role of carotenoids in vivo will be discussed in Section 5.4.


photochemical reactions of Chi in vitro proceed through its lowest (ΤΓΤΓ*) triplet excited state. Considerations (see Livingston and Pugh, 1959) of photochemical yields and collision intervals in solution, and of reaction kinetics, show that the photooxidations and reductions involve a long-lived excited state of Chi: much longer than the singlet lifetime, which is around 10~8 sec. A state of several milliseconds' lifetime is pro-duced in high yield in illuminated Chi in solution. This metastable state has an absorption spectrum to be expected for transitions from the lowest excited triplet state (Linschitz and Sarkanen, 1958). The meta-stable state is quenched strongly by 0 2 (Livingston, 1960) ; this indi-cates its triplet (paramagnetic) character. I t is also quenched efficiently by carotenoids, and by ascorbic acid in wet (but not in dry) pyridine. Carotenoids not only quench the long-lived (presumably triplet) excited state of Chi; they also interfere with photooxidations, reductions, and isomerizations sensitized by Chi (Claes and Nakayama, 1959). Effective interference occurs if the carotenoid contains a conjugated system of at least seven double bonds. The carotenoid appears to compete with 0 2 and electron donors for interaction with Chi in its triplet state.

Although the importance of the triplet state in photochemistry seems well established, a word of caution may be appropriate: The possibility is just beginning to be explored that the highly polarized ηπ* singlet states of organic molecules may be extremely reactive as electron transfer agents (Kasha, 1960b). Comparatively little is known about the yields and lifetimes of these states.

5. Evidence for States and Reactions of Chlorophyll In Vivo

5.1 General Remarks

On the whole, the properties of Chi as predicted theoretically and as observed in vitro are reflected not too strikingly in the behavior of Chi in living cells. In the photochemistry of Chi in solution, for example, the triplet state and reduced Chi play conspicuous roles. But the reac-tions observed in plants and photosynthetic bacteria suggest that a specialized form of Chi becomes oxidized in the light. Furthermore, the evidence for the triplet state in vivo is meager (see Section 5.4). A happier situation prevails with regard to the transfer of excitation energy. Conceived theoretically and demonstrated in vitro by experi-ments showing sensitized fluorescence, this process has provided an acceptable basis for the functioning of a photosynthetic unit having a reaction center. As we shall see, there is evidence that energy transfer does proceed effectively in photosynthesizing cells.


5.2 Evidence for the Transfer of Excitation Energy In Vivo

The primary excited state of Chi in vivo has a lifetime, as measured directly by Brody and Rabinowitch (1957), of 1.6 X 10-9 sec. Energy transfer in molecular aggregates proceeds as rapidly as 1016 transfers/sec, so as many as 107 transfers could occur during the excited state life-time of Chi. Because the absorption spectrum of Chi in vivo is similar to that in vitro, it has been argued (Franck and Teller, 1938) that a very close coupling of Chi molecules does not prevail and that the transfer rate must be less than the frequency of nuclear vibrations (1012 to 1013

per sec). On that basis only 103 to 104 transfers could occur during the excited-state lifetime ; this is barely enough to ensure that the excita-tion will reach a reaction center in a unit of 300 molecules. This restric-tive argument is not too compelling, as there is considerable uncertainty about the nature and degree of spectral shifts that accompany close electronic coupling (see Section 3.3).

Heterogeneous energy transfer, from accessory pigments (phyco-bilins and carotenoids) to Chi and BChl in living cells, has been estab-lished beyond question (Emerson and Lewis, 1942; Arnold and Oppen-heimer, 1950; Dutton and Manning, 1941). Light absorbed by accessory pigments promotes photosynthesis and Chi fluorescence; the efficiency of energy transfer ranges from <20% to >90% (Duysens, 1952; Rabinowitch and Govindjee, 1961; Clayton, 1962d; see also Chapter 7).

Homogeneous energy transfer, among Chi molecules and finally to a trace constituent in the Chi system, is shown most convincingly by the high quantum efficiencies of light-induced reactions of these minor con-stituents. The P700 of green plants, which comprises about 0.3% of the total Chi, is oxidized with an efficiency of one molecule per not more than 5 quanta of light absorbed at 680-700 τημ by the "bulk" Chi (Kok and Gott, 1960). The primary light-reacting cytochrome in Chro-matium is present to the extent of about three molecules per 100 BChl molecules. Even at 77°K, the absorption of light by BChl causes oxida-tion of this cytochrome, in whole cells, with an efficiency approaching one electron transfer per quantum (Chance and Nishimura, 1960; J. M. Olson, 1962). The BChl2 of photosynthetic bacteria, comprising about 3% of the total BChl, is altered (apparently oxidized) when the major component of BChl absorbs light. The quantum efficiency of this reaction in chromatophores is estimated conservatively to be about 0.3, even at 1°K (Clayton, 1962c).

These findings leave little doubt that energy absorbed in a Chi unit is transferred efficiently to a reaction center in photosynthetic tissues. The occurrence of this transfer at extremely low temperatures


probably rules out the transfer of energy via excited triplet states (M. Kasha, verbal communication).

A final piece of evidence for homogeneous energy transfer in vivo is that when Chi in Chlorella is excited with polarized light, the fluorescent light is depolarized (Arnold and Meek, 1956). The degree of depolarization is comparable to that with 0.1 M Chl in viscous solution.

5.3 The State of Aggregation of Chlorophyll and Bacteriochlorophyll In Vivo

From investigations of the structure and optical properties (dichroism and birefringence dispersion) of chloroplasts it appears that Chl exists in vivo as a two-dimensional aggregate, probably a monolayer sand-wiched between protein and lipid layers (Goedheer, 1957; Granick, 1957; Thomas et al, 1957).

After an exhaustive physicochemical investigation of bacterial chromatophores, Bergeron and Fuller (1961) have concluded that BChl in vivo also forms a monolayer between protein and lipid layers.

I t was mentioned earlier (see Section 2.3 for references) that Chl a occurs in vivo in two (and perhaps more) forms associated with two photochemical systems. The shorter-wave form, Chl a670, is one of the components of "System I I " (Fig. 3) ; far-red Chl a is identified with System I. Excitation energy in phycobilins is transferred preferentially to Chl a67o> whereas the transfer of energy from carotenoids is mainly to far-red Chl a. These properties suggest that far-red Chl a is in a lipoidal environment, protected from water, and Chl a670 is in a position exposed to water. The two forms are then analogous to Chl a in dry and in wet benzene, and the properties of the in vitro system (relative ηπ* and ττπ* levels, triplet yield, and photochemical reactivity) become of potential interest for photosynthesis. Thus Franck (1958) has proposed a theory (prior to the one mentioned at the end of Section 2.3) in which singlet and triplet states cooperate in a two-quantum photochemical reaction. The protected and exposed chlorophylls, absorb-ing light, contribute different proportions of triplet and singlet excitation ; this provides a basis for understanding the Emerson red drop and en-hancement effects. According to this theory the exposed (photochemically active) Chl is a lesser fraction that acts as a sink for excitation energy absorbed in the protected Chl. This is in accord with the conclusion that the 7Γ7Γ* singlet state is of lower energy in wet benzene (corresponding to exposed Chl) than in dry benzene. But this relation in benzene is just the opposite of what is found in vivo, where the far-red Chl a, having the lower energy level, is the form associated with a lipoidal (dry) environment.


If Chi exists in viva as a two-dimensional aggregate, one may ask whether the aggregation is orderly (crystal-like) or amorphous. To this end investigators have compared absorption spectra of natural and arti-ficial Chi aggregates. For Chi a in dilute solution the main red absorp-tion band is located at 660 to 666 m/x, depending on the solvent. Layers of Chi a prepared at water-air interfaces and then dried are of two kinds. Monolayers described by Jacobs et al. (1954) as amorphous show the red band at 675 ηΐμ, in good correspondence with the location of this band in vivo. Thicker layers (two molecules or more in thickness) described as crystalline absorb at 735 νημ, as do microcrystals precipi-tated from solution. Colmano (1962) has deposited Chi a, Chi b, and carotene layers on water surfaces and measured their spectra without drying them; with suitable proportions of the three substances the spectra correspond closely to the in vivo spectrum of Chlorella. I t can only be concluded from these investigations that Chi exists in a highly con-densed state in vivo; inferences as to the orderliness of the arrange-ment seem premature.

Recent experiments by R. A. Olson et al. (1961) have shown that in chloroplasts the molecules absorbing at about 705 τημ (probably P700) are oriented with respect to the entire chloroplast, whereas the bulk of the Chi a molecules are not. Chloroplasts were exposed to un-polarized exciting light; the fluorescence bands at about 690 τημ (from Chi a) and 720 πΐμ (from the 705 τημ pigment; see also Brody, 1958) were examined separately. The 720 πΐμ fluorescence was found to be strongly polarized while the 690 ηΐμ, fluorescence was not. Moreover the 705 πΐμ pigment, but not the "bulk" Chi a, exhibits strong dichroism (R. A. Olson et al., 1962). These findings are of special interest in connection with the idea that P700 is part of a photochemical reaction center.

Evidence for energy transfer in vivo was presented in the last sec-tion. Chi a in a condensed state is nonfluorescent, implying that energy is transferred efficiently to vibrational sinks where it is dissipated in radiationless de-excitations. The occurrence of fluorescence in vivo (with a yield of 2.4% according to the data of Latimer et al., 1956) then shows the absence of adventitious (parasitic) energy sinks in the living system.

The absorption bands of BChl occur in vivo at 800, 850, and 870^-890 ναμ (cf. 770 ταμ for BChl in methanol or ether) ; crystals and solid films of BChl show bands in the range 800-900 πΐμ (Jacobs et al., 1954). The band at 850 τημ in vivo is correlated with the presence of colored carotenoids (Griffiths et al., 1955), but it is not known whether this represents a direct interaction between BChl and carotenoid molecules


or a secondary structural consequence of the presence of carotenoids. In Rhodopseudomonas sphéroïdes the BChl bands are at 800, 850, and 870 ιπμ (a carotenoidless mutant lacks the 850 τπμ band). Chromatophores of this organism can be broken, by exposure to deoxycholate, into two kinds of subunits. One contains the 850 m/x-absorbing component and the other the 870 πΐμ component. Both fractions retain carotenoids and both have an absorption band at 800 τημ. From these observations (Clayton, 1962b), and also those of Bril (1958), it appears that the 850 and 870 m/A bands represent two different states of aggregation in different parts of the chromatophore. The 800 ιημ absorption may arise from an addi-tional transition of the BChl molecules that is common to both states of aggregation.

BChl in vivo is probably protected from contact with water, as the absorption spectrum of R. sphéroïdes chromatophores is the same whether they are suspended in water, dried as films on glass plates, or subjected to exhaustive dessication (R. K. Clayton, unpublished observations).

5.4 Primary Photochemical Reactions In Vivo

Photochemical reactions in plants and photosynthetic bacteria are generally classified as "primary" if they involve some form of Chi or if they occur at or near the temperature of liquid N2 (77°K). They are observed as spectrophotometric manifestations and identified, as far as possible, by comparisons with spectral and oxidation-reduction properties of known pigments. Identifiable reactions in this category, caused by illumination of photosynthetic tissues, are the following:

1. Bleaching and red-shift of the absorption bands of carotenoids (Chance and Smith, 1955).

2. Reduction of Chi, identified as such on the basis of a band that appears at 520 τημ (Coleman and Rabinowitch, 1959; Witt and Moraw, 1959).

3. Oxidation of P700 in plants and of BChl2 in bacteria, characterized mainly by bleaching of the long-wave absorption band (Kok, 1961; Clayton, 1962b).

4. Oxidations of cytochromes subsequent to the oxidation of P700 or BChl2 (Witt et al, 1961; Clayton, 1962d).

The carotenoid reaction occurs reversibly in bacterial chromatophores at 1°K (Arnold and Clayton, 1960). In intact cells its onset is as rapid as the onset of BChl2 and cytochrome reactions (Smith et al., 1960). In spite of these "primary" attributes, the reactions of carotenoids can-not be essential to the photosynthetic mechanism, at least in bacteria. Carotenoidless mutants of R. sphéroïdes, lacking even the colorless poly-ene precursors of carotenoids, grow photosynthetically as vigorously as


the normally pigmented parent strain as long as 0 2 is not present (Clayton and Smith, 1960).

Carotenoids do protect cells against photooxidative injury in the presence of light and 02, and there is evidence that this protection results from the ability of carotenoids to quench the excited triplet state of Chi (Section 4.2). Thus the protective action of carotenoids may be construed as signifying the occurrence of some triplet excitation in vivo.

A more convincing case for triplet excitation in vivo (Witt et al., 1960) can be built on the reaction (light-induced absorption increase at 520 m/x and decrease at 430 τημ) that suggests the formation of reduced Chi. This reaction is linked with System I I of Fig. 3 (or Chln in Fig. 4) ; it occurs at — 150°C. It is inhibited by paramagnetic gases (02 and NO) ; these gases quench the excited triplet state of Chi in vitro. The inference is that the "520 m/x" reaction signals formation of reduced Chi via the excited triplet state, in analogy to the photoreduction of Chi in vitro. In support of this identification is the finding that structurally deranged chloroplasts (treated with digitonin or acetone) exhibit, instead of the 520 m/x reaction, a spectral change that resembles the in vitro Chi triplet spectrum. The spectrum of the Chi triplet state has not been observed in "normal" (undenatured) plant tissues (J. L. Rosenberg et al., 1957) ; it can of course be argued that the triplet state is a precursor of all primary electron transfer acts in photosynthesis, but that its rapid utilization precludes the possibility of observing it (Kasha, 1959).

The 520 τημ reaction in vivo is obscured by a different spectral change, an absorption increase at 515 τημ, that is caused by oxygénation as well as by light (Spruit, 1956). Neither the 515 nor the 520 ηΐμ reaction has been identified with a photochemical reaction center. Both are distinct from the light-induced oxidation of P700, which is associated with the far-red system in green plants and algae.

The reactions of P700, BChl2, and associated cytochromes have been discussed in Sections 2.4 and 5.2. To recapitulate briefly, these pigments appear to be specialized Chi and BChl types that serve as reaction centers in photosynthetic units. In this view, excitation energy promotes the transfer of an electron from P700 (or from BChl2) to an unidentified acceptor. Subsequently an electron is transferred from Cyt f to P700, or from a bacterial cytochrome to BChl2. As a result, an exciton in the Chi unit, acting through P700 or BChl2 as an electron transferring agent, affords the formation of oxidized cytochrome and reduced acceptor. Light-induced absorbancy changes in bacterial chromatophores (Clayton, 1962b) and in chloroplasts (Klingenberg et al., 1962) suggest that the acceptor might be a quinone. Coenzyme Q7 in Chromatium, and Q9 in


Rhodospinllum, are major components of chromatophores, there being some 20 molecules of the quinone for every 100 BChl molecules (Fuller et al, 1961). In plants the quinone/Chl ratio is 10 to 20% (Bishop, 1959).

Light-induced ESR signals have been observed in plants and photo-synthetic bacteria by several investigators (Commoner et al., 1956; Sogo et al, 1957; Calvin, 1959b; Allen et al., 1961) ; their correlation with specific photochemical reactions is just beginning to be successful. Beinert et al. (1962) have shown convincingly that a light-induced signal of g value 2.0025, band width about 8 gauss, and no hyperfine structure is associated with the oxidation of P700. The signal is irreversible at —150°C, whereas in Rhodospirillum rubrum the light-induced ESR signal is reversible (Calvin, 1959b). These observations are in harmony with an outstanding difference between the light-induced oxidations of P700 and BChl2: The former is irreversible at — 150°C; the latter is reversible at temperatures down to 1°K (cf. Witt et al., 1961; Arnold and Clayton, 1960; and Clayton, 1962b). The difference in reversibility of the two systems suggests differences in the mechanism by which the acceptor molecule (quinone ?) receives and holds electrons.

Another difference between the plant and bacterial systems has to do with the size of the photochemical unit. There are about 400 Chi molecules for every molecule of P700 in chloroplasts (Kok, 1961), but only about 40 BChl molecules for every BChl2 molecule in chromato-phores (Clayton, 1962b). These ratios are in striking agreement with the size of the photosynthetic unit in plants and in bacteria, as determined from the maximum yield of a brief flash of light (Section 2.2).

To sum up, there is ample evidence that P700 and BChl2 act as electron-transfer agents in the reaction centers of photosynthetic units, but this is of course only a partial description of the primary events in photosynthesis. Reactions associated with the shorter wave System II and with 02 evolution must be considered. Especially one should keep in mind the cogent arguments of Gaffron (Section 2.3) to the effect that every primary photochemical reaction, in photosynthetic bacteria as well as in green plants and algae, is a 2-quantum process that involves the splitting of a water molecule. Details of such a 2-quantum process have not yet been formulated in such a way as to account for the observed primary reactions. But it can readily be imagined (Tollin, 1962) that the observed one-electron reaction of P700 (or of BChl2) is only part of a coordinated 2-quantum event.

5.5 Evidence for Photoionization and Conduction In Vivo

There remains to be described the evidence that photosynthetic tissues behave like organic semiconductors. This evidence has been


assembled mainly by Arnold and collaborators; its application to theories of photosynthesis has been championed in recent years by Calvin (1959a, 1961).

1. The electrical resistance of dried chloroplasts varies with temper-ature in the manner typical of an intrinsic semiconductor (Arnold and Sherwood, 1957). But the energy gap for this semiconduction is 2.1 ev (cf. 1.44 ev for crystalline Chi).

2. Dried chloroplasts and dried bacterial chromatophores are photo-conductors (Arnold and Maclay, 1959; Arnold and Clayton, 1960). The sensitizing pigment is Chi or BChl.

3. Dried films of chromatophores show a light-induced increase in their dielectric constant, signifying that light has set free electric charge that can be polarized by a weak potential gradient (Arnold and Clayton, 1960). This is taken as evidence for photoionization in the chromato-phores. The effect decays in darkness with roughly the same kinetics as the BChl2 reaction as observed spectrophotometrically.

4. Dried chloroplasts exhibit thermoluminescence after they have been illuminated (Arnold and Sherwood, 1957). Analysis of "glow curves" obtained by heating these specimens shows a multiplicity of activation energies.

5. All kinds of photosynthetic tissues emit delayed light (Strehler and Arnold, 1951; Tollin et al., 1958a,b). The kinetics of this light re-emission again shows a distribution of activation energies (Arnold, 1957). Delayed light and thermoluminescent light have the same spec-trum as fluorescent light from the same material. These luminescences are best interpreted as resulting from the untrapping of trapped electrons in the Chi unit. The trapping is much attenuated at 77°K (Arnold and Sherwood, 1957).

6. The kinetics of the light-reaction (oxidation) of BChl2 in dried chromatophores (Arnold and Clayton, 1960) also suggest the involve-ment of electron trapping that does not occur at low temperatures: Around room temperature the decay after illumination is predominantly first-order and slow (several seconds' lifetime), becoming slower as the temperature is lowered. But below about 150°K the decay abruptly becomes second-order, rapid (about 50 msec lifetime), and independent of temperature down to 1°K.7

Of the six phenomena in this list, only the BChl2 reaction has been shown to proceed with high quantum efficiency (Clayton, 1962c). I t appears unlikely, therefore, that photoionization and conduction are major events in the whole Chi unit. The channeling of energy to a reaction center probably occurs by migration of excitation energy and

7 The resemblance of this kinetic pattern to that of the light-induced ESR signal in Rhodospinllum rubrum (Calvin, 1959b) is most suggestive.


not of separated electrons and holes. But the events at the reaction center may well involve ionization and electron trapping in a crystalline system.

6. Evaluation and Outlook The reader may now appreciate why this chapter has been written

in a hesitant fashion characterized by the liberal use of such words as "probably" and "apparently." A great wealth of theoretical material provided by the quantum chemists, plus a fascinating variety of experi-mental observations, has engendered a proliferation of detailed hy-potheses for the mechanism of photosynthesis (see Tollin, 1962). Truly definitive experiments continue to be the rarest commodity in this complicated field. One result is that almost any point of view finds enough support to warrant its further exploitation; another is that the most popular models are open to serious criticism.

The most profound bifurcation is found in the conflict between "1-quantum, one-electron transfer" schemes, exemplified by Fig. 3, and models in which 2 quanta cooperate in a concerted photochemical event that splits water. These two approaches lead to entirely different interpre-tations of the Emerson red drop and enhancement phenomena (see Section 2.3; also Tollin, 1962) and of the relation between bacterial and plant photosynthesis.

The 2-quantum schemes are upheld by convincing arguments but suffer in that detailed formulations are not supported compellingly by direct spectrophotometric observations. The primary events that have been observed directly (reactions of P700, BChl2, and cytochromes) are interpreted simply and plausibly as 1-quantum, one-electron reac-tions. Whether these are the only primary reactions remains open to question (consider for example the apparent formation of reduced Chi), but they are certainly high-yield processes reflecting the transfer of excitation energy to a reaction center.

The mechanisms of these primary reactions remain to be established. The conversion of an exciton into a state in which charge is separated might involve as an intermediate the long-lived triplet state or the highly polarized η-π* singlet state of Chi (e.g., of P700 or BChl2). Elec-tron transfer might involve an orderly charge transfer state between excited Chi and an acceptor, followed by the transfer of an electron from cytochrome to oxidized Chi. Alternatively the mechanism sug-gested by the phthalocyanine-chloranil system (Section 4.1) might pre-vail. In a literal use of this model, an ensemble of Chi molecules is adjacent to a layer of quinone molecules (recall that in chromatophores there is one quinone molecule for every five BChl molecules). The


quinone provides electron affinity sufficient to dissociate excitons, leaving conducting holes in the Chi layer. These find their way to cytochrome molecules.

With its imputed high reactivity and relatively long lifetime, the η,π* singlet state is an intriguing candidate for important roles in photo-chemistry. A reaction between 7i7r*-Chl and 02 has already been visual-ized by Franck as the basis of the Emerson phenomena; further con-ceptual use of this state in mediating photosynthetic electron transfer can be anticipated. On the observational side many exciting experi-ments suggest themselves. The quantum yield of P700 fluorescence, using exciting light absorbed by Chi a or accessory pigments, ought to be measured. Also the relative yields of fluorescence from Chi a and P700 should be determined as functions of wavelength and intensity of the exciting light. It should be possible to establish the identity of electron acceptors associated with P700 and BChl2 through careful observation of absorption spectrum changes induced by light and by chemicals; a beginning in this direction has been made (Clayton, 1962b; Klingenberg et al., 1962). The whole area of flashing-light experiments and photo-synthetic units should be investigated in terms of the Emerson phe-nomena, using flashes or pairs of flashes designed to excite the far-red and/or the shorter-wave system.

There is much to be done, both by theoreticians and experimentalists. The former continue to provide exciting ideas as to what is possible, and the latter continue to press the former into a re-evaluation of these ideas.

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1542-1550. Losada, M., Whatley, F. R., and Arnon, D. I. (1961). Nature 190, 606-610. McClure, D. S. (1960). Radiation Research Suppl. 2, 218-242. McGlynn, S. P. (1960). Radiation Research Suppl. 2, 300-323. McRae, E. G., and Kasha, M. (1958). J. Chem. Phys. ,28, 721-722. Myers, J., and French, C. S. (1960). Plant Physiol. 35, 963-969. Nelson, R. C. (1962). In "Progress in Photobiology" (B. C. Christensen and

B. Buchmann, eds.), pp. 266-270. Elsevier, Amsterdam. Olson, J. M. (1962). Science 135, 101. Olson, J. M., and Chance, B. (1960). Arch. Biochem. Biophys. 88, 26-39. Olson, R. A., Butler, W. L., and Jennings, W. H. (1961). Biochim. et Biophys. Acta

54, 615-617. Olson, R. A., Butler, W. L., and Jennings, W. H. (1962). Biochim. et Biophys. Acta

58, 144-146. Platt, J. R. (1956). In "Radiation Biology" (A. Hollaender, ed.), Vol. I l l , pp. 71-

123. McGraw-Hill, New York. Rabinowitch, E. (1956). "Photosynthesis and Related Processes" Vol. II, Part 2.

Interscience, New York. Rabinowitch, E. (1959). Plant Physiol. 34, 213-218. Rabinowitch, E., and Govindjee (1961). In "Light and Life" (W. D. McElroy and

B. Glass, eds.), pp. 378-386. Johns Hopkins Press, Baltimore, Maryland. Rice, F. O., and Teller, E. (1949). "The Structure of Matter," pp. 240-242. Wiley,

New York. Rieke, F. F. (1949). In "Photosynthesis in Plants" (J. Franck and F. W. Loomis,

eds.), pp. 251-272. Iowa State College Press, Ames, Iowa. Rosenberg, B. (1958). / . Chem. Phys. 29, 1108-1118. Rosenberg, J. L., Takashima, S., and Lumry, R. (1957). In "Research in Photo-

synthesis" (H. Gaffron et al, eds.), pp. 85-88. Interscience, New York. San Pietro, A., and Lang, H. M. (1958). / . Biol. Chem. 231, 211-229. Simpson, W. T., and Peterson, D. L. (1957). J. Chem. Phys. 26, 588-593. Smith, L., Baltscheffsky, M., and Olson, J. M. (1960). / . Biol. Chem. 235, 213-218. Sogo, P. B., Pon, N. G., and Calvin, M. (1957). Proc. Natl. Acad. Sei. U. S. 43,

387-393. Spruit, C. J. P. (1956). Rec. trav. chim. 75, 1097-1100. Stanier, R. Y. (1961). Bacteriol. Revs. 25, 1-17. Strehler, B. L., and Arnold, W. A. (1951). J. Gen. Physiol. 34, 809-820. Suzuki, I., and Werkman, C. H. (1958). Arch. Biochem. Biophys. 77, 112-123. Terenin, A. N., and Ermolaev, V. L. (1956). Trans. Faraday Soc. 52, 1042-1052. Thomas, J. B., Blaauw, O. H., and Duysens, L. N. M. (1953). Biochim. et Biophys.

Acta 10, 230-240. Thomas, J. B., Minnaert, K., and Elbers, P. F. (1957). Acta Botan. Neerl. 6,

345-350. Tollin, G. (1960). Radiation Research Suppl. 2, 387-406. Tollin, G. (1962). / . Theoret. Biol. 2, 105-116. Tollin, G., Fujimori, E., and Calvin, M. (1958a). Nature 181, 1266-1267. Tollin, G., Fujimori, E., and Calvin, M. (1958b). Ann. N. Y. Ahad. Sei. 74, 310-328. van Niel, C. B. (1935). Cold Spring Harbor Symposia Quant. Biol. 3, 138-150. van Niel, C. B. (1941). Advances in Enzymol. 1, 263-328.


van Niel, C. B. (1949). In "Photosynthesis in Plants" (J. Franck and F. W. Loomis, eds.), pp. 437-496. Iowa State College Press, Ames, Iowa.

Vishniac, W., and Ochoa, S. (1953). / . Biol. Chem. 195, 75-93. Witt, H. T., and Moraw, R. (1959). Z. physik. Chem. (Frankjurt) [N.S.] 20, 253-

282 and 283-298. Witt, H. T., and Müller, A. (1959). Z. physik. Chem. (Frankfurt) [N.S.] 21, 1-23. Witt, H. T., Moraw, R., Müller, A., Rumberg, B., and Zieger, G. (1960). Z. physik.

Chem. (Frankfurt) [N.S.] 23, 133-138. Witt, H. T., Müller, A., and Rumberg, B. (1961). Nature 192, 967-969.

Chapter 7

ACCESSORY PIGMENTS AND PHOTOSYNTHESIS

L. R. Blinks

Hopkins Marine Station, Stanford University Pacific Grove, California

Chlorophyll has been traditionally considered the essential photo-synthetic pigment, since all photosynthetic organisms contain at least one form of it: all algae and higher plants have chlorophyll a, (appar-ently without exception) and bacteria have one or another special bacterial chlorophyll. But, almost equally invariably, there are other pigments present in the photosynthetic apparatus (plastids or chromato-phores)—quite aside from incidental and possibly insignificant pigments in oil drops, elaeoplasts, vacuoles, and other nonphotosynthetic organ-elles. Thus ^-carotene is found in all plants, and certain other caro-tenoids in bacteria (Strain, 1938, 1949, 1958).

Chlorophyll has quite properly attracted the attention of many physiologists, biochemists, and physicists, and great strides have recently been made in understanding its role in photosynthesis (as summarized in Chapters 5 and 6). But fewer workers have investigated the so-called "accessory pigments" (see Haxo, 1960a) ; these are considered in the pres-ent chapter.

1. Chemical and Physicochemical Nature of Accessory Pigments

The chief and probably the only pigments or biochromes now clearly implicated as photosynthetic accessories are (1) carotenoids (including carotene itself), and (2) phycobilins. The former are widespread in all plants and bacteria; the latter are restricted, as the name implies, to algae. These pigments differ markedly in both their chemistry and their light absorption.

1.1 Carotenoids

These substances are fat soluble. /^-Carotene has the formula: 199

200 L. R. BLINKS

CH3 ? H CH, CH3 ÇH3H3C CH3

^CC=CC=CC=CC=CC=CC=CC=CC=CC=CC, CH2

3

H2 3 H 2

The carotenoids (or xanthophylls) display varying degrees of oxidation of this unsaturated polyene skeleton. Most of these compounds have well-known structures; though curiously enough fucoxanthin (C4oH5606), one of the most important carotenoids in the photosynthesis of both fresh-water and marine brown algae (diatoms and kelps) is not well-characterized (see Karrer and Jucker, 1950). The absorption spectrum of fucoxanthin is most strongly influenced by conjugation with protein; as with some animal carotenoids such as the astaxanthin of lobsters, it changes considerably on heating or extraction with methanol. This con-jugation may well have a bearing upon its very active participation in photosynthesis (see below). Many special carotenoids are found in photo-synthetic bacteria; spirilloxanthin and rhodopsin are examples. Most carotenoids absorb at 400 to 500 m//,.

1.2 Phycobilins

These substances are water-soluble protein pigments whose chromo-phore has the fundamental structure of the animal bile pigments, varied in considerable degree by substituted side chains. For example, phycoerythrobilin (derived from phycoerythrin) shows the following characteristics (Lemberg, 1949) :

M E M P P M M E

H H2 H H H

Where M is methyl, E ethyl and P propyl

It may be noted, that (as in the carotenoids), there is no metal in the chromophore. (Calcium, sometimes reported as present, is probably attached to the associated protein.) The conjugation link to the protein (contrary to carotenoid bonding) is extremely strong, the chromophore being hydrolyzed from its protein only by drastic reagents such as hot HC1. Several types of phycobilins are known, with varying absorption spectra; R-phycoerythrin from the Florideae (higher red algae) is per-haps the most abundant, with absorption maxima at 495, 540 and 565 πΐμ. B-Phycoerythrin, from the rather primitive genus Smithora (Bang-iales) lacks the maximum at 495 ηΐμ, while C-phycoerythrin, from

7. ACCESSORY PIGMENTS AND PHOTOSYNTHESIS 201

Cyanophyceae has only a single maximum at about 550 τημ. Jones and Fujimori (1961) recently showed that the height of the maxima in a typical R-phycoerythrin is subject to considerable variation by agents (heat, urea, oxidants, reductants, enzymes, metals, etc.) which affect the protein or its linkage to the chromophore. Although no such change of absorption has been reported in vivo under physiological or light control, this effect may well account for the differences that often occur between species.

The protein complex has a molecular weight of about 300,000 (de-pending on the source) and an isoelectric point of about pH 4.7 (often crystallizing or precipitating at lower values). The complex is very stable, and fluoresces a brilliant yellow in solution. When crystallized it chromatographs uniformly on calcium phosphate. Freshly extracted solu-tions, however, often show several bands, not only of phycoerythrin, but also of phycocyanin, the uppermost of which seem to be highly polar and elute only in rather basic solution. These fractions display very rapid electrophoresis as anions, with an isoelectric point at pH 1 or lower. Only on standing for several days, usually in the presence of other cell mate-rial, does the normal isoelectric point appear. Whether this alteration is due to the hydrolysis of phosphate (or possibly sulfate), is not yet known, nor is its physiological significance clear (Airth and Blinks, 1957).

In addition to the C-phycocyanin of the blue-green algae, with an absorption maximum around 620 m/x, there is a red algal R-phycocyanin with a second peak at 550 m/x, and an allophycocyanin with peak at 650 m/x, overlapping well into the chlorophyll absorption region (for curves of all these, see 0 hEocha, 1960).

I t is apparent from the chemistry of the chief accessory pigments that it would be desirable to know more about the topography of these substances, their spatial arrangement in the plastid or chromatophore, and particularly their linkage with chlorophyll. But aside from specula-tion, nothing can really be said about this important matter, even after twenty years of electron microscopy.

2. The Role of the Accessory Pigments

How do the accessory pigments work? Information on their mode of action has been sought from several types of study:

1. Primarily, action spectra, the principles of which have already been discussed in Chapter 4.

2. Quantum efficiency determination, which is somewhat more pre-cise and meaningful. The first type of study is usually relative, the second absolute.

202 L. R. BLINKS

3. Indications of energy transfer—usually by fluorescence. Here may also be included the "after light" (see Arnold and Thompson, 1956) though its mechanism is probably different.

4. The so-called "two-light" phenomenon, producing enhancement or the Second Emerson Effect.

5. "Chromatic transients" (observed on illuminating cells successively with two different wavelengths). These are closely related to the Emer-son Effect.

6. Solarization and protection against photooxidation (often studied in mutants deficient in one or another pigment).

2.1 Action Spectra

The first systematic work of this type was made by that masterly investigator Engelmann (1883, 1884) who employed his ingenious motile bacteria method to indicate the production of oxygen when microspectra were used to illuminate algal strands or filaments. Engelmann confirmed that in the green algae (Fig. la), as expected, most of the oxygen was released at the absorption peaks of chlorophyll. In brown algae (Fig. lb) there was increased activity in the blue-green region toward the middle of the spectrum (in agreement with the presence of fucoxanthin), while in blue-green algae (Fig. lc) there was a corresponding increase of photosynthesis in the orange region (corresponding to the presence of phycocyanin). Most striking of all perhaps was the action spectrum of a red alga (Fig. Id) which showed its highest photosynthetic rate in the middle of the spectrum, the green light being absorbed by the red pig-ment phycoerythrin. Although relatively few points were actually mapped by Engelmann, his method, if photographically recorded, might still lend itself to highly precise determination. His "biological sensing" instrument is probably capable of as good resolution as many of our more sophisticated modern methods.

Indeed, Engelmann's work was not surpassed for half a century, during which several other investigators (Montfort, 1934, 1936, 1940; Schmidt, 1937; Seybold, 1934; Wurmser, 1921) used rather broad spectral regions isolated by glass filters or colored solutions, and measured photosyn-thesis by pH or dissolved oxygen determinations. The best that can be said of such work is that it confirmed Engelmann's general conclusions, though some peculiar results (at higher light intensity) were reported by Montfort, and are still not understood. The first real improvement came with the utilization of a larger number of Jena glass filters by Levring (1947) who was able to plot some 13 points along the spec-trum. Brown and red algae definitely showed a higher photosynthetic rate in the middle of the spectrum than did green algae, although there

7. ACCESSORY PIGMENTS AND PHOTOSYNTHESIS 2 0 3

was also some oxygen evolution (determined by Winkler titration) in the chlorophyll absorption region in all the algae studied by Levring. This investigator has recently discussed these findings, again with special reference to depth (Levring, 1960).

Much more elegant were the quantum efficiency determinations made by Emerson and Lewis (1942, 1943) at a large number of points through-out the visible spectrum. Very narrow spectral regions (isolated by a powerful monochromator), illuminated suspensions of Chlorella and Chroococcus, measurements of gas exchange being made by Warburg manometiy. The quantum efficiency remained at about 0.11 throughout much of the longer wavelength region, but it fell to 0.08 and less in the shorter. This drop in quantum efficiency was interpreted as due to ineffective absorption by some or all of the carotenoids. Most striking of all, although not further investigated at that time, was the extremely low efficiency (0.05 or less) observed in the far end of the chlorophyll absorption (near 700 ταμ). This "red drop" was tentatively ascribed to the low-energy quanta involved, an explanation scarcely in keeping with the good efficiency of infrared at 800 to 880 τημ in photosynthetic bac-teria. Actually this low efficiency at 700 τημ became the clue to a much better understanding of both chlorophyll and accessory pigments, when Emerson later resumed a study of these problems. Emerson and Lewis (1943) were also the first to measure quantum efficiency in the region of phycocyanin absorption; this was shown in the blue-green alga Chro-ococcus to be as great at 620 τημ as at the chlorophyll maximum of 675 ιπμ. (Efficiency fell off, however, in the blue end of the spectrum, just as in Chlorella.)

Concerning the role of accessory pigments, Emerson and Lewis clearly implicated phycocyanin as photosynthetically active, but raised the question as to whether all the carotenoids were active. A little later, and using the same methods, Tanada (1951) worked with the diatom Navicula in which the characteristic brown algal pigment fucoxanthin absorbs farther into the green region of the spectrum than do other carotenoids. This region showed essentially the same efficiency as did red light, indicating that fucoxanthin, at least, is effective in photosyn-thesis. However, the mechanism by which the carotenoid acts was not elucidated, although other work, discussed below, gives an indication of what takes place.

A quite different method for studying the action spectra of photo-synthesis with special reference to accessory pigments was used by Haxo and Blinks (1950). The stationary platinum electrode previously employed for oxygen determination (Blinks and Skow, 1938), was ap-plied directly in contact with very thin thalli of marine algae (some-

204 L. R. BLINKS

100

90

80

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FIG. 1. The first photosynthetic action spectra (done with the motile bacteria method). Open circles: absorption spectra; filled circles: action spectra, (a) Green alga, (b) brown alga, (c) blue-green alga, and (d) red alga. [Reproduced from Engelmann (1883, 1884).] Wavelengths (abscissas) are in ο,μ (τημ χ 10).

times only one-cell thick) and illuminated with narrow spectral bands (10-20 τημ) derived from a grating monochromator. Such thalli have great advantages from the standpoint of both optics and diffusion of


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gases and other solutes; the method, while probably capable of absolute calibration, is mostly used for relative measurements, and to record rapid changes in rate or transients. Haxo (1960b) has recently improved the Blinks and Skow electrode for unicellular algae. Figures 2 and 3 indicate relative action spectra for several green, brown, blue-green, and red algae. The first two algae show very close correspondence between

206 L. R. BLINKS

thallus absorption and photosynthetic rate (when adjusted for equal incident quanta and for reflection from the electrode surface). There is a deviation in the region of 700 τημ, confirming the red drop of Emerson. On the other hand, the carotenoids in these green algae (Ulva, Chlorella) appear to be nearly as active as the chlorophyll; otherwise the close agreement at the blue absorption peak of the latter could scarcely occur, since about half the absorption in that region is due to carotenoids. The deviation at ca. 480 ιημ on the other hand may represent some carotenoid (or perhaps, chlorophyll b) inactivity. Certainly this situation does not represent all green algae, for Ulva at times shows much lower blue efficiency (especially at its reproductive periods), and other green algae (Monostroma, Enteromorpha) may show consistently lower photosyn-thesis in the blue end of the spectrum. This tendency toward inactive carotenoid absorption finds its extreme in some of the "brick red" Chlorophyta such as Dunaliella and Trentepohlia, which are crowded with oil droplets containing carotene. These algae show practically no photosynthesis in the blue end of the spectrum ; sometimes they may even show photooxidation. However, this is to be expected from the topog-raphy of the cell, since the carotene is outside the plastids. But perhaps the plastid itself may at times segregate carotenoids from the photo-synthetic apparatus (e.g., in Ulva during reproduction).

Confirming carotenoid participation in photosynthesis, the dino-flagellate Gonyaulax and the brown alga Coilodesme showed good ac-tivity throughout the spectrum, with close correspondence between absorption and photosynthetic rate (Figs. 2b, 3b). In particular the comparison with green algae is instructive, since these show (like Engel-mann's curve, Fig.lb) increased absorption and photosynthesis toward the middle of the spectrum in the blue-green region (480-540 τημ). This agrees with the fucoxanthin absorption region (in vivo). Although there is some deviation from exact correspondence of the two curves in the blue region, it is evident that there is good activity of other carotenoids as well. All told, the brown algae show the most efficient utilization of light throughout the spectrum, and it is perhaps not surprising that diatoms are predominant as phytoplankton while kelps are the most massive of marine algae. (Sargassum also belongs here.)

The red algae do indeed fill in the absorption gaps more effectively, phycoerythrin nicely complementing both chlorophyll and carotenoid by absorbing in the middle of the spectrum. However, as Figs. 2c, d and 3d show, the overall photosynthetic efficiency is not as good as the absorp-tion might indicate. There is good activity in the green region, but a marked fall away toward the red, and notably near the blue end of the spectrum. Particularly in Figs. 2c and 3d the photosynthetic rate


plunges toward a low level just as the absorption by chlorophyll rises to its peak. In some cases there is a plateau, occasionally a slight "foot-hill" at the chlorophyll absorption maximum (Fig. 2d) ; there is clearly some activity due to this pigment, but perhaps not over a third to a half of the activity due to phycoerythrin (although with about equal absorp-tion). Brody and Emerson (1959) found the efficiency to be fairly high at 644 τημ (where, however, allophycocyanin might be absorbing).

This remarkable result was for some time unexplained, save for the rather obvious conclusion that part of the chlorophyll a is inactive for oxygen evolution. Whether such chlorophyll exists in different form or a different position (e.g. shielded from enzymes) could not be decided; but the red algae clearly show, (even more drastically than does Chlorella) Emerson's "red drop," which occurs here at 650 to 675 m/x instead of at longer wavelengths. Undoubtedly this phenomenon is cor-related with the absence of chlorophyll b in red algae; its degree may depend upon greater amounts of allophycocyanin (absorbing at 650 τημ) in some red algae than in others. Whatever the reason, the red algae show two outstanding characteristics: (1) The relative ineffective-ness of chlorophyll a at its peak of absorption; (i.e., the most con-spicuous "red drop" known), and (2) the high activity of a so-called "accessory pigment" which may be two or three times as great as that of chlorophyll a, at equal absorption.

It should be mentioned that this observation was corroborated by a totally different method, namely volumetric determination of 02 gas produced photosynthetically (Yocum and Blinks, 1954). The quantum efficiencies found in these measurements are summarized in Table I.

TABLE I NUMBER OP ABSORBED QUANTA REQUIRED TO RELEASE ONE MOLECULE OP OXYGEN"

Wavelength (πΐμ)

Algae 436 500 560 620 675

Green Ulva lobata U. lactuca

Brow ii

Ilea fascia Reu

Porphyra Nereocystis P. naiadumh

Delesseria decipiens

14 16

10

37 35 26

18 14

11

16 24 16

23 13

11

14 13-17

16

16 15

11

15 18 20

12 15

10

25 25 32

° After Yocum and Blinks, 1954. * Now known as Smithora naiadum.

208 L. R. BLINKS

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FIG. 2. Absorption spectra and action spectra of several marine algae (thalli). (a) green (Ulva), (b) brown (C oilodesme), (c) red (Porphyra Nereocystis), and (d) red with more phycocyanin (P. perforata). (From Haxo and Blinks, 1950.)

Duysens (1952), using the polarographic method of Haxo and Blinks, found the peak of activity in a blue-green alga (Oscillatoria) to cor-respond with the absorption by phycocyanin (620 πΐμ), with considerably lower photosynthesis in the chlorophyll region. However, Haxo (1960b)


100

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Wavelength (m/*)

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σ

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(d)

FIG. 2 (continued).

has recently reported good chlorophyll activity in another blue-green alga (Phormidium), as shown in Fig. 3c. This activity resembles the high efficiency found in Chroococcus by Emerson and Lewis (1943). Apparently blue-green algae vary.

In both the red and blue-green algae very low efficiency in the blue end of the spectrum is found. While part of this low efficiency no doubt corresponds to the low chlorophyll a activity, it probably also is due to

210 L. R. BLINKS

IOO| I I I I I I I I I

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(a)

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Cell absorption

o—o Action spectrum For photosynthesis

400 500 600 Wavelength (m/x)

(b)

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Fia. 3. Absorption spectra and action spectra of several unicellular algae, (a) Chlorella (green), (b) Gonyaulax (dinoflagellate), (c) Phormidium (blue-green) and (d) Porphyndium (red). (From Haxo, 1960b.)

inactive carotenoid absorption as well as the absence of either chloro-phyll b or c. The chief accessory pigments in both these groups are apparently phycobilins, not carotenoids or other chlorophylls.


100 Phormidium ectocarpi

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Wavelength {π\μ)

(c)

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Porphyridium aerugineum

Cell absorption o—o Action spectrum

Aqueous extract

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Wavelength (πιμ) id)

Fia. 3 (continued).

700

2.2 Mechanisms of Action of Accessory Pigments: Energy Transfer

The action spectra and quantum efficiency findings in red algae just discussed were rather unexpected, and contrary to the accepted notion that chlorophyll was the principal photosynthetic pigment. Conse-

212 L. ß. BLINKS

quently there were early efforts to explain these results by some mech-anism such as a transfer of the energy absorbed by the carotenoids or phycobilins to the presumably essential chlorophyll. The means for detecting this transfer are rather limited, and depend mostly upon ex-citation of specific chlorophyll fluorescence when other pigments are the chief light absorbers. Dutton et al. (1943) early showed that when diatoms were illuminated at the mercury green line (546 τπμ, light absorbed largely by fucoxanthin) the chlorophyll fluorescence was as intense as when red light was absorbed. Very efficient transfer was evi-dently occurring, as was later confirmed by Wassink and Kersten (1946). It remained for Arnold and Oppenheimer (1950) to give a sound physical meaning to such transfer; they considered the following mechanisms: (1) molecular collision, (2) emission and reabsorption and, (3) internal conversion or resonance transfer. These investigators concluded that the latter mechanism was the most probable, since it occurred with an efficiency of nearly 97% in blue-green algae (from phycocyanin to chloro-phyll). This efficiency could hardly result from mechanisms 1 or 2, and depends upon very close packing—of the order of a half wavelength of light. Actually the distance between chlorophyll molecules in the plastid laminations is much less than this—perhaps 50 Â, although the spacing of accessory pigments with relation to the chlorophyll is not known.

However, an anomaly was found by French and Young (1952) in such transfer studies; namely, that chlorophyll fluorescence in red algae was more readily excited by green light (absorbed by phycoerythrin) than by red light (absorbed by chlorophyll itself). In fact, the action spectrum for the excitation corresponded to the absorption spectrum of phycoerythrin, rather than to that of chlorophyll. This is still unex-plained—except perhaps on the basis that two kinds of chlorophyll exist —one which can absorb but not carry out fluorescence (or photochemical work), and another which can. (There is, perhaps, something slightly illogical in equating fluorescence with photochemical activity, since it is evident that any molecule which actually fluoresces can scarcely be accomplishing other photochemical work with the same quantum.) Arnold and Thompson (1956) also found that the production of the "after light" can also be excited by accessory pigments.

However, this and other problems of transfer became less difficult to understand in the light of new developments in the photophysiology of chlorophyll and accessory pigments, which will now be described.

2.3 Photosynthetic Enhancement (the "Emerson Effect")

The "red drop" and the role of accessory pigments in photosynthesis was remarkably clarified about five years ago when Emerson and


Chalmers (1958) reported that concurrent light of shorter wavelength greatly increased the effectiveness of the longer wavelengths. This en-hancement, soon called the "Second Emerson Effect" (the first being the carbon dioxide "gush" at the onset of illumination) has become the subject of intensive research in several laboratories in the succeeding years. Briefly, simultaneous exposure to wavelengths absorbed by other pigments make the poor quantum yield of the far-red (at ca. 700 ηΐμ,) rise to almost "normal" values. The two light sources are thus more than additive, and the enhancement factor E may rise 50 to 100% above predicted simple addition. (Fig. 4). This effect has not only been

100

_c Q) (A I 50 C

400 500 600 700

Wavelength (π\μ)

FIG. 4. Action spectra of photosynthetic enhancement. The supplementary light is added to far-red (690 ιημ). Open circles, red alga (Porphyridium) ; black circles, green alga (Chlorella). (From Haxo, 1960b, after the data of Emerson.)

found in Chlorella, but in brown, blue-green, and red algae in which it is especially striking. Its action spectrum closely resembles the absorption of other pigments. In other words the accessory pigments, when excited along with chlorophyll, raise the activity of the latter in spectacular fashion. In this sense, chlorophylls b and c are to be regarded as "ac-cessory" to chlorophyll a, and it even seems that some forms of chlorophyll a which absorb at shorter wavelengths are accessory to the form (which may be a dimer) absorbing at 700 τημ (French, 1961).

One explanation of the low activity of chlorophyll a based on in-creased respiration has recently been proposed (French and Fork, 1961a). This is best understood in the light of transient effects, the discovery of which slightly antedated that of the enhancement effects; see Blinks, 1957.)

-

-

rw—

1

-J ^-o-

1

- 1 Λ

1 ,<* /

/

f

1

\ 1 \ \ I I I \ \ \ I \ \ \ \ \

1

1 1

1 " " - - - o l

-

-

-

214 L. R. BLINKS

2.4 Chromatic Transients

While induction effects, C02 gushes and "gulps," post-illumination respiration, and fluorescent transients have long been known to occur during the first moments of illumination (or darkness), these phenom-ena had not been related to different pigments until fairly recently, when it was shown (Blinks, 1957, 1959) that the time course of oxygen evolution (as well as of consumption afterward) differed at different wavelengths. Thus, in red algae chlorophyll absorption (red light) gave rise to a slower, asymptotic rise of 02 evolution (Fig. 5) than did

J I I I L. 3 6 9 12 15

Minutes

FIG. 5. Time course of photosynthetic oxygen production by a red alga (Porphyra) in red light (675 τημ) and green light (560 ιαμ).

absorption by phycoerythrin, which was characterized (Fig. 5) by an abrupt rise, to a cusp (a), a subsequent depression (b) and a final recovery to a steady state (c). There were also differences in the sub-sequent dark respiration, as shown.

In a green alga it was suggested (Blinks, 1960b) that the slow rise due to chlorophyll a is accounted for by an increase of respiration during the first moments of illumination, as indicated by dark respiration values after brief illumination (Fig. 6a) whereas in the region of the accessory pigment (here chlorophyll b) this increased respiration was somewhat delayed, giving the cusp a, already described (Fig. 66).

The same time courses prevail, or become even more conspicuous, when two such wavelengths are alternated, instead of being interrupted by dark periods. Figure 7 shows an example of a green alga where photosynthetic rates have been adjusted to be equal at 688 τημ (ab-sorbed by chlorophyll a) and 640 τημ. (largely absorbed by chlorophyll


Fia. 6. Time course of oxygen evolution and subsequent respiration in the dark, by a green alga (Enteromorpha), (a) in far-red light (700 m/i, absorbed by chlorophyll a) and (b) near-red (645 m/i, absorbed by chlorophyll b). The light is interrupted at 15, 30, 45, 60, 90, 120 sec etc., in order to show the respiration after various parts of the transient.

b). The latter shows the same abrupt rise (a) depression (b) and re-covery (c) that characterized the time course with phycoerythrin absorption; conversely, the depression (d) on return to chlorophyll absorption corresponds to the increased respiration which caused the slow initial rise of Fig. 6a. A schema accounting for these curves in terms of the respiration changes was given by Blinks (1960c). A some-what similar interpretation has been offered by French and Fork

216 L. R. BLINKS

640 640

688 688

580 560

FIG. 7. Chromatic transients of oxygen evolution, (a) In a green alga (Ulva) ; (b) in a red alga (Porphyra) on alternating exposures to wavelengths shown; (c)

•both chromatic transients and enhancement in Porphyra.

(1961a), involving greatly increased respiration during chlorophyll absorption.

The transients persist when one wavelength is added to another, given enhancement (Fig. 7) ; here the cusps characteristic of alternation are still seen, while a new depression (e) following enhancement (during the accessory pigment absorption alone) becomes extremely large—in some cases going well toward the base line (dark value) before re-covering. This is probably due to the large respiration following chloro-phyll absorption.

Whatever the biochemical basis of these effects (see below) the bare facts indicate an appreciable duration to the time courses of the pig-ment participations—far longer than would be expected from simple photochemical considerations where singlet and triplet lifetimes are small fractions of a second. Here we are clearly dealing with durations of several minutes—and only chemical products with lifetimes (or dif-fusion times) of that order of magnitude could account for these effects.

It is not surprising therefore that some carry-over from one wave-length persists into the next, even when an appreciable dark period in-tervenes. The cusps characteristic of phycoerythrin are still prominent


400 500 600

Wavelength (m/i)

700

FIG. 8. Action spectra for enhancement and chromatic transients in Chlorella. The former shows the effectiveness of other wavelengths added to 700 m/x; the latter, the magnitude of the transient as per cent of the steady rate on alternating 700 m/i with the indicated wavelengths. (From Myers and French, 1960.)

after a minute or more of darkness, following chlorophyll activation (red light). Then they tend to die away, only to reappear after 5 or 10 minutes of darkness. I t seems possible that some respiratory process might be restoring the necessary precursor (e.g. by oxidative phos-phorylation). Some such carry-over undoubtedly also accounts for the flicker effect, the partial enhancement found (see French, 1961) in Chlorella on rapid alternation of two wavelengths, causing fusion of transients. This flicker effect has been observed in red algae as well (Blinks, unpublished). I t seems quite likely that the same mechanism accounts for both enhancement and the chromatic transients—a hypothe-sis well borne out by Fig. 8, which shows practically identical action spectra for the two phenomena in Chlorella. The same is true for a red alga (Fig. 9).

2.5 Biochemical Mechanism of the Accessory Pigments

The markedly different time courses of their transients strongly indicate that chlorophyll a and the accessory pigments (including chloro-phylls b and c) are participating in different photochemical reactions,

2 1 8 L. R. BLINKS

1.6

E 1.4

1.2

400 500 m 600 700 m/x (a)

30

t% 20

10

400 500 600 700 m/x (b)

FIG. 9. Action spectra for enhancement (E) and chromatic transients (t) in a red alga (Porphyra). Reference beam is (a) 702 m/i, (6) 566 m/t. (From Blinks, 1960a.)

each perhaps necessary for complete photosynthesis. What are these characteristic photochemical reactions? Identification has been at-tempted in at least two studies: Arnon (1961) reported that photophos-phorylation in plastids showed peak activity at 678 πΐμ (indicating chlorophyll a) while the evolution of oxygen showed a peak at 644 τημ (chlorophyll b absorption peak). TPN reduction had a different action spectrum from either, although no peak was apparent in the narrow spectral region studied. However, Hoch and Kok (1961) found the ac-tion spectrum for photophosphorylation mediated by PMS (phenazine methosulfate) to peak at 700 to 710 τημ where only chlorophyll a ab-sorbs, although Jagendorf and Forti (1961) and Fork (1961a) found the maxima lower (668 to 680 πΐμ) where both chlorophylls absorb; there was still high activity at 650 m/x, near the chlorophyll b peak. Appar-ently both chlorophylls are active in phosphorylation although evidence indicates chlorophyll a to be the more effective (Arnon, 1961).


However, enhancement has definitely been shown in another photo-physiological process, the Hill reaction (the release of 02 in the pres-ence of an oxidant, such as quinone). Rabinowitch and Govindjee (1961) found a definite Emerson Effect on adding shorter wavelengths to far-red light (700 ηΐμ). The action spectra of such effects indicate that chlorophyll "a-670" and chlorophyll b in Chlorella, as well as phyco-cyanin in Anacystis, can enhance the far-red activity with respect to the Hill reaction.

On the other hand, no enhancement of growth, or of acetate assimila-tion, was found in the photosynthetic bacterium, Rhodospirillum rubrum, either between different regions of chlorophyll absorption, or between these and the carotenoid region (Blinks and van Niel, 1963). This organ-ism, of course, evolves no oxygen.

It would appear therefore that the enhancement effect takes place in the oxygen-evolving, rather than the C02-fixation part of the photo-synthetic process. Just exactly how the pigments are involved with the Hill reaction remains unexplained, although enhancement studies upon TPN reduction as well as phosphorylation will undoubtedly have been announced before this review appears.

2.0 Protective Action of Accessory Pigments

A quite different role for some accessory pigments has been proposed by Sistrom et al. (1956). Certain purple photosynthetic bacteria (Rhodo-pspeudomonas sphéroïdes) normally tolerate oxygen in the light, but a "green" mutant, deficient in colored carotenoids, was found to be rapidly injured when illuminated in the presence of oxygen. (It grew well aerobically in the dark, or with light anaerobically.) Stanier has sug-gested that the carotenoids protect against photooxidation—presumably by becoming oxidized themselves. A somewhat similar case was reported in Chlorella (Claes, 1954). It is an intriguing fact that several green algae, such as "red snow" (Haematococcus), Dunaliella (brine flagel-late) and Trentepohlia (arboreal alga), exposed to very bright light all have great accumulations of /3-carotene. As pointed out above, however, the carotene is usually in oil droplets; whether these droplets would have a protective action when isolated from the plastid is not certain. Dunaliella, however, can be cultured with much less carotene, and it might be possible to test its tolerance of bright light by this means. Per-haps the carotenoids of red algae (which seem to be inactive photo-synthetically) serve some such protective function in the presence of the highly fluorescent (hence possibly photooxidative) phycoerythrin. In this sense they may not be as indifferent as the action spectra indi-cate. Platt (1959) has made another interesting suggestion about the

220 L. R. BLINKS

carotenoids; namely, that they might serve to facilitate energy transfer by a resonance mechanism along their conjugated double-bond "back-bone." Calvin (1955) has also made a somewhat similar suggestion in-volving the carotenoids in the normal energy pathway of the cell, with close complexing between the carotenoids and chlorophyll. In this sense the color of the carotenoids might be less significant from the point of view of energy absorption than as an indicator of their oxidation and reduction, with a transfer of charge along the conjugate chain. Loss of such essential links would of course expose other sensitive cellular (per-haps plastid) components to photooxidation. In this connection Fork has recently shown that red algal plastids leached free of phycoerythrin show photooxidation rather than photosynthesis.

REFERENCES

Airth, R. L., and Blinks, L. R. (1957). J. Gen. Physiol. 41, 77-90. Arnold, W., and Oppenheimer, J. R. (1950). / . Gen. Physiol. 33, 423-435. Arnold, W., and Thompson, J. (1956). J. Gen. Physiol. 39, 311-318. Arnon, D. I. (1961). Bull Torrey Botan. Club 88, 21&-259. Blinks, L. R. (1954). Ann. Rev. Plant Physiol. 5, 93-114. Blinks, L. R. (1957). In "Research in Photosynthesis" (H. Gaffron, ed.), pp. 444-

449. Interscience, New York. Blinks, L. R. (1959). Plant Physiol. 34, 200-203. Blinks, L. R. (1960a). Proc. Natl. Acad. Sei. U. S. 46, 327-333. Blinks, L. R. (1960b). Science 131, 1316. Blinks, L. R. (1960c). In "Comparative Biochemistry of Photoreactive Systems"

(M. B. Allen, ed.), pp. 367-376. Academic Press, New York. Blinks, L. R., and van Niel, C. B. (1963). In "Studies on Micro-algae and Photo-

synthetic Bacteria," pp. 297-307. Univ. Tokyo Press, Tokyo. Blinks, L. R., and Skow, R. K. (1938). Proc. Natl. Acad. Sei. U. S. 24, 420-427. Brody, M., and Emerson, R. (1959). / . Gen. Physiol. 43, 251-264. Calvin, M. (1955). Nature 176, 1215. Claes, H. (1954). Z. Naturforsch. 9b, 461. Dutton, H. J., and Manning, W. M. (1941). Am. J. Botany 28, 516-526. Dutton, H. J., Manning, W. M., and Duggar, B. M. (1943). / . Phys. Chem. 47,

308-313. Duysens, L. M. N. (1952). "Transfer of Excitation Energy in Photosynthesis,"

Doctoral Dissertation, Utrecht University. Duysens, L. M. N., Amesz, J., and Kamf, B. M. (1961). Nature 190, 510-512. Emerson, R. (1959). Ann. Rev. Plant Physiol. 9, 1-24. Emerson, R., and Chalmers, R. V. (1958). Phycol. Soc. Am. News Bull. 11, 51-6. Emerson, R., and Lewis, C. M. (1942). J. Gen. Physiol 25, 579-595. Emerson, R., and Lewis, C. M. (1943). Am. J. Botany 30, 165-178. Engelmann, T. W. (1883). Botan. Ztg. 41, 1-13. Engelmann, T. W. (1884). Botan. Ztg. 42, 81-93 and 97-105. Fork, D. C. (1961a). Carnegie Inst. Wash. Yearbk. 00, 363-366. Fork, D. C. (1961b). Carnegie Inst. Wash. Yearbk. 00, 369-370. Fox, D. L., and Sargent, M. C. (1938). Chem. & Ind. (London) 57, 1111. French, C. S. (1961). In "Light and Life" (W. D. McElroy and B. Glass, eds.),

pp. 447-471. Johns Hopkins Press, Baltimore, Maryland.


French, C. S., and Fork, D. C. (1961a). Fifth Intern. Biochem. Congr., Moscow Symposium No. 6, Preprint 73 (15 pp).

French, C. S., and Fork, D. C. (1961b). Biophys. J. 1, 669-681. French, C. S., and Fork, D. C. (1961c). Carnegie Inst. Wash. Yearbk. 60, 351-357. French, C. S., and Young, V. K. (1952). / . Gen. Physiol. 35, 873-890. Haxo, F. T. (1960a). In "Handbuch der Pflanzenphysiologie" (W. Ruhland, ed.),

Vol. 5, Part 2, pp. 349-363. Springer, Berlin. Haxo, F. T. (1960b). In "Comparative Biochemistry of Photoreactive Pigments"

(M. B. Allen, ed.), pp. 339-360. Academic Press, New York. Haxo, F. T., and Blinks, L. R. (1950). Λ Gen. Physiol. 33, 389-422. Hoch, G., and Kok, B. (1961). Ann. Rev. Plant Physiol. 12, 155-194. Jagendorf, A. T., and Forti, G. (1961). In "Light and Life" (W. D. McElroy and

B. Glass, eds.), pp. 576-586. Johns Hopkins Press, Baltimore, Maryland. Jones, R. F., and Fujimori, E. (1961). Physiol. Plantarum 14, 253-259. Karrer, P., and Jucker, E. (1950). "Carotenoids," p. 311. Elsevier, New York. Lemberg, R. (1949). "Hematin Compounds and Bile Pigments," pp. 128-130. Inter-

science, New York (contains references to Lemberg's original papers on phycobilins).

Levring, T. (1947). Göteborgs K. Vetenskaps Vitterhets-Samhäll. Handl. IV. Ser. B5(6), 1-90.

Levring, T. (1960). Botan. Manna 1, 67-73. Montfort, C. (1934). Jahrb. wiss. Botan. 79, 493-592. Montfort, C. (1936). Jahrb. wiss. Botan. 83, 725-772. Montfort, C. (1940). Z. physik. Chem. A186, 57-93. Myers, J., and French, C. S. (1960). / . Gen. Physiol. 43, 723-736. 0 hEocha, C. (1960). In "Comparative Biochemistry of Photoreactive Systems"

(M. B. Allen, ed.), pp. 181-203. Academic Press, New York. Platt, J. R. (1959). Science 129, 372-374. Rabinowitch, E., and Govindjee (1961). In "Light and Life" (W. D. McElroy and

B. Glass, eds.) pp. 378-386. Johns Hopkins Press, Baltimore, Maryland. Sager, R. (1961). Carnegie Inst. Wash. Yearbk. 60, 374-376. Sager, R., and Zalokar, M. (1958). Nature 182, 98^100. Schmidt, G. (1937). Jahrb. wiss. Botan. 85, 554r-591. Seybold, A. (1934). Jahrb. wiss. Botan. 79, 593-654. Sistrom, W. R., Griffiths, M., and Stanier, R. Y. (1956). / . Cellular Comp. Physiol.

48, 473 -515. Stanier, R. Y. (1958). Brookhaven Symposia in Biol. 11, 43-53. Strain, H. H. (1938). Carnegie Inst. Wash. Publ. 490, 147 pp. Strain, H. H. (1949). In "Manual of Phycology" (G. M. Smith, ed.), pp. 244-262.

Chronica Botanica, Waltham, Massachusetts. Strain, H. H. (1958). "Chloroplast Pigments and Chromatographie Analysis." Phi

Lamda Upsilon, Pennsylvania State Univ., University Park, Pennsylvania. Tanada, T. (1951). Am. J. Botany 38, 276-283. Thomas, J. B., and Govindjee (1960). Biophys. J. 1, 63-72. Thomas, J. B., and Govindjee (1961). In "Light and Life" (W. D. McElroy and

B. Glass, eds.), pp. 475-478. Johns Hopkins Press, Baltimore, Maryland. Wassink, E. C , and Kersten, J. A. H. (1946). Enzymologia 12, 2-32. Witt, H. T., Müller, A., and Rumberg, B. (1961). Nature 191, 194-195. Wurmser, R. (1921). Arch. Physiol. Biol. pp. 33-141. Yocum, C. S., and Blinks, L. R. (1954). J. Gen. Physiol 38, 1-16.

Chapter 8

PHOTOTROPISM IN HIGHER PLANTS1

Winslow R. Briggs

Department of Biological Sciences, Stanford University, Stanford, California

1. Introduction When growing plants are illuminated from one side with certain

wavelengths of light, a characteristic growth response is frequently in-duced. Because there is a difference in the growth rate between the il-luminated and shaded sides, curvature develops. Although the plane of the curvature is determined by the direction of the incident light, the direction may be either directly toward the light (positive curvature) or directly away from it (negative curvature). This growth response, if allowed to continue until elongation ceases, is permanent and irrevers-ible, and is called phototropism. Even if the light is turned off during development of curvature, the plant can only be induced to grow erect if a new differential growth is induced (either by light or gravity) in the opposite direction. Thus, phototropism is not to be confused with photonasty, which applies to movements of plant parts in response to a light stimulus, but with the direction of movement independent of the direction of the stimulus, freely reversible, and dependent not upon growth but upon rapid changes in the water relations of the cells in-volved in the movement.

A great majority of studies on phototropism in higher plants have utilized the coleoptile of the grass seedling. The coleoptile is the very first portion of the shoot to emerge from the germinating seed. It is hol-low and cylindrical, usually closed at the top, and within it, at its base, is found the shoot apex. From the shoot apex, the primary leaf extends up into the cylinder, ultimately breaking through the coleoptile, the growth of which is usually complete within a week of germination.

Coleoptiles present several advantages for phototropic studies. First, 1The work of the author and his associates which is discussed in this article

was supported by grants from the National Science Foundation and a grant from the Research Corporation. The author is extremely grateful for this aid.

223

224 WINSLOW R. BRIGGS

they contain only very small amounts of the photosynthetic pigments, even after illumination, and thus serious screening of phototropic pig-ments by photosynthetic ones is not a problem (but, see Section 5). Second, virtually all cell division has taken place by the time the coleoptiles are usually used (at about 72 hours after soaking for germi-nation), and thus any light-induced growth response is primarily a con-sequence of alteration of cell elongation (Tetley and Priestley, 1927; Avery and Burkholder, 1936). Third, coleoptiles of certain species can be grown in large numbers with a high degree of uniformity, which permits quantitative results with a small sample. Finally, if the coleop-tiles are grown in darkness, they are extremely sensitive to unilateral illumination. Thus, although reference will be made below to studies with radish (Raphanus), bean (Phaseolus), and certain other plants, a large amount of the work cited has involved the coleoptile of oat (Avena sativa L.) or corn (Zea mays L.) seedlings. Since the coleoptile is a fairly short-lived organ found within the grass family only, conclusions derived from its study should be applied to other plants only with great caution. I t is quite possible that some of the mechanisms described be-low are unique to coleoptiles, and are replaced by other mechanisms in other species and families of higher plants.

Modern study of phototropism was initiated by the simple but ele-gant studies of Charles and Francis Darwin (1881) demonstrating that for certain dark-grown grass coleoptiles the site of sensitivity for photo-tropic induction is the coleoptile apex. If coleoptiles were unilaterally illuminated with all but the extreme apex shielded from light, curva-ture nevertheless developed normally and then migrated down into the shielded regions. The migration of the development of curvature led to the demonstration by Boysen-Jensen (1910) that the influence causing curvature could be transmitted across a gelatin-filled wound gap, and was, therefore, probably chemical in nature. This type of investigation was continued by other workers, and led ultimately to the well-known demonstration by Went (1928) that the coleoptile apex produces a growth hormone, auxin, which is transported strictly basally to promote the growth of the rapidly elongating regions below. Went found that if oat coleoptile tips were placed on small blocks of agar, auxin was trans-ported into the blocks and could be accumulated there. Then, if the blocks were placed on the stumps of decapitated coleoptiles, in contact with just one side, they would induce curvature. This curvature was, within limits, proportional to the concentration of auxin in the blocks. Went thus developed what still remains the most sensitive bioassay for auxin. In the course of his studies he also collected separately the auxin transported from the shaded and illuminated sides of unilaterally ir-

8. PHOTOTROPISM IN HIGHER PLANTS 225

radiated coleoptiles and found that the shaded side invariably trans-ported more auxin into the agar than the illuminated side (as long as the light dosage was such that positive curvature was induced in control plants). Thus, much of the early study of phototropism was closely tied to the study of plant growth hormones, and much fundamental informa-tion concerning auxin arose from essentially phototropic studies.

Following the early work, an enormous number of experiments were done during the first quarter of the present century, culminating in what is now known as the Cholodny-Went theory of tropisms (Cholodny, 1924; Went, 1926) This theory, applied to phototropism, stated that phototropic curvature of plant organs was a consequence of a light-induced lateral diversion of auxin in the apical region of the organ. Since auxin transport was found to be strictly basipetal, at least in the Avena coleoptile, the amounts of auxin reaching the shaded and il-luminated sides of rapidly elongating regions would be expected to differ. Therefore, the growth rates of the two sides would differ, and curvature would result. Experimental evidence for the Cholodny-Went theory for phototropism, provided by Went (1928), has already been cited above. Similar evidence for the lateral diversion of auxin by geo-tropic induction was provided soon after by Dolk (1936).

Another theory which held prominance before the development of the Cholodny-Went theory, and indeed was for a while its chief competi-tor, was that invoking a so-called light-growth reaction (Blaauw, 1914, 1915, 1918). This theory in its broadest sense stated that light impinging upon a plant cell in some way affected its growth rate, either by stimu-lating or suppressing it. Thus in a unilaterally illuminated organ, the existence of a light gradient as a result of absorption within the organ, would produce a light-growth reaction gradient, and curvature would result. The direction of this curvature would depend on whether the par-ticular light-growth reaction involved growth stimulation or growth in-hibition. Whereas the Cholodny-Went theory invoked the influence of one part of the organ on another, and hence involved a correlation phenomenon, the Blaauw theory implied that each cell of the organ acted independently, and that curvature was the consequence of the summation of separate and independent light-growth reactions. I t should be pointed out that Blaauw used primarily Helianthus, in which evidence for lateral transport is lacking, for his higher plant studies. The present status of the Blaauw theory will be considered later.

A third and somewhat more recent theory, actually a specialized case of Blaauw's theory, was that light of a certain wavelength inactivated auxin within the phototropically responsive organ. As a consequence of the light gradient across the unilaterally illuminated organ, more auxin


would be inactivated on the illuminated than on the shaded side, and curvature would result. This theory is based on careful in vitro studies of light-induced destruction of auxin with various enzyme preparations or flavin pigments present as photosensitizers. Galston and co-workers (Galston, 1949; Galston and Hand, 1949; Galston and Baker, 1949a, 1951; Galston et al., 1953) presented the first direct experimental evi-dence in vitro, and Reinert (1953) and Brauner (1952, 1953) provided further such evidence. A number of earlier experiments, including the classic ones of Went (1928), are frequently cited as supplying in vivo evidence, and these experiments will be considered in detail later.

A fourth theory, proposed by Galston in 1950 and then discussed in more detail in 1959, is that light inactivates some enzyme or co-factor which limits the synthesis of auxin in the apical region of a particular plant organ. As a consequence, an auxin precursor would accumulate on the illuminated side, where more of the inactivation would take place, and would eventually diffuse randomly to the shaded side where less inactivation had occurred, and where it would be then converted to auxin for transport to the lower regions. Galston and Baker (1949b) had indeed shown that certain enzymes were inactivated by visible light in the presence of riboflavin.

In view of these rather different and in some cases mutually exclu-sive theories, for each of which some experimental support could be martialed, it is not surprising that the study of phototropism has given rise to a considerable amount of controversy. The present chapter is an attempt to explore the various lines of evidence, resolve, if possible, some of the controversy, and supply the author's view of the present status both of the physiological and photochemical aspects of the field. No attempt is made to provide an exhaustive coverage of the literature. The early work has been elegantly reviewed by Boysen-Jensen (1936a) and by Went and Thimann (1937) ; two very complete recent reviews are also available (Galston, 1959; Thimann and Curry, 1960). Other pertinent reviews are those of Van Overbeek (1939), Galston (1950), Schrank (1950), Brauner (1954), Went (1956), and Reinert (1959). The author is currently preparing a review which treats in somewhat more detail some of the current problems discussed in the present chapter (Briggs, 1963c).

2. Dosage-Response Curves Many of the earlier workers were aware that the phototropic re-

sponse was a highly complex one that might well involve more than one pigment or mechanism. This awareness stemmed in part from the curious shape of the log dosage-response curve. In 1934, du Buy and


Nuernbergk, assembling both their own data and that of other workers for oat coleoptiles, presented a phototropic log dosage-response curve with a rather distorted sinusoidal shape. This curve, reproduced in Fig. 1, has at least three maxima and two minima, one of which was actually

FIG. 1. Phototropic dosage-response curve for oat coleoptiles. From du Buy and Nuernbergk (1934), after Went and Thimann (1937).

negative. The curve shown was first presented in graphical form by Went and Thimann (1937). The relative heights of the maxima and minima should not be regarded as providing any quantitative informa-tion since quite different conditions were used to obtain different regions of the curve. Furthermore, the unilateral light used to obtain the curve was incandescent, so nothing can be said about wavelength dependence. However, parts of the curve, particularly those including the first maxi-mum and minimum, have been obtained for oat coleoptiles using blue (4358 Â) or long UV (3650 Â) light (cf. du Buy, 1933; Thimann and Curry, 1960) and Thimann and Curry (1961) obtained the first portion of the curve leading to the second maximum. Briggs (1960) obtained a

FIG. 2. Phototropic dosage-response curve for corn coleoptiles. Dosages ad-ministered at high light intensity. (Briggs, 1960.)


log dosage-response curve for corn coleoptiles with unilaterally applied white light; the curve was similar to that reported for oats (it in-cluded only dosages up to those yielding the second maximum), with the exception that the first minimum was not negative (Fig. 2). Recently, Asomaning and Galston (1961) have obtained a log dosage-response curve for barley coleoptiles in which the first maximum and minimum are apparently entirely missing. Evidence that this may indeed be the case will be considered below.

The various peaks and valleys of the oat dosage-response curve have been called, respectively, first-positive, first-negative, second-posi-tive, indifferent, and third-positive curvature, reading from threshold to higher dosages. This terminology is a little confusing, since, as will be shown below (Section 6), it is possible to obtain curvature responses that represent approximately the algebraic sum of several components, with this sum either positive, negative, or zero. Therefore, throughout the remainder of this article, first-positive, first-negative, and second-positive curvatures will be called systems I, II, and III respectively, after Zimmerman and Briggs (1963b). Since little quantitative work has been done in the indifferent region or on third-positive curvature, these regions of the curve will not be considered in detail.

Before further analysis of the dosage-response curve, it is necessary to consider certain physical facts about the phototropic responses of coleoptiles. The first of these concerns the precise localization of sensi-tivity. The Darwins had shown that the coleoptile apex is the most sen-sitive region for light reception, but they had provided no real quantita-tive information. Data of a more quantitative nature were given by Sierp and Seybold (1926) and Lange (1927) who irradiated coleoptiles in various ways with narrow beams of light. These workers all deter-mined the minimum amount of energy necessary to elicit threshold curvature for various regions of the oat coleoptile, and thus were clearly working only with system I. Sierp and Seybold reported the apical half-millimeter to be 36,000 times as sensitive as a comparable half-milli-meter region between 1.5 and 2.0 mm from the apex. Lange, using a finer light beam, localized the region of greatest sensitivity to the most apical 50 microns.

These two papers tell us little about the localization of sensitivity of systems II and III. However, Zimmerman and Briggs (1963a) ob-tained normal systems II and III curvature, illuminating at most the top 3 mm of oat coleoptiles. Curvature so induced migrated normally down into the shaded regions, implying basipetal transport of the con-sequences of phototropic induction. An equally clear case for system II curvature is made by Briggs (1963a, his Table 8) where the negative


curvature induced under certain conditions continued to migrate down the coleoptile for a full 220 minutes. Thus, although the exact limits of the region of sensitivity for systems I I and I I I curvature are not closely defined, these limits clearly encompass the apical region. Evidence that, at least for system III , the limits extend further down the corn coleop-tile than system I is provided by the auxin studies discussed in Section 3.

The next matter to be considered is the validity of the Bunsen-Roscoe law of photochemical equivalence, or the reciprocity law as it is also known (Bunsen and Roscoe, 1862). This law states that as long as the product of light intensity and exposure time remains constant, the photochemical effect of the light should also remain constant. The law was one of the very first to be investigated for phototropism. Fröschel (1908) and Blaauw (1909) found the law to be valid for photo-tropic curvature of Lepidium (mustard) and Avena seedlings, respec-tively, over an enormous range of intensity-time combinations, at least with the very low dosages necessary to elicit threshold curva-ture. Other authors (Arisz, 1915; Haig, 1935) found the law to be valid at least through maximum system-I curvature for oat coleoptiles (Haig used unilateral "violet" light), confirmed by Briggs (1960) for both corn and oat coleoptiles. Following the earlier studies, there was a tendency to regard the law as valid for phototropism in general, with-out regard for dosage, although Arisz (1915) clearly demonstrated that it could not be considered valid for system-II (first-negative) curva-ture, and Bremekamp (1918) concluded that it was valid neither for systems I I nor I I I .

In a recent study of the phototropic reciprocity relationships of corn coleoptiles, Briggs (1960) found that for dosages beyond those yielding maximum system-I curvature, the reciprocity law could not be con-sidered valid. His criterion for validity was the induction of equal amounts of curvature by a given light dosage, regardless of the com-bination of intensity and time used. Under certain conditions, the amount of curvature obtained seemed purely a linear function of exposure time with no direct relationship to total dosage (see particularly Briggs's Figs. 7 and 8 for corn and Figs. 13 and 14 for oats). The shape of the dosage-response curve could be greatly altered by using only very low intensities and long exposure times to achieve all dosages beyond those giving maximum system-I curvature. The curve, under those conditions, was step-shaped, with the first and second increments corresponding to the first- and second-positive curvatures discussed by the earlier work-ers, and separated by a region in which increase in dosage did not par-ticularly alter response magnitude (Fig. 3, cf. Fig. 2). Briggs, thus, con-


ω LU UJ 30 a: o LU ° . 2 0 UJ Q:

S» >

1 2 3 4 5

LOG MCS

FIG. 3. Phototropic dosage-response curve for corn coleoptiles. Dosages ad-ministered at low light intensity. (Briggs, 1960.)

eluded that the reciprocity law was valid for the higher dosages only when the intensity of incident light was low and the exposure time was long.

At the same time, Thimann and Curry (1960), reviewing all of the available data, including some of their own from oat coleoptiles, con-cluded that systems I and II showed reciprocity validity when the in-tensity of incident light was high, while system III did not obey the reciprocity law under any conditions, but was probably dependent only upon length of exposure. These apparently conflicting conclusions have been resolved by Zimmerman and Briggs (1963a). These workers ob-tained for oat coleoptiles a detailed series of dosage-response curves by using for each curve a single intensity of monochromatic light (4358 Â), and by varying the dosage by changing the exposure time. For the first time, all of the necessary information could be obtained from a single series of experiments done under uniform conditions. The results of these experiments are shown in Fig. 4.

If we consider for the moment only Figs. 4a, 4b, and 4c, an explana-tion for the conflict is now clear. The reciprocity law is apparently valid, as suggested by Thimann and Curry, for systems I and II, as long as light intensity is high and exposure time short. However, with decreasing light intensity and increasing exposure time, progressively more system-Ill curvature is obtained for a particular dosage. It is not possible to determine, then, whether the law is valid for systems I and II or not, but Zimmerman and Briggs (1963b) showed indirectly that it might be. Assuming that it was valid, they subtracted a theoretical curve for systems I and II (see Section 6) from the various experi-mental curves, and plotted the remaining curvatures purely as a func-tion of exposure time. The results, shown in Fig. 5, gave an approxi-

» ■ »


Minus Red Light Plus Red Light

b.M.4xlO"12

c.M.4xlO-"

d.M.4xlO*13

e.I«l.4xlO"12

f.I-l.4xlO"n

-13 -12 -II -10 -9 -8 -13 -12 -II -|0

LOG (Ixt), EINSTEINS CM-2 at 4358 Â -9 -8

FIG. 4. Phototropic dosage-response curves for oat coleoptiles at three intensi-ties of monochromatic light. Left-hand curves (a-c), no red light pretreatment. Right hand curves (d-f), 2 hours red light pretreatment. The peaks for system I curvature are denoted by the two vertical lines. Intensities (7) are in Ein-steins/cm2/sec at 4358 Â. (Zimmerman and Briggs, 1963a.)

mately straight line, regardless of the initial light intensity used. Since the straight line was obtained whether or not the original curvature contained some system-I or -II curvature, it seems reasonable that their original assumption of complete reciprocity validity for systems I and II must be justified. Thus, when Briggs (1960) considered the reciprocity law invalid, he was presumably studying only the increase


LIGHT /

* S E C " · /

/ 8

MINUS RED LIGHT '

K-2.95XIO"4 SEC"1

500 1000 1500 ' EXPOSURE TIME, SECONDS

FIG. 5. Net phototropic response obtained when curvature predicted for systems 1 and II (Section 6) is subtracted from total experimental curvature (Fig. 4) and plotted as a function of exposure time. Intensities (/) in Einsteins/cmVsec at 4358Â. Open circles: J = 1.4 X 10"12, 2 hours red light. Solid circles: / = 1.4 X 10"n, 2 hours red light. Open squares: I = 1.4 X 10"12, no red light. Solid squares: / = 1.4 X 10"11, no red light. K refers to the slope. (Zimmerman and Briggs, 1963b.)

in the system-Ill component with decreasing intensity and increasing exposure time. For corn, when the exposure time became longer than a certain value, perhaps some physiological factors other than photo-tropic induction became limiting. No further increase in curvature could be obtained, and Briggs then considered the reciprocity law as valid. With oats, his experiments showed only an increase in system-Ill curva-ture with increasing exposure time and, thus, he concluded that the reciprocity law was not valid at all.

Figure 4 provides certain other types of information which will be briefly mentioned here and then discussed more fully in Section 4. Fig-ures 4a, 4b, and 4c represent results obtained from plants which re-ceived the same treatment as those from which the results in Figs. 4d, 4e, and 4f, respectively, were obtained, with respect to unilateral il-lumination. However, the experiments illustrated by the first three graphs were done with plants that had had no exposure to red light immediately prior to phototropic induction, while those illustrated by the second three graphs represent plants that had had two hours of red-

I.O

LÜ Φ Z

2 IÜI.2 O Û. O

.8 o X Q_ LÜ > I -< . 4 LU OC

I

[

[

I

L · O Q M O 0 / ^ o 7 m

À o

y· ^

"1

%

.1

1

PLUS RED

K- 8.27xl0"<

o


light treatment just before blue light exposure. The differences between these two sets of curves are dramatic. Red light clearly reduces the sen-sitivity of systems I and I I to blue light, as indicated by the shift of the respective peaks almost a full log unit to the right. Such a shift for system-I curvature had previously been described by Curry (1957). At the same time, red light increases the sensitivity of system I I I to blue light, indicated by a shift of the system I I I curve almost half a log unit to the left. Consideration of possible explanations for these shifts must await discussion of other red-light effects (Section 4), of the pigment problem (Section 5), and of a possible kinetic scheme for phototropic curvature of oat coleoptiles (Section 6).

3. Auxin Relationships Three of the four theories for phototropic curvature mentioned

earlier (Section 1) depend upon a difference between the amount of auxin reaching, or present in, the lighted and shaded sides of the photo-tropically induced organ. These theories include: (1) the light induc-tion of lateral transport of auxin, (2) the inactivation of auxin, and (3) the inactivation of some component of the auxin-synthesizing sys-tem. The fourth theory, which invokes light-growth reactions, depends upon light-induced changes in tissue sensitivity to auxin, rather than upon an auxin differential, but this theory might well represent a proc-ess superimposed upon any of the other three suggested. Therefore, a detailed consideration of auxin relationships following phototropic in-duction will be presented next. I t may then be possible to evaluate the relative contributions, if any, of each of the proposed mechanisms.

The original evidence for a light-induced auxin differential (Went, 1928) has already been mentioned (Section 1). This evidence consisted of the separate collection in agar of auxin being transported from the illuminated and shaded sides of oat coleoptile tips. Went used light dosages which induced either system-I curvature or a curvature near zero degrees, in the region of the log dosage-response curve between maximum system-I and system-II curvature. About ten years later, Asana (1938) repeated these split-tip experiments again with oat coleoptiles, utilizing both dark controls and plants unilaterally illumi-nated to yield substantial system-II (negative) curvature. Wilden (1939) also repeated the experiments, utilizing dosages of light inducing system-I, -II, or -III curvature. In all of these cases an auxin differential was obtained, with the greatest amount of auxin coming from the side of the tip which would correspond to the most rapidly elongating side of the curving organ. The Cholodny-Went theory depends fundamentally upon these experiments.


The theory gained added support from some intriguing experiments by Boysen-Jensen (1928), who demonstrated that physical continuity was necessary between the illuminated and shaded sides of the oat coleoptile tip for the development of curvature following induction. By making a vertical slit in the coleoptile tip and inserting a small frag-ment of platinum foil into the slit, he was able to inhibit curvature de-velopment if the slit and foil were in a plane at right angles to the incident illumination. Brauner (1957) repeated Boysen-Jensen's experi-ments and found that as the light dosage was gradually increased, the amount of inhibition of curvature by the inserted barrier (a small piece of cover slip) decreased. Briggs (1960) did similar experiments with corn coleoptiles, using light dosages inducing either maximum system-I or system-Ill curvature, and, like Brauner, found that the slit and barrier completely prevented curvature development only when the light dosage was low. There was only partial inhibition with higher light dosages.

On the basis of the above results, one might well conclude that lateral transport of auxin (or a precursor or some factor limiting syn-thesis) mediates system-I curvature. As far as system-II curvature is concerned, the evidence is suggestive but incomplete, and for system III, if lateral transport occurs, it must occur partially below the inserted barrier. In any case, direct light-induced inactivation would seem highly unlikely for system I, and improbable for system II, in which the greatest amount of auxin is found being transported down from the illuminated side.

However, let us reexamine the evidence from direct auxin studies. How much auxin is being transported from the various phototropically induced tips in comparison with yields from dark controls? Many re-views which have cited the evidence from split-tip experiments, have presented tables showing only the relative percentages of auxin trans-ported out of the illuminated and shaded sides in relation to the total obtained from the induced tips, with no mention of dark controls. Therefore, the evidence is presented again in Table I, including, where possible, information concerning auxin produced by dark controls. The picture now becomes somewhat altered, and the evidence for the Cholodny-Went theory is weakened. In Went's classical experiments there was usually a light-induced reduction in the total amount of auxin obtained. Wilden (1939) did not include any dark controls in her experiments. Only Asana (1938) obtained what appears to be 100% recovery of auxin from illuminated tips.

In an effort to resolve this problem, Briggs and co-workers (Briggs et al, 1957; Briggs, 1963a) have repeated and extended the split-tip


TABLE I THE PERCENTAGE DISTRIBUTION OF AUXIN TRANSPORTED PROM ILLUMINATED

AND SHADED SIDES OF OAT COLEOPTILE TIPS

Per cent recovery

Dark Lighted Shaded (of dark Experiment control side side control) Author

System-I light dosage

1 2 3 4 5

Average

1

100 100 100 100 100 100 —

38 26 6 32 33 27 17

57 51 62 60 57 57 83

95 77 68 92 90 84 —

Went

Went Went Went

Went Went Wilden0

Light dosage yielding system-II curvature

1 1 2 3 4 5 6 7 8 9 10 11 12

Average

— 100 100 100 100 100 100 100 100 100 100 100 100 100

38 28 34 31 55 61 46 79 90 68 41 79 90 58.6

62 24 26 32 65 45 32 47 53 41 25 50 63 41.9

— 52 60 63 120 106 78 126 143 109 66 129 153 100.5

Wilden« Asana6

Asana6

Asana6

Asana6

Asana* Asana6

Asana6

Asana6

Asana6

Asana6

Asana6

Asana6

Asana6

Light dosage yielding system-Ill curvature

1 — 36 64 — Wilden« a Since Wilden did not include dark controls, her results are expressed as percentage

of the auxin yield from the illuminated tips. 6 Asana included dark controls with only two of his experiments, although he meas-

ured yield from dark controls on six separate occasions. Thus the 100% yield for the dark controls represents the average of all six of these tests. Controls included on the same day as illuminated tips did not yield significantly more auxin (by Student's t test) than the illuminated tips.

experiments with corn coleoptiles. These investigators included both unilluminated and illuminated intact controls, the usual split tips, and a further modification—tips that were entirely bisected longitudinally, with the halves then replaced in normal orientation on either side of


a thin glass barrier for illumination and auxin collection. Light dosages inducing either maximum system-I (Briggs, 1963a) or system-Ill (Briggs et al., 1957; Briggs, 1963a) curvature were used and the results of these experiments are summarized in Tables II and III. Examination of auxin yields from dark and illuminated intact controls reveals that light does not affect the amount of auxin being produced in any sig-nificant way. Examination of results from partially split tips indicates that the expected auxin differential has been produced, and that the amount of auxin emerging from the shaded side is substantially greater than that from the dark control (on a per tip basis). Finally, results with totally split tips, show that interposition of a complete barrier eliminates the differential both for system-I and for system-Ill curva-ture. In these experiments, then, the possibility that light inactivates auxin or directly inhibits synthesis is eliminated, since in either case, a differential should appear despite the barrier. Thus, in corn at least, something must be laterally translocated in both system-I and system-Il l curvature, and these systems are indeed correlation phenomena as suggested by Went and Thimann (1937).

Leaving for the moment the problem of precisely what is translocated, and deferring possible explanations for the reported light-induced re-duction in the amount of auxin produced by oat coleoptile tips, we should consider several other observations concerning corn coleoptiles (Briggs, 1963a). First, for both system I and III, the region of lateral transport is primarily the apical half-millimeter. If the coleoptiles are partially split to within a half millimeter of the apex, a barrier inserted from below, and the tips illuminated from one side, a clear difference is still found between the amounts of auxin transported out from lighted and shaded sides. Only when continuity is completely broken between the two coleoptile halves is the difference eliminated. Thus, both for system-I and system-Ill curvature, the region of lateral transport coincides roughly with the region of maximum phototropic sensitivity. Second, if coleoptiles are given a dosage of unilateral light under con-ditions in which the exposure time is short, so that no system-Ill curva-ture is obtained, but the total dosage is sufficiently high that neither system-I nor system-II curvature is induced, lateral translocation does not occur. Furthermore, there is no light-induced alteration in auxin production; in the absence of curvature, the differential is absent. Third, if the coleoptiles are exposed to an enormous light dosage (106 Mes), given in an extremely short time, there is a slight but probably signifi-cant reduction in auxin production. The fact that the coleoptiles some-times curve a few degrees toward the light under these conditions suggests that differential auxin inactivation or inhibition of synthesis


TABLE II LATERAL DISTRIBUTION OF A U X I N 0 FROM CORN COLEOPTILE TIPS FOLLOWING

1,000 MCS UNILATERAL LTGHT6

Experiment

12 13 14 15

16

17

18 20

Averages Per cent dark

control, per tip basis

Averages

Auxin yield (degrees Avena curvature)

Intact tips

Dark

26.5 28.2 23.6 26.3

—

27.3

23.8 25.0 25.8

100.0

Light

26.2 25.8 21.6 27.3

—

27.0

— —

25.6

99.3

Partially split tips

Shade Light

27.5 31.8 29.9 32.0

27.8

32.4

29.1 35.1 30.7

118.0 90.4

15.6 19.0 9.8

19.7

13.3

18.6

14.4 19.0 16.2

62.8

Totally sp]

Shade

13.3 26.4 13.8 19.8 23.3 23.5 27.3 27.5 —

25.6 22.3

86.4 87.8

lit tips

Light

17.3 27.8 16.9 19.5 21.6 28.3 25.1 25.3 —

25.1 23.0

89.2

α Auxin collected in agar for 3 hours, 3 tips or 6 half-tips per block. 6 Light intensity 53.8 MC (5 ft-candles), exposure time 18.5 sec, except experiment

12 (40 MC, 25 sec).

TABLE III LATERAL DISTRIBUTION OF AUXIN" FROM CORN COLEOPTILE TIPS FOLLOWING

232,000 MCS UNILATERAL LIGHT*

Experiment

5

7

Per cent dark control, on per tip basis

Averages

Auxin yield (degrees

Intact tips

Dark

20.5 21.8C

25.5 25.8C

100

Light

19.8 19.4C

26.3 23.3C

94.8

Avena curvature)

Partially split tips

Shade Light

25.5 —

31.0 —

122.0

13.5 —

12.5 —

55.6

88.8

Totally e

Shade

20.9 —

23.0 —

93.8

split tips

Light

19.6 —

24.8 —

94.8

94.3 a Auxin collected in agar for 3 hours, 3 tips or 6 half-tips per block. 6 Light intensity 21.5 MC, exposure time 3 hours. c Totally split tips, halves recombined and placed on single block.


may be occurring, but critical experiments to test this hypothesis have not been done.

Returning now to the earlier experiments which indicated a real reduction by low dosages of unilateral light in the amount of auxin obtainable in agar, and hence the possibility of either the inactivation of auxin or the inhibition of its synthesis, or both, is there any other way in which one might account for these results? In 1936, Van Over-beek demonstrated that the orange "safelight" usually used in darkrooms can reduce the auxin production of oat coleoptiles by as much as a third. More recently, Blaauw-Jansen (1959) has shown that red light (6,600Â, 700 ergs/cm2) reduces the total solvent-extractable auxin con-tent of oat coleoptiles by about 50%. Briggs (1963b) has studied the effect of red light on auxin production by corn coleoptiles and found that, in these also, red light (3,300 ergs/cm2/sec for periods up to 2 hours) reduces auxin production by about 50%, as measured by collec-tion in agar. The reduction seems to start immediately upon the start of red treatment, and to continue for over an hour until an equilibrium is reached and no further change is found. After two hours of red treatment, auxin production stays at the low level for approximately an hour (25°C) before beginning a slow rise back to the initial value of the unirradiated plants. A very significant aspect of this effect, however, is that it was obtained only if the coleoptiles were intact when they were treated with red light. Red illumination was without effect on auxin production if directed upon excised 5-mm coleoptile tips.

It is now possible to make a suggestion concerning the earlier experi-ments in which a clear light-induced reduction in auxin production was apparently demonstrated. In all of the cases cited thus far (Went, 1928; Asana, 1938; Wilden, 1939), the coleoptiles were irradiated with uni-lateral light before the tips were excised, split, and placed on agar for auxin collection, while in Briggs's experiments (1963a) irradiation occurred only after the tips were excised. Since the earlier authors were using polychromatic light, which contained phototropically ineffective red as well as phototropically effective blue wavelengths (see Section 5 for action spectra), it is conceivable that, in addition to inducing lateral transport, the unilateral illumination also induced the red-mediated reaction leading to reduction of auxin production. Even if this is not the case, one would expect that coleoptiles which were unilaterally ir-radiated, whose tips had been individually excised and split, and which were then placed carefully over devices for collection of auxin separately from lighted and shaded sides, might, on the average, remain intact under the darkroom "safelight" somewhat longer than dark controls in which the tips were simply excised and set on agar blocks. That the red-induced


suppression of auxin production could not account even in part for phototropic curvature, and thus does not occur following a light gradient, is demonstrated by the fact that red light alone is photo-tropically ineffective, which even the very early workers knew (Blaauw, 1909). Further discussion of the possible role of this red-light effect in modifying phototropic curvature will be postponed to the following section (Section 4).

In addition to the red-light effect, and the effect of high-intensity visible light, there is a third mechanism whereby light might reduce auxin production. Burkholder and Johnston (1937) have shown that fairly high-intensity UV irradiation is capable of inactivating auxin in vivo. Whether or not this inactivation follows a gradient in the plant, and could therefore be responsible for phototropic curvature, is not known. However, Curry et al. (1956) have shown that the action spectrum for phototropic curvature of oat coleoptiles in response to short wavelength UV light, with the coleoptile tips shielded from illu-mination, looks remarkably like the absorption spectrum of indole-3-acetic acid (IAA). [According to Wildman and Bonner (1948), Reinert (1950), and Shibaoka and Yamaki (1959), IAA is the principal auxin in Avena]. This spectrum in turn matches the action spectrum for in vitro inactivation of IAA. The action spectrum for curvature, however, has its major features shifted approximately 100Â toward longer wavelengths, in comparison with the other two spectra. Curry et al. suggest this to be the consequence of light absorption, not by free IAA, but rather by some biologically active complex of auxin, with an absorption spectrum somewhat modified and shifted. Again, the crucial experiments testing the distribution of auxin inactivation, or indeed demonstrating its occurrence under the conditions producing curvature in intact coleoptiles have not been done.

In view of the above discussion, it would seem that lateral transport provides a plausible mechanism to account both for system-I and system-Ill curvature in corn, and possibly all three systems in oats. Though auxin inactivation may occur under conditions of high light in-tensity or certain conditions of UV exposure, it has not been conclusively demonstrated to play a role in phototropism. Certainly it cannot for systems I through III, but it may for the so-called third-positive curva-ture. The experiments of Curry et al. provide the only real evidence for this suggestion. However, any hypothesized or demonstrated effect of light on auxin production or content must take into account all of the various possibilities; it must clearly separate them and demonstrate, if implications for phototropism are suggested, that the effect is indeed a differential one within the intact plant. A demonstration of auxin in-


activation in vitro, even accompanied by a highly suggestive action spectrum, is insufficient to invoke the process in phototropic curvature. The auxin destruction literature in this context is considered in more detail elsewhere (Briggs, 1963a, 1963c).

A final line of evidence which opened to serious question not only the lateral transport concept but the concept of auxin inactivation in phototropism as well remains to be considered. This evidence is pro-vided by careful observations on the distribution of exogenously supplied OMabeled IAA following either geotropic or phototropic induction of oat or corn coleoptiles (Bünning et al, 1956; Gordon and Eib, 1956; Reisener, 1957, 1958; Ching and Fang, 1958; Reisener and Simon, 1960; Gillespie and Thimann, 1961). With the exception of Gillespie and Thimann, none of these authors detected any significant lateral trans-location of the applied labelled auxin following tropic induction, nor, indeed, any modification of the amount of label in the tissue. Only Gillespie and Thimann, using corn coleoptiles, found a lateral differ-ential (following geotropic induction). Their investigation differed from all of the others in that they alone determined not only total tissue radioactivity, but the amount of radioactivity emerging into agar blocks placed against the upper and lower halves of the geotropically stimulated coleoptile section. Thus it was in the transported auxin that the clear differential was found, and Gillespie and Thimann calculated that even with apical application of the labeled auxin, only some 20% of it actually entered the auxin transport system. Since all of the experi-ments determining the distribution of native auxin described above were concerned with auxin in transit rather than total tissue content, it seems probable that the recurrent failure by various investigators to de-tect a tropically induced differential may be traced to their measurement of total tissue radioactivity or total activity extractable with organic solvents. If only auxin in transit is affected, any differential so induced would be masked by the large amount of auxin entering the tissue but not the transport system, and therefore not subject to redistribution. That it is auxin in the transport system which is critical for growth was suggested by Went (1956) and this has been reemphasized by Scott and Briggs (1960) and Steeves and Briggs (1960).

Gillespie (unpublished) has done further experiments with corn coleoptiles, using a phototropic instead of a geotropic stimulus. If the labelled auxin was applied to the intact tips and light dosages inducing system-I curvature were used, a clear differential was found in the amount of auxin recoverable in agar blocks from the bases of shaded and illuminated sides. Still more interesting was the observation that if the coleoptiles were decapitated, a section removed, labeled auxin ap-


plied in agar to the apical end (now minus the primary site both for photoreception and lateral transport), and light dosages inducing sys-tem-Ill curvature were used, a small but significant differential in transported activity was still obtained.

It is now possible to present a reasonable picture of the auxin rela-tionships of the phototropic curvature of coleoptiles. System-I curvature involves light-induced lateral transport in the extreme apex, and Gil-lespie's phototropic experiments strongly suggest that it is auxin itself that is laterally displaced. The region of lateral transport coincides roughly with the region of maximum phototropic sensitivity (Briggs, 1963a). Similarly system-Ill curvature involves light-induced lateral redistribution of auxin. Again, this lateral transport occurs primarily in the upper half-millimeter of the apex (Briggs, 1963a), but Briggs's earlier experiments (1960) suggest that substantial although not maximal curvature may occur even if the apical portion of the coleoptile is split and an impermeable barrier inserted down to a depth of approximately 2 mm. Furthermore, Gillespie (unpublished) has shown that regions of the corn coleoptile below the apex are capable of some lateral transloca-tion of labelled auxin under suitable illumination conditions, in the absence of the apex. In Briggs's experiments with endogenous auxin, light dosages for system-Ill curvature induced about 5% more lateral translocation of auxin than light dosages for system I (Briggs, 1963a). It is interesting, then, that Gillespie found the regions of the coleoptile below the apex to be capable of translocating just 5% of the labeled auxin entering the transport system. It seems reasonable, then, that the pigment systems for systems-I and -III curvature must at least partially overlap spatially in the extreme apex, while the pigment system of system-Ill curvature must extend substantially below that for system I. For system-II curvature, there are only the earlier observations on the lateral differential in auxin transported from oat coleoptiles following phototropic induction under conditions which yield negative curvature. Although Wilden (1939) did not include dark controls, Asana (1938) did, and found that light under these conditions did not reduce the auxin yield. Therefore, at the moment, there is no reason to invoke any mechanism other than lateral transport for system II, with the direction of transport toward rather than away from the light stimulus. Although it seems fairly clear that the pigment for system II is located within the top 2 or 3 mm of the coleoptile, it is not possible at the present time to state its spatial distribution more precisely.

Up to this point, little has been said concerning the phototropic responses of organs of higher plants other than grass coleoptiles. Van Overbeek (1933), in a detailed study of Raphanus hypocotyls, found a


clear light-induced auxin differential in the amount of auxin in the transport system. Curiously he obtained full recovery of auxin from the illuminated plants, in comparison with the dark controls, only when he excised hypocotyl cylinders and allowed them to transport auxin obtained in agar from other Rapharms seedlings. The auxin-containing agar blocks were applied to the apical ends of the cylinders, and auxin was collected as usual from illuminated and shaded sides of the base. If, instead, he used hypocotyls with their apices intact, and therefore col-lected the endogenous auxin itself, light induced a substantial loss. Whether this loss is related to phototropism or simply reflects a red light-induced suppression of auxin synthesis or something else is not known. Boysen-Jensen (1936b), studying Phaseolus hypocotyls, found a light-induced differential in the amount of auxin extractable with chloro-form from the illuminated and shaded sides, but did not include dark controls. There are other studies, but none of them provide a really con-clusive picture of the role of lateral transport versus other mechanisms in phototropic curvature.

4. Influence of Red Light on Phototropic Sensitivity and Related Phenomena

In the last few years, a number of authors have become aware that red light, although itself phototropically inactive, may exert a profound influence upon subsequent phototropic sensitivity. Thus, Curry (1957) first observed that preirradiation of oat seedlings with red light for one hour produced a clear decrease in their phototropic sensitivity to low dosages of unilateral blue light (4358 Â). Curry was investigating sys-tem-I curvature only, and his criterion for decrease in sensitivity was a shift of the entire log dosage-response curve almost a full log unit to the right along the log dosage axis. However, Blaauw-Jansen (1959) reported that red light induced a subsequent increase in the phototropic sensitivity of oat coleoptiles to dosages of blue light inducing first-positive (system-I) curvature, and Asomaning and Galston (1961) re-ported for oat coleoptiles that small dosages of red light induced an increase while larger dosages induced a rather substantial decrease in subsequent phototropic sensitivity to blue light dosages inducing system-I curvature. To confuse the picture further, Asomaning and Galston reported that even with very large dosages of red light, subsequent phototropic sensitivity of barley coleoptiles was increased only. As mentioned previously (Section 2) Zimmerman and Briggs (1963a) found that red light induced a decrease in the phototropic sensitivity of sys-tems I and II, using the same criterion as Curry (1957), but a sub-stantial increase for system III. The present section is an attempt to


draw these apparently conflicting reports together and to suggest an hypothesis that could account for the apparent discrepancies mentioned above.

There are three known ways in which red light might induce changes in subsequent phototropic sensitivity. First, it might in some way alter auxin relationships. Evidence that it does at least suppress auxin pro-duction has already been discussed (Section 3). Second, it might in some way alter either the photochemical efficiency or the amount of the various phototropic pigments. In fact, Asomaning and Galston (1961) have shown that under their conditions of red-light treatment, red light does induce an increase both in the flavin and carotenoid complements in both oat and barley coleoptile tips. Finally, it might alter tissue sensi-tivity to endogenous auxin. Liverman and Bonner (1953) have shown that red-light treated coleoptile sections elongate significantly more in response to nongrowth-limiting concentrations of auxin than do sections previously kept in the dark.

Unfortunately, each of the above investigations involved a different schedule for red-light treatment, and thus comparison of one set of experiments with another is at best risky. However, certain generaliza-tions can be made at this point. If red light affects phototropism either through suppression of auxin production or through alteration of tissue sensitivity to auxin, red irradiation should produce a change in the amount of curvature obtained following induction with a particular dosage of blue light, whether the red-light treatment preceded or fol-lowed phototropic induction. The persistence of the lateral transport mechanism long after the conclusion of phototropic induction was first well-documented by Went (1928), and has since been studied in detail by von Guttenberg (1959). In fact this persistence is frequently cited as excellent evidence that the primary effect of light is not inactivation of auxin, since the auxin differential may be obtained as much as 8 hours after the end of phototropic induction (von Guttenberg, 1959). Briggs (1963b) has shown that the red light-induced suppression of auxin production in corn coleoptiles is complete within about an hour and twenty minutes. Therefore, if the red-light alteration of phototropic sensitivity is directly related to the auxin changes, one would expect at least some change in the magnitude of the phototropic response when red light follows blue-light treatment.

The same arguments may be applied for a red light-induced change in tissue sensitivity to auxin. Liverman and Bonner (1953) obtained a significant effect with only a half-hour red-light treatment. Unfortu-nately kinetic studies are not available to determine whether the red light-induced reaction had really gone to completion within the 30


minutes, or whether it continued to develop for a matter of hours after the end of the treatment. Certainly the fact that the reaction is still reversible with far-red light (see below) 45 minutes after the end of red-light treatment (at least as determined by total section growth at the end of 6 or 16 hours) suggests that it does continue to develop for some time. However, the crucial question as to how soon the effect of red light on tissue sensitivity is actually expressed is not answered. A small growth rate stimulation very early during red illumination, later re-versed by far-red light, might easily escape detection in sections measured after several more hours of growth in the dark, but might well effect phototropic response. In either case, however, one would expect some measureable effect of red light on the magnitude of the curvature re-sponse obtained following phototropic induction. If, however, the primary effect of red light is an alteration of pigment status or amount, red light administered after phototropic induction should be totally ineffective in inducing any change. The importance of this discussion should become clear shortly.

Another generality that should be made is that whereas alterations in auxin level or tissue sensitivity to auxin could be invoked to explain red light-induced changes in the magnitude of curvature obtainable from a given blue-light dosage, they cannot explain the pronounced shifts in the dosage-response curves shown in Fig. 4. However, a change either in the content or photochemical efficiency (for whatever reason) of a given pigment system would be expected to alter the position of the dosage-response curve. It is therefore necessary to examine in detail the various reports of red light-induced phototropic sensitivity change to determine whether or not any of the three possibilities, or, indeed, any combination of them, can account for the observed results.

The phototropic sensitivity changes described by Curry (1957) and Zimmerman and Briggs (1963a) are similar in that both involve con-tinuous red-light exposures of an hour or more and both are reflected in shifts in the various features of the dosage-response curve. Thus it is unlikely, as discussed above, that a change in auxin production or tissue sensitivity to auxin could account for the changes observed. A red light-induced change involving the pigment is much more likely. Further evi-dence for this suggestion is found in the observation that red light administered to coleoptiles after phototropic induction is without any effect on subsequent development of curvature (Briggs, 1963b). The plants were indistinguishable from those receiving no red light at all.

There remain, however, the reported increases in sensitivity men-tioned both by Blaauw-Jansen (1959) and by Asomaning and Galston (1961) for oats, and by the latter authors for barley. Considering the


oat coleoptile experiments first, it seems probable that Blaauw-Jansen was not observing the same type of change as that described above. She irradiated her plants with red light for 15 minutes or less and then immediately gave them phototropic induction. Briggs (1963b) has shown that whatever changes are occurring to shift the dosage-response curve have only just begun after 15 minutes, and would normally continue for almost an additional hour. According to Curry (1957) and to Zimmer-man and Briggs (1963a), these shifts should result in a decrease rather than an increase in the amount of curvature obtained for the dosages of blue light used by Blaauw-Jansen. It seems likely, then, that what the investigator may have observed was the effect of red light upon tissue sensitivity to auxin. This effect should be expressed in the form of in-creased curvature in response to a particular dosage of unilateral illu-mination, but no shift in the dosage-response curve threshold or maximum. Although her data are a little scant for one to be certain, they certainly suggest this sort of an increase. Although the same argument might be used to suggest that auxin production changes are responsible for the shifts that Blaauw-Jansen observed, a lowering of available auxin should decrease rather than increase the amount of curvature obtained, and the auxin changes follow a time-course similar to that for the phototropic dosage-response curve shift; these changes have only just begun by the end of the first 15 minutes of red treat-ment (Briggs, 1963b). If tissue sensitivity changes were to occur more rapidly than auxin production changes, one would expect to be able to detect them very early during red-light treatment. However, after more extended red-light irradiation, their combined effects on phototropism might approximately cancel each other and one would only observe the dosage-response curve shift. Additional evidence that Blaauw-Jansen was observing a phenomenon different from that observed by Curry or Zimmerman and Briggs is provided by her observation that for obtain-ing the reported sensitivity increase, red light was just as effective after phototropic induction as before, in direct contradiction to Briggs's (1963b) experiments.

The above hypothesis might also apply to the small sensitivity in-crease observed by Asomaning and Galston following low dosages of red light, but the situation is hardly as obvious. These authors ad-ministered red light at 24-hour intervals on each of one, two, or three days before phototropic induction, and the last red-light exposure was never less than about 14 hours before induction. Briggs (1963b) found that the red light-induced changes he was studying, namely the dosage-response curve shift and depression of auxin production, remained maxi-mal for about an hour after the end of red-light treatment. They then


decayed, disappeared entirely after an additional two hours, and did not reappear according to a circadian rhythm. Briggs (1963b) has questioned the statistical significance of the increase reported by Aso-maning and Galston, and it should certainly be verified.

However, there is no question that what Asomaning and Galston refer to as a decrease in phototropic sensitivity following their higher red-light dosages represents a shift of the log dosage-response curve for system-I curvature to the right (see their Fig. 3). Perhaps repeated red-light treatment either induces a rhythm not normally brought about by a single exposure, or some permanent change similar to the temporary change described by Briggs (1963b). In fact, Ball and Dyke (1954) have found that changing seedlings from red light to darkness induces an endogenous rhythm in the growth rate of the oat coleoptile with a first maximum occurring 16 to 17 hours later. Asomaning and Galston may have done their experiments at just about the right time to include this maximum, and Briggs (1963b) may have missed it completely. These various experiments must be compared with caution, however, in view of the divergent schedules used to administer red light.

All of the cases cited so far have concerned only sensitivity changes for system-I curvature following red-light treatment. However, as men-tioned earlier, Zimmerman and Briggs (1963a) have shown that under their conditions the sensitivity of system II is decreased by almost exactly the same factor as system I (Fig. 4). Either these two systems are physically related in some way or else both are affected in the same fashion by some other factor modified by red light.

Figure 4 clearly shows, however, that system III is actually made more sensitive to blue light by previous exposure to red light. In addi-tion, Asomaning and Galston (1961) showed that the only phototropic curvature they could obtain from barley coleoptiles required very large dosages of unilateral illumination, and this curvature system, like system-Ill curvature in oats, also responded to red treatment by increase in sensitivity. It is tempting to assume that the phototropic system in barley is homologous with system-Ill curvature, and that barley is lacking both systems I and II, and Asomaning and Galston make just this suggestion. The energy range required to induce curvature is ap-proximately the same both for barley and for system III of oats; red light increases the phototropic sensitivity of both systems, and, perhaps most striking, the reciprocity law is not valid in either case. Both appear to be a function of exposure time, although in the case of barley there is not enough information available to know within what limits this generality applies.

Zimmerman and Briggs (1963b) have proposed a kinetic scheme for


the phototropic mechanisms of oat coleoptiles which will be discussed in detail in Section 6. The scheme provides a convenient hypothesis to ac-count for the various red light-induced shifts in the dosage response curve. To consider system I I I first, these investigators point out that the sensitivity increase could be accounted for mathematically either by a change in certain rate constants or an increase in effective pigment concentration. However, they feel that it is more reasonable to assume that red light induces a change in pigment concentration rather than an intrinsic alteration in molecular structure that would result in a rate constant shift. Zimmerman and Briggs cite the red-induced increase both in flavin and carotenoid content in the coleoptile tips (Asomaning and Galston, 1961) as supporting evidence.

An appealing feature of this hypothesis is that it can also be used to account for the sensitivity decreases for systems I and I I following red-light treatment. If the pigments for the three systems are so located spatially that system-Ill pigment partially screens the pigments for systems I and II , an increase in system-Ill pigment should produce as a consequence a decrease in the sensitivity of systems I and II . Less of the incident light is now reaching them. Furthermore, if all three pig-ments (assuming that there are three) had approximately the same absorption spectrum, it would be extremely difficult to detect an action spectrum shift for system I due to increased screening by the pigment for system III . The reasons are as follows: (1) the latter might change the relative heights of the peaks and valleys but would not significantly shift the positions of peaks, and (2) the variability of the oat curvature response would render any small changes virtually undetectable. (For a full discussion of screening effects, see Thimann and Curry, 1960.) That such physical overlapping of the various pigment systems may occur is suggested by Briggs's (1963a) studies on the localization of the region of lateral transport for systems-I and -III curvature, and Gillespie's (unpublished) observation that regions of the coleoptile below the ex-treme apex may mediate a little lateral transport for system III .

In summary, the red light-induced changes in phototropic sensitivity which are expressed as shifts of various portions of the log dosage-response curve to the left or to the right can be reasonably accounted for by the hypothesis that red light induces an increase in the amount of active pigment for system I I I which partially screens the pigment sys-tems for systems I and II . (The possibility that it may also induce increases in other nonphototropic pigments does not invalidate the hypothesis.) The small increases in sensitivity for system I, reported in-duced by small or at least short dosages of red light, are possibly re-flections of red light-induced changes in tissue sensitivity to endogenous

2 4 8 WINSLOW R. BRIGGS

auxin. If the kinetics of this latter reaction are such that it goes to completion within a few minutes of the beginning of red-light irradia-tion, it might well be detected immediately following very short red-light exposures. It would appear as an increase in the curvature obtainable from a particular blue-light dosage, but not as a shift in the threshold or maximum of the dosage-response curve. After somewhat more than an hour, increase in tissue sensitivity might well be cancelled by decrease in auxin production, at least in terms of their relative effects on the magnitude of phototropic response within a fixed time for curvature development. In this case the only noticeable effects would be dosage-response curve shifts caused by the hypothesized pigment changes.

A final case must be considered. Curry et al. (1956) found that the curvature obtained in response to short-wavelength (2800 Â) UV light was reduced by about 50% if the oat coleoptiles used were first irradi-ated for an hour with red light. The suggestion of these workers that this type of curvature might be mediated by light-induced inactivation of an active auxin complex has already been mentioned (Section 3). Since the pigments for systems I, II, and III are probably not involved in this response (largely confined to the base of the coleoptile near the node) it seems reasonable that the red light-induced change found may be purely a function of suppression of auxin production. Observations by Curry et al. on the distribution of growth along the coleoptile before and after red-light treatment support this contention. Although ulti-mately the growth of the apical portions of the coleoptile is enhanced, that of the lower regions, in which curvature occurs in this case, is sup-pressed. Perhaps the red light-induced increase in tissue sensitivity to auxin occurs primarily in the apical and more rapidly growing regions, which more than compensates for the decreased amount of auxin avail-able there.

So far nothing has been said about the photochemical nature of the various red light-induced reactions. The dosage-response curve shifts found by Curry (1957) and by Zimmerman and Briggs (1963a) are clearly partially reversible by far-red light (Briggs, 1963b), suggesting mediation by the red, far-red, photoreversible pigment phytochrome. (For a detailed account of phytochrome, see Chapter 10.) However, of the three known consequences of red-light irradiation which might affect phototropic sensitivity, only increased tissue sensitivity to auxin has been tested for far-red reversibility (Liverman and Bonner, 1953), and under their conditions it appeared fully reversible. Briggs (1963b) did not look for photoreversibility of the auxin changes with far-red light. (Although dark decay of the red light-induced change to the level of the dark controls followed roughly the same time course as dark decay of


far red-absorbing phytochrome to the red-absorbing form, these parallel changes are hardly conclusive evidence.) Furthermore, Asomaning and Galston (1961) did not look for reversibility of the red light-induced changes in pigment content. Thus, presence or absence of far-red reversi-bility of a particular process can not be used as a criterion to determine whether or not the process could mediate the phototropic sensitivity changes. The picture is further complicated by the fact that in most cases far more red-light energy was used than is normally sufficient to saturate phytochrome (about 3 X 104 ergs/cm2 is normally enough; see Borthwick and Hendricks, 1960.) Thus, in no case can the minimum energy requirement be stated and the reactions separated on this basis. Finally, Asomaning and Galston have reported that preillumination of oat or barley coleoptiles with blue light induces the same sensitivity and pigment changes as preirradiation with red light (although they give no data for the influence of blue light on oats).

At the moment, there is insufficient information available to tie all of these various reports together with any absolute certainty. Neverthe-less, from what is known it should be possible to separate the various reactions and to determine which, if any, are of primary importance in affecting phototropic sensitivity.

5. Pigments and Action Spectra A prime requirement for a clear understanding of any photobio-

logical process is positive identification of the receptor pigment. Without this identification, it is extremely difficult to determine very much about the process at a molecular level except in a most indirect fashion. Unfortunately at present there is no conclusive evidence as to the nature of the light receptor molecules for phototropism. As will become clear below (Section 6), several pigments may be involved in mediating photo-tropic responses in coleoptiles, and at least in some cases, different pig-ments may act independently. Since the current status of the pigment problem has recently been discussed in detail by Thimann and Curry (1960, 1961) and, from a somewhat different standpoint, by Galston (1959), the present section is in no sense intended as an exhaustive review.

Almost all of the action spectrum work done with coleoptiles has dealt with first-positive (system-I) curvature. Virtually nothing has been done with system II, and the small amount of work on system III will be discussed at the end of this section. The very earliest workers were aware that blue light was the most effective in inducing system-I curvature, that long-wavelength UV light was somewhat less effective, and that red light was virtually inactive (cf. Blaauw, 1909). Bachmann


and Bergann (1930), followed by Johnston (1934), and then Galston and Baker (1949a) all obtained action spectra for system-I curvature. All of these spectra showed two general peaks in the blue portion of the spectrum. Only very recently, however, have action spectra become available with sufficient points to show fine structure in the visible and long-UV regions (Shropshire and Withrow, 1958; Thimann and Curry, 1960). Thimann and Curry's action spectrum is redrawn in Fig. 6.

3400 3800 4200 4600 5000

WAVELENGTH, A

FIG. 6. Action spectrum for system-I curvature of oat coleoptiles. (From Thi-mann and Curry, 1960.)

There is a clear peak at about 3700 Â, a shoulder near 4250 Â, and two distinct peaks at 4450 Â and 4740 Â respectively. Shropshire and Withrow, using a slightly different technique, obtained approximately the same action spectrum.

Once a good action spectrum becomes available, one should nor-mally try to find a pigment molecule with a corresponding absorption spectrum. Voerkel (1933) first suggested that a carotenoid might be the photoreceptor. With the discovery by Wald and du Buy (1936) that oat coleoptiles contained a carotenoid component, with an absorption spec-trum corresponding roughly with the early action spectra in the blue region, carotenoids became the prime candidates. Bünning (1937a, 1937b) next investigated the spatial distribution of carotenoids in the oat coleoptile, using the ingenious microchemical method of Molisch whereby characteristic crystals are formed in situ, Bünning found that the greatest amount of carotenoids was located near the apex, with progressively less in the lower regions of the coleoptile. Thus, at least superficially, the distribution of carotenoids appears to parallel the distribution of phototropic sensitivity discussed earlier (Section 2). Brauner (1955) also noted that the more apical regions of the coleop-


tile contained the greatest amount of carotenoids. Unfortunately, how-ever, Bünning had found that the extreme apex, reported by Lange (1927) to be the most photosensitive region, was almost devoid of crystals. Bünning (1955) actually determined the carotenoid content of the various regions of the coleoptile. The section from 4 to 8 mm below the tip contained about 20 /Ag/gm of carotenoid per dry weight, while the apical 4-mm region contained 35.5 ftg. Further subdividing the apical region, Bünning found that the apical 2-mm contained approximately 64 /Ag/gm of carotenoid per dry weight. Finally Mrs. Sorokin (see Thimann and Curry, 1961) confirmed the earlier observations that although carotenoids were present in greatest abundance near the coleop-tile apex, the extreme tip was devoid of them; she also used the Molisch method. On the basis of this evidence, one must conclude either that carotenoids are actually absent from the extreme apex, or that they are present in amounts too small to be detected by the method used.

Meanwhile, another substance was presented as a plausible candidate for the role of photoreceptor. In 1949, Galston presented clear evidence that riboflavin could sensitize the photooxidation of a number of indole compounds including IAA. The same year, Galston and Baker (1949a) obtained an action spectrum for the process, and an action spectrum for the light-sensitized destruction of IAA by a clear brei obtained from etiolated pea epicotyls. These action spectra corresponded with the absorption spectrum of riboflavin, and also, roughly, with the action spectrum for phototropic curvature, mentioned above. Galston and Baker went on to demonstrate that riboflavin not only could sensitize the photooxidation of auxin collected in agar from oat coleoptiles, but that free riboflavin is abundant and evenly distributed throughout the oat coleoptile, with an average content of about 30 μ-g/gm dry weight. Very soon thereafter, Reinert (1953) showed, first, that ß-carotene would not sensitize the photolysis of IAA; and, second, that if the carotenoid were included in a solution containing riboflavin and IAA, it would actually protect the IAA from the photolytic action of riboflavin. These experiments gave rise to a theory which gave carotenoids the role of an internal filter in the coleoptile apex, causing a light gradient across the apex, and thus protecting the auxin on the shaded side from photoinactivation. This theory is discussed in detail by Brauner (1954), and we shall return to it shortly.

There appeared to be two appealing arguments for riboflavin, or at least a flavoprotein as the actual photoreceptor. The first argument was that a mechanism was apparently available, namely sensitization of the photoinactivation of auxin, which might account for the auxin gradient across the tip. The second was that riboflavin has an absorption spec-


trum with a broad band in the long-UV region, in just the appropriate position for the corresponding peak of the action spectrum. A serious problem, however, was that riboflavin does not possess the characteristic two-peaked absorption in the visible region, as required for a pigment to match the action spectrum, and proponents of the carotenoid hypoth-esis were quick to point this out. This aspect of the problem will be discussed in a later section.

Evidence against the carotenoid hypothesis (although not necessarily for the riboflavin hypothesis) has also been offered on rather different grounds, namely a study of the pigment content and phototropic sensi-tivity of various so-called carotenoidless mutants. The first of these (Bandurski et al, 1950, cited by Reinert, 1959), concerning a corn mutant in which the coleoptiles were deficient in carotenoids but photo-tropically sensitive, appeared as an abstract of a paper presented before the Western Section of the American Society of Plant Physiologists. A little more detail appeared in a note by Bandurski and Galston (1951) which stated that the mutant contained approximately 0.1% of the carotenoid fraction normally found in the wild type, although photo-tropic sensitivity was still within 50-80% of the wild type (see Galston, 1959). Galston (1959) also cites unpublished experiments by Labouriau and Galston (1955, again only an abstract is available) suggesting that an albino strain of barley (Colsess) seemed to show normal light sensi-tivity for system-I curvature, in the virtual absence of carotenoids. Finally, Wallace and Schwarting (1954) reported a white mutant of sunflower (Helianthus) which was "totally lacking in carotenoids" but showed "apparently normal phototropic and geotropic responses" and Wallace and Habermann (1958) come to the same conclusion regarding the pigment content of the mutant, referring to various unpublished analyses which failed to detect carotenoids. In no case is sufficient experimental data presented to allow one to examine the results critically.

On the other side of the ledger, the evidence is just as incomplete. Went (1956), referring to the experiments of Bandurski et al. (1950), states that he examined the various albino corn seedlings, and in all cases, found at least minute traces of carotenoids, but does not state what regions of the coleoptiles he examined. Thimann and Curry (1961), referring to the Helianthus mutant of Wallace and Schwarting (1954), state that its phototropic sensitivity is abnormally low, but detailed data are not given. Virgin (1957) reports some quantitative informa-tion on the carotenoid content of a white barley mutant (albina), finding the amount to be about 5% of normal, but unfortunately does not present any observations on phototropic sensitivity!


There is only a single paper in which sufficient data are available for a critical evaluation. Asomaning and Galston (1961) reported that an albino mutant of Colsess barley had about one-third the phototropic sensitivity of the normal seedling, showing at the same time that the coleoptile tips of the albino contained approximately one-third the carotenoid content of the normal tips, but approximately the same amount of flavin. In the same study, they showed that red-light treat-ment increased both the flavin and carotenoid content of oats and another barley variety (Oderbrucker), but that these changes did not seem to match the red-light-induced phototropic sensitivity changes found in the two plants.

Thus, studies with albino mutants or plants which have had their pigment content altered by previous light treatment do not as yet pro-vide reliable clues as to the nature of the light receptor molecule for system-I curvature. In considering albino mutants, another point should be considered. Bünning (1937b) calculated that the number of quanta actually involved in inducing first-positive (system-I) curvature was very small. (It appeared too low by a factor of 104 to account for the auxin differential observed on the basis of auxin destruction, assuming a quantum yield of unity.) Subsequently Galston (1959) and Thimann and Curry (1960) made estimations of the quantum efficiency of sys-tem-I curvature. In both of these papers, a number of assumptions are made, but both agree that the quantum yield is far above unity (Galston calculates it to be about 4700, and Thimann and Curry find 400, both papers conceding that the values are at best approximations). Despite slightly different assumptions about the area of the receptor surface, etc., both Galston and Thimann and Curry agree that about 2 X 108 quanta are sufficient to induce system-I curvature (5° for Thimann and Curry, maximum system-I curvature for Galston). This number refers in both cases to the actual number of absorbed, and therefore effective, quanta following corrections for scattering, etc. Thus, if the quantum yield is unity, 2 X 108 pigment molecules must be involved. Dividing this figure by Avogadro's number, we find that only 3 X 10"16 moles of pigment are necessary. Assuming that this pigment occupies a volume of approximately 0.3 mm3, the molar con-centration is about lO^ikf. It would be extremely surprising if this minute amount of pigment could be detected in an albino mutant with-out extracting an enormous number of tips. Furthermore, if in a par-ticular mutant all of the carotenoid except that involved in photo-reception for phototropism were lost, it would seem quite plausible that the total carotenoid concentration might be drastically reduced while phototropic sensitivity remained unchanged.


That coleoptile tips may actually contain some carotenoids is shown by three separate lines of evidence. First, Thimann and Curry (1960) extracted 500 oat coleoptile tips (exact size not noted) with hexane and obtained a carotenoid fraction with the absorption spectrum shown in Fig. 7 (solid line). Second, Briggs (unpublished) extracted 1000 0.5-mm tips of corn coleoptiles in acetone and water, and transferred the pig-ment to petroleum either, obtaining a carotenoid fraction with the absorption spectrum shown in Fig. 7 (dashed line). The positions and

3400 3800 WAVELENGTH, A

4600

FIQ. 7. Solid line: absorption spectrum of hexane extract of 500 oat coleoptile tips (from Thimann and Curry, 1960). Dashed line: absorption spectrum of extract of 1000 corn coleoptile tips in petroleum ether. Ordinate arbitrary.

heights of the peaks and valleys of these two extracts relative to those of the action spectrum, are shown in Table IV. Clearly, at least in the visible region of the spectrum, the coincidence is fairly good. Third, Thimann and Curry (1960, 1961) measured the absorption spec-trum of 32 oat coleoptile tips in vivo, using the ingenious opal glass technique developed by Shibata et al. (1954). These investigators found clear evidence for a yellow pigment in these tips, with peaks at about 4500 Â and 4800 Â. There was also a peak at about 4250 A, but, in the authors7 own words: "Unfortunately, the interesting 370 τοημ region is completely obscured by the very steep rise in the curve" due, pre-sumably, to scattering and "end-absorbing" materials. None of these three studies, however, answers the crucial question concerning whether or not carotenoids are found in the very extreme apex, although they indicate that this is a possible condition.

Thus far, little has been said about the action spectrum peak in the long-UV region. A number of workers have looked either for a flavin derivative with a double peak in the visible or a carotenoid with a


TABLE IV CHARACTERISTICS OF THE ACTION SPECTRUM FOR SYSTEM I PHOTOTROPIC CURVATURE

COMPARED WITH THOSE OF THE ABSORPTION SPECTRA OF EXTRACTS OF 500

Avena COLEOPTILE TIPS (IN HEXANE) AND OF 1000 CORN COLEOPTILE TIPS

(IN PETROLEUM ETHER)

Location of maxima and Ratio of height to height of minimum (Â) principal peak

Action spectrum

3700 4250 4450 4620 (min.,

est.) 4740

Oat tip extract

4220 4420 4600 (min.,

est.) 4720

Corn tip extract

— 4190 4420 4590

4720

Action spectrum

0.53 0.70 1.00 0.73

0.82

Oat tip extract

0.26 0.78 1.00 0.70

0.88

Corn tip extract

0.22 0.78 1.00 0.76

0.85

respectable peak in the long-UV region. Neither the pigment extracted from oat coleoptile tips by Thimann and Curry nor that extracted from corn coleoptile tips by Briggs shows an appreciable absorption in this region. Although certain possible compounds have been found both among carotenoids and flavins, none of them really fits very well to the action spectrum. Even if they did, there would remain the problem of demonstrating that the compounds occurred within the phototropically sensitive region of the coleoptile. (For a detailed discussion of this matter, see Thimann and Curry, 1960.) Thus although in time this sort of study may prove fruitful, it has not at present solved the problem.

Let us now consider other current hypotheses. On the basis of Reinert's (1953) experiments with pigment mixtures and IAA in vitro, the screening hypothesis mentioned briefly above was developed. This hypothesis proposed that riboflavin was the primary photoreceptor for system-I curvature, accounting for the broad UV peak in the action spectrum. It then suggested that in the visible region, the action spec-trum was modified by the presence of carotenoids, known to absorb in that region to give the characteristic two-peaked structure. Although this theory is ingenious, it must be discarded for two reasons: first, it assumes photoinactivation of auxin, something not found for system-I curvature (see Section 3) ; and second, Thimann and Curry (1960) have presented an elegant mathematical analysis showing that the kind of screening suggested could not possibly transform the action spectrum in the manner proposed. Therefore, we must turn to other possibilities.

2 5 6 WINSLOW R. BRIGGS

There are a number of ways to account for the action spectrum. First, the spectrum might correspond directly to a pigment with the appropriate absorption spectrum. At present, such a pigment has not been isolated. Second, it might be the consequence of conjugation of a pigment [such as the carotenoid extracted by Thimann and Curry (1960) or Briggs] with a protein or some other moiety in the cell which would confer upon it an absorption band in the long-UV region. Indeed, Nishimura and Takamatsu (1957) have demonstrated a carotenoid-protein complex in spinach leaves and have compared its absorption spectrum (in water) with that of the ß-carotene (in hexane) which could be separated from it. There appear to be shifts in absorption peaks, but this particular example does not provide a peak in the long-UV region. Third, the action spectrum may be the consequence of activation not of one but of two pigments.

This last suggestion may be subdivided into three possibilities. First, the two pigments, absorbing respectively in the UV and the visible regions, may mediate lateral transport independently. This seems un-likely, in view of the similar shapes of the dosage-response curves at 3650 Â and 4360 Â, but at the moment, this possibility cannot be con-clusively eliminated. Second, one pigment may be the primary photo-receptor, directly linked to the lateral transport mechanism. The other may be closely associated with it, absorbing light energy and passing it to the first by resonance transfer or some other means. Such a trans-fer is well-known in photosynthesis (see, for instance, Duysens, 1951; Franck et al., 1941). At the moment, there is no direct evidence either for or against this hypothesis. Third, one pigment may be the primary photoreceptor and may receive energy from the other by radiant trans-fer, perhaps over relatively long distances. Shropshire and Withrow (1958) have suggested this possibility on the basis that they have observed that if coleoptiles are irradiated with high-intensity light at 3650 Â, blue fluorescent areas are clearly visible near the tip. Thimann and Curry (1960) also comment on the possibility of absorption of UV light by a substance which fluoresces in the visible region, citing as evidence the occurrence of a large number of fluorescent substances in plants.

Recent experiments in the author's laboratory support the last possi-bility mentioned. The top centimeter of 90 72-hour-old oat coleoptiles, with primary leaves removed, was ground in a 1/15 M, pH 6.2 phos-phate buffer. The brei obtained was centrifuged at 30,000 g for 20 minutes and the clear supernatant, made up to 6.5 ml with buffer, was transferred to a cuvette for spectrofluorimetric assay. (A Farrand spectrofluorimeter was kindly made available by Dr. Victor Burns of


the Biophysics Laboratory of Stanford University.) With an excitation wavelength of 3300 Â, the fluorescence spectrum shown in Fig. 8 was

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^ „ ^ s ^*"-

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1 1 1 1 1 1 1 1 1 1 1 i 4000 4500 5000

EMISSION WAVELENGTH, A

FIG. 8. Fluorescence emission spectrum for buffer extract of oat coleoptiles excited by light of wavelength 3300 Â. Dashed line roughly represents scattering curve. Ordinate arbitrary.

obtained. Since the supernatant being tested scattered some light, the steeply descending curve represents scattered light, progressively less reaching the photosensitive element as the wavelength at which emis-sion is being measured recedes from the excitation wavelength. Despite this scattering curve, however, a clear peak in the neighborhood of 4500 Â is visible. This fluorescence peak is just where phototropic sensi-tivity is near maximum according to the action spectrum. Then, to determine which wavelengths were most effective in exciting fluores-cence, emission at 4500 Â for different exciting wavelengths, was deter-mined with the results shown in Fig. 9. Again, the steep curve, as the excitation wavelength approaches the wavelength at which emission is being measured, is a consequence of scattering, but between 2900 Â and 3900 Â, there is an unmistakable broad peak, in the right region to account for the action spectrum for phototropism. Although experi-ments must be done to identify the fluorescent component or com-ponents and to localize these substances in the coleoptile, the results mentioned above are at least informative. In summary, the pigment for system-I phototropic curvature is probably a carotenoid, possibly the one noted by Thimann and Curry (1960) or Briggs (unpublished). Both of these pigments are fully able to account for the action spectrum in


en if)

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EXCITATION WAVELENGTH, Â

FIG. 9. Action spectrum for excitation of fluorescence from buffer extract of oat coleoptiles. Fluorescence measured at 4500 Â. Dashed line roughly represents scattering curve. Ordinate arbitrary.

the visible region of the spectrum. In the long-UV region, however, another unknown pigment (possibly a flavin, since many flavins are highly fluorescent) absorbs the light energy, fluorescing at a longer wavelength close to the maxima in the visible region for the action spectrum, and therefore inducing curvature. The numerous studies of phototropic sensitivity of "carotenoidless" mutants do not really negate this argument, since only extremely small amounts of pigment would be sufficient to confer normal sensitivity, and these amounts might well remain virtually undetected by the various carotenoid assays used. Furthermore, even if all but 0.1% of the carotenoid complement of a particular plant is missing in the mutant, this 0.1% might well be sufficient to account for phototropic sensitivity if it were all localized in the appropriate photosensitive region.

Concerning system-Ill curvature, very little can be said about pig-ments and action spectra. Haig (1934) presented an action spectrum for this system, but his curve shows little detail except steep decline between 4000 Â and 5000 Â. Asomaning and Galston (1961) have re-cently published an action spectrum for the phototropic curvature of barley coleoptiles. Their curve also slopes down sharply between 4000 Â and 5000 Â (additional evidence for their suggestion that barley possesses only system-Ill curvature, see Section 2). Since the reci-procity law is not valid for system-Ill curvature, and the magnitude of the response apparently depends upon irradiation time only (see Sections 2 and 6), an action spectrum, as these workers justly point


out, is very difficult to obtain. Asomaning and Galston attempted to standardize the response by using identical irradiation times and inten-sities at each of the different wavelength values, and then calculating the number of quanta necessary to obtain a given fixed response at each wavelength station. Since their calculations are not shown, it is not possible to know whether these investigators arrived at their final values by assuming that response is proportional to dosage or to exposure time. Nevertheless, their action spectrum does show peaks at roughly 4250, 4500, and 4800 Â, again suggestive of a carotenoid. Clearly further investigation is needed before any more specific conclusions can be drawn.

6. Kinetic Studies The detailed phototropic dosage-response curves obtained for oat

coleoptiles by using a series of fixed intensities of monochromatic light (4358 Â) have already been mentioned (Section 2, Fig. 4, see Zimmer-man and Briggs, 1963a). For each intensity, dosage was varied by varying exposure time, and curves were obtained both for plants with prior red-light treatment and for those without. On the basis of these curves, Zimmerman (1962) and Zimmerman and Briggs (1963b) have proposed a kinetic model for the various phototropic responses. This model will now be considered in some detail.

Zimmerman (1962) originally assumed that the phototropic re-sponses mediated by systems I and II were proportional to the number of pigment molecules excited on the lighted side minus the number excited on the shaded side. If, as originally suggested on qualitative grounds by Briggs (1960), an activated pigment molecule could be subsequently inactivated by absorption of a second quantum, Zimmer-man's assumption provides for the production of positive and negative curvature with a single pigment. With fairly high light dosages, all of the molecules on the lighted side might be inactivated while some on the shaded side were still activated, and negative curvature might be expected. Zimmerman idealized the shape of the coleoptile tip to a hemispheroid with major and minor axes of 200 and 100 microns in length respectively. He further assumed that the kinetics of the process obeyed a simple one hit photochemical conversion of one molecular species to another in a constant flux of photons. Other assumptions were that the attenuation of light across the coleoptile tip obeyed an ex-ponential law, that the coleoptile was homogeneous, and that the pigment distribution within the tip could be described as a function of the three coordinates of the hemispheroid. Zimmerman then calculated theoretical dosage-response curves for a number of different absorption


FIG. 10. Shadowgraphs illustrating the various types of phototropic curvature of oat coleoptiles. Intensities (I) are in Einsteins/cm3/sec at 4358 Â. Upper left, system-I curvature (/ = 1.4 X 10"u, t = 175 sec, 2 hours red light). Upper right, system-II curvature (J = 1.4 X 10"1*, t = 325 sec, no red light). Lower left, system-


coefficients for the apex, including those both far larger and far smaller than actually measured. He used two extremes of pigment distribution, either uniform surface or uniform volume, and a number of possible kinetic mechanisms. Unfortunately none of the theoretical curves even approximated the experimental dosage-response curves shown in Fig. 4. The measured light gradient across the tip was too small to account for the width of the experimental curves.

Zimmerman (1962) then rejected the assumption that the light gradient determined the magnitude of the response, and proposed in-stead that it determined only the direction of response. Brauner (1955) also concluded that lateral transport of auxin in the extreme apex was independent of the light gradient. Magnitude would then simply depend upon the number of pigment molecules activated. Zimmerman (1962) also suggested that systems I, I I , and I I I might be the consequence of three separate and possibly distinct mechanisms. Evidence for this latter proposal (Zimmerman and Briggs, 1963a) is, first, that red light increases the sensitivity of system I I I to blue light, but decreases the sensitivity of systems I and II . Evidence that systems I and I I may in addition be independent is based on the observation of plants that should contain components of both types of curvature. Figure 10 shows typical examples. Plants exposed to light dosages inducing both posi-tive and negative components, whether the positive component is from system I or system III , are distinctly S-shaped. Thus it is hard to believe that negative curvature is simply a consequence of reversal of the mechanism for positive. Net curvature is the sum of two com-ponents, and both clearly can develop simultaneously in a single coleoptile.

The first ascending portion of the dosage-response resembles the curve one would expect from a greater-than-zero Poisson distribution, or from the simple conversion of one molecular species to another with the rate proportional to the concentration of the first (Curry, 1957; Zimmerman, 1962; Zimmerman and Briggs, 1963b). This curve may be described by the following equation:

R = K(l - e~kIt) (1)

where R is phototropic response, K and k empirical constants, I the intensity of blue light, and t the exposure time. This equation actually

III curvature ( / = 6.6 X 10"12, t = 1710 sec, 2 hours red light). Lower right, curvature resulting from sum of system II plus system III curvature (/ = 1.4 X 10"13, t = 1000 sec, no red light). Net curvature was about 14° positive (Zimmerman and Briggs, 1963a).


describes the simplest mechanism by which light can convert an inactive molecule to an active species.

It was reasoned that the whole dosage-response curve for systems I and II might be described by a combination of similar terms, each corresponding either to the formation of an active species for system I or II, or to subsequent inactivation of these active species by absorp-tion of a second quantum. On this basis, the following empirical equa-tion was presented:

R = KM1 - er*") - (1 - e-*2l0] - KJL0- - e~Ä3l0 - (1 - er*")]} (2) The four terms correspond, respectively, to activation and inactivation of system I pigment, and activation and inactivation of system II pigment. A function of this form was successfully fitted to the existing dosage-response curves for systems I and II alone (see Figs. 11 and 12, lowest curves).

FIG. 11. Theoretical phototropic dosage-response curves for oat coleoptiles at three intensities of monochromatic blue light. Plants given no red light treatment. Experimental points for these intensities (from Fig. 4) shown for comparison. In-tensities (I) in Einsteins/cmVsec at 4358 Â. [From Zimmerman and Briggs (1963b).]

Next the two following kinetic schemes were proposed: hv hv

System I : x —» y —> z kil Jc2l

System II: x ' 4 y ' 4 z ' ksl kd

where x and x' represented the original unactivated pigments for systems I and II respectively, y and y' the activated forms, and z and z' inacti-


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FIG. 12. Theoretical phototropic dosage-response curves for oat coleoptiles at three intensities of monochromatic blue light. Plants given 2 hours red light. Experimental points (from Fig. 4) shown for comparison. Lowest curve, system I and II alone. Intensities (7) in Einsteins/cm2/sec at 4358 Â. [From Zimmerman and Briggs (1963b).]

vated forms. The authors assume that pigment recovery following in-activation, or de novo pigment synthesis, is unimportant over the period of the experiment. Briggs (1960) presents evidence that there is at least some justification for this assumption. He exposed corn coleoptiles to high light dosages which did not induce curvature (presumably because x and x7 had all been converted to z and z' respectively). After this treatment, a full 20 minutes were required for the recovery of normal phototropic sensitivity. Briggs suggested that the high dosages com-pletely inactivated the phototropic pigment, and that the 20 minutes were required for this pigment either to be restored or replaced.

Considering for the moment system I only, the following differential equations are readily obtained from the kinetic scheme (Zimmerman, 1962; Zimmerman and Briggs, 1963b):

dx Ί T

Ht = ~hIx

dy dt

= kilx — k2Iy

Î - hIy

(3)

(4)

(5)


Zimmerman (1962) has shown that solutions for these equations are readily obtainable by standard procedures. Since the amount of y formed in a given exposure time is what presumably determines the phototropic response, the solution for y is given:

= *(0) (j^k) [(1 - e~klIt) - (1 - e-™')] (6)

where y(t) is the concentration of y at time t after the start of illumination, and x(0) is the concentration of x at the start of illumina-tion. Thus phototropic response is assumed to be proportional to the concentration of y, namely y(t), and the equation is precisely the form required by the empirical curves [Eq. (2)]. A similar set of equations can be solved for system II, for ^ ( i ) , with the net photo-tropic response from both systems equal to y —y'. The authors deter-mined the values for the various constants which best fitted the empirical curves, and on the basis of these values the curves shown in Figs. 11 and 12 were drawn. One of the most interesting observations is that pretreatment with red light decreased by a factor of four the values of all four constants for the two kinetic schemes. (The implica-tions of these red-induced changes have already been discussed in Section 4.)

The independence of system III from total dosage, and its de-pendence upon exposure time has already been mentioned (Section 2, Fig. 5). Empirically, system III phototropic response simply equals Kt. If the theoretical curves for systems I and II are subtracted from the various experimental curves, as mentioned earlier, and the remainder simply plotted as a function of time, two straight lines are obtained, the slope being steeper for the red light-treated and more sensitive plants (Fig. 5).

Once again a kinetic scheme was proposed (Zimmerman, 1962; Zimmerman and Briggs, 1963b) :

ail a%

System III : u ^± v —» w a— J

where w is the phototropically active component, and u and v are pre-cursors. At the start of illumination, u is presumed to go rapidly to equilibrium with v, the reaction being strongly photoreversible. Then, v would decay thermochemically to w, the phototropically active moiety. Differential equations were obtained which led to the following approx-imate solution for w(t) for sufficiently large values of t:

8. PHOTOTBOPISM IN HIGHER PLANTS 265

««) « «(0) (-ψ-) t (7) \ai -f- a-i/

where w(t) is the amount of w formed after time t in the light, and u(0) is the initial concentration of u, presumably the primary photo-receptor. This equation is just the R = Kt required by the empirical observations. The authors acknowledge that the same simple relation-ship could be derived from a number of other functions, but point out that this particular scheme fits the observed dosage-response curves quite closely. The form Kt may be the limiting case of -4(1 — e-**) which becomes Aat for small values of αέ, as determined by a power series expansion. In this case, w(t) would have the behavior of a single ex-ponential term, independent of intensity, as required by the empirical curves.

This last kinetic scheme predicts several other things which can be tested experimentally. It predicts, first, that flashing light should enhance the amount of curvature obtained from a given dose and intensity, over the amount obtained from the same dosage given con-tinuously. The authors cite flashing light experiments by Briggs (1960) to support this contention, and give a detailed mathematical treatment for flashing light experiments. It is obvious that if v can only decay in the dark to w, and not to u, administering a given dosage of light in flashes should increase the amount of w formed per unit dosage. Un-fortunately an insufficient number of flashing-light experiments have been done to date to give the hypothesis a critical test.

The last kinetic scheme also predicts certain temperature effects. Of all of the reactions in the kinetic schemes for systems I, II, and III, only the decay of v to w, governed by the constant a2, is a dark re-action. One would therefore expect this constant to be temperature-de-pendent. If plants were phototropically induced at different temperatures, and then all allowed to develop curvature at, say, 25°C, only that curvature mediated by system III should show temperature sensitivity. Zimmerman and Briggs (1963b) cite unpublished experiments by Bar-bara Koch, in the present author's laboratory, showing that system-Ill curvature induction is indeed temperature-sensitive, but not system I or II. Experiments at 5°C are consistent with the interpretation that «2 is reduced by a factor of about three from its value at 25°C.

Zimmerman and Briggs (1963b) point out that any finite, continuous, single-valued function can be described either by a Fourier series or a power series if a sufficient number of terms are taken and the constants adjusted appropriately. Since the kinetic model discussed above depends upon a number of constants, it might well be criticized on these grounds.


Furthermore, although it is consistent with virtually all of the experi-mental data assembled in the author's laboratory, there is one case (Briggs, 1963b) in which lateral transport of auxin was found in corn coleoptiles in the absence of any appreciable phototropic curvature. This observation is not in keeping with the assumption that phototropic curvature is a direct function of the amount of auxin translocated, which in turn is a direct function of the number of pigment molecules excited.

Although the model is quite possibly an oversimplification, there exist the following points in its favor: it can readily be derived from kinetic schemes representing first-order photochemical reactions; it provides excellent fit for the experimental dosage-response curves for oat coleop-tiles, except when light intensity is extremely low and exposure time long, in which case factors other than pigment activation may well be-come limiting; it is consistent with the red light-induced shifts in the phototropic dosage-response curve described earlier (Section 4) ; it predicts certain flashing-light and temperature effects which preliminary experiments have confirmed; and, finally, it presents a number of oppor-tunities for experimental test. On this last basis alone, even if the model represents a gross oversimplification, it at least provides a direction for further experimentation.

7. Discussion A schematic summary of the general features of systems I, II, and

III curvature is given in Fig. 13. There remain, however, several loose ends which require brief treatment. First, little has been said in the present article either about the Blaauw theory for phototropism or about the theory proposing the action of light on the auxin synthesis mechanism. Most of the arguments that are applicable against auxin destruction are equally applicable against the latter theory. In view of the persistence of lateral transport long after the end of the light stimulus, and the absence of change in auxin production following ir-radiation, it seems highly unlikely that an effect of light on auxin syn-thesis is involved in systems I, II, or III. However, the third-positive curvature mechanism discussed briefly at the beginning of the article has not been given the same detailed investigation as the three systems above, and light-sensitized inactivation of auxin or of one of the enzymes for its synthesis might well be involved. So many of the earlier reports did not take into account either the failure of the reciprocity law for system-Ill curvature, or red-light effects on auxin production or tissue sensitivity to auxin, that this author finds the literature ex-tremely difficult to interpret.

For the light-growth reaction of Blaauw, there may well be several


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FIG. 13. Schematic summary of systems I, II, and III curvature of oat coleoptiles. A phototropic dosage-response curve obtained with high intensity monochromatic light. Definitions: x, x', u (primary photoreceptor molecules); y» y'» w (phototropically active forms) ; z, z' (light-inactivated forms) ; v (light-activated form of u; may be photochemically reconverted to u or decay thermo-chemically to w) ; It (light reaction) ; dk (dark reaction) ; RL (red light).

components. Already discussed are the effects of red light on tissue sensitivity to auxin and on auxin production. Illumination of coleoptiles with white light would undoubtedly activate both of these reactions, either or both of which might modify growth rate, but neither of which would contribute to phototropic curvature. However, there is increasing evidence that there are other photomorphogenetic reactions which are induced by relatively high energies of blue or far-red light (see Mohr, 1962). The problems of sorting out these various reactions and determining which, if any, contribute to phototropic curvature, have simply not been solved.

Perhaps the beginning of an understanding of the curvatures medi-ated by systems I, I I , and I I I has been established, but a great many questions remain unanswered. Are these various systems unique to coleoptiles or do they occur widely throughout the plant kingdom? By what mechanism can activation of a pigment molecule affect the trans-port of an auxin molecule? Will the present kinetic model withstand rigorous testing?


There is a large literature on the phototropic responses of the sporangiophores of various fungi which has not been covered in the present chapter (see Banbury, 1959; Curry and Thimann, 1960; Page, 1962; Shropshire, 1963). The two most widely studied genera in this literature have been Phycomyces and Pilobolus. There is no question that light-growth reactions occur in both genera. Blaauw (1918) and Buder (1918) suggested that phototropic curvature of the Phycomyces sporangiophore was at least partially attributable to its light-focusing properties. Unilateral light is focused in such a way that the intensity of the light on the side of the sporangiophore distal to the light source is greater than on the proximal side. Thus unequal positive light-growth reactions cause positive phototropic curvature. Buller (1934) extended this lens hypothesis to the more complex system found in Pilobolus.

Unfortunately, careful studies of the light-growth reactions of Phy-comyces sporangiophores show that they are transient. The organs quickly adapt to changes in light intensity and resume their original growth rate. Phototropic curvature, however, may continue to develop over long periods of time, and one is forced to the conclusion that the tropic response is not just a reflection of differential light-growth reac-tions. Thus even in these relatively simple structures, at least two reactions to light occur (their action spectra are quite similar) and the problems of unravelling them are manifold. A detailed discussion of Phycomyces is to be found in the review by Shropshire (1963).

For very young sporangiophores of Pilobolus (as opposed to more mature ones) even the lens hypothesis does not seem tenable. Upon unilateral illumination, these organs first stop growing (negative light-growth reaction). They then respond by initiation of growth on the illuminated side of the tip. Page (1962) discusses the phototropic be-havior of these structures as well as that of mature sporangiophores in detail. In neither Phycomyces nor Pilobolus has the phototropic pigment been conclusively identified. Are the response mechanisms for these sporangiophores homologous, analogous, or entirely unrelated to those of coleoptiles? There is no evidence that auxin plays a role either in growth or tropic responses of the fungi. Thus, although phototropism represents a relatively old area of photobiology, much remains to be done, both on higher and on lower plants.

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Bachmann, F., and Bergann, F. (1930). Planta 10, 744-755. Ball, N. G., and Dyke, I. J. (1954). / . Exptl. Botany 5, 421-433. Banbury, G. H. (1959). In "Handbuch der Pflanzenphysiologie" (W. Ruhland,

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New York. Bünning, E. (1937a). Planta 27, 148-158. Bünning, E. (1937b). Planta 27, 583-610. Bünning, E. (1955). Z. Botan. 43, 167-174. Bünning, E., Reisener, H. J., Weygand, F., Simon, H., and Klebe, J. F. (1956).

Z. Naturforsch, l ib , 363-364. Bunsen, R., and Roscoe, H. (1862). Ann. Phys. Chem. 117, 529 -562. Burkholder, P. R., and Johnston, E. S. (1937). Smithsonian Inst. Misc. Collections

95, No. 20, 1-14. Ching, T. M., and Fang, S. C. (1958). Physiol. Plantarum 11, 722-727. Cholodny, N. (1924). Ber. deut. botan. Ges. 42, 356-362. Curry, G. M. (1957). "Studies on the Spectral Sensitivity of Phototropism." Ph.D.

Dissertation, Harvard University, Cambridge, Massachusetts. Curry, G. M., Thimann, K. V., and Ray, P. M. (1956). Physiol. Plantarum 9, 429-

440. Darwin, C , and Darwin, F. (1881). "The Power of Movement in Plants." Appleton-

Century, New York. Dolk, H. E. (1936). Rec. trav. botan. néerl. 33, 509-585.


du Buy, H. G. (1933). Rec. trav. botan. néerl. 30, 798-925. du Buy, H. G., and Nuernbergk, E. (1934). Ergeb. Biol. 10, 207-322. Duysens, L. N. M. (1951). Nature 168, 548-550. Franck, J., French, C. S., and Puck, T. T. (1941). / . Phys. Chem. 45, 1268-1300. Fröschel, P. (1908). Sitzber. kais. Akad. Wiss. Math-naturw. Kl 107, 235-256. Galston, A. W. (1949). Proc. Natl. Acad. Sei. U. S. 35, 10-17. Galston, A. W. (1950). Botan. Rev. 16, 361-378. Galston, A. W. (1959). In "Handbuch der Pflanzenphysiologie" (W. Ruhland, ed.),

Vol. 17/1, pp. 492-529. Springer, Berlin. Galston, A. W., and Baker, R. S. (1949a). Am. J. Botany 36, 773-780. Galston, A. W., and Baker, R. S. (1949b). Science 109, 485-486. Galston, A. W., and Baker, R. S. (1951). Am. J. Botany 38, 190-195. Galston, A. W., and Hand, M. E. (1949). Am. J. Botany 36, 85-94. Galston, A. W., Bonner, J., and Baker, R. S. (1953). Arch. Biochem. Biophys. 42,

456-470. Gillespie, B., and Thimann, K. V. (1961). Experientia 17, 126-129. Gordon, S. A., and Eib, M. (1956). Pfant Physiol. 31, xiv (abstract). Haig, C. (1934). Proc. Natl Acad. Sei. U. S. 20, 476-479. Haig, C. (1935). Biol. Bull. 69, 305-324. Johnston, E. S. (1934). Smithsonian Inst. Misc. Collections 92, No. 11, 1-17. Labouriau, L. G., and Galston, A. W. (1955). Plant Physiol. 30, xxii (abstract). Lange, S. (1927). Jahrb. wiss. Botan. 67, 1-51. Liverman, J. L., and Bonner, J. (1953). Proc. Natl. Acad. Sei. U. S. 39, 905-916. Mohr, H. (1962). Ann. Rev. Plant Physiol 13, 465-488. Nishimura, M., and Takamatsu, K. (1957). Nature 180, 699-700. Page, R. (1962). Science 138, 1238-1245. Reinert, J. (1950). Z. Naturforsch. 5B, 374-380. Reinert, J. (1953). Z. Botan. 41, 103-122. Reinert, J. (1959). Ann. Rev. Plant Physiol. 10, 441-458. Reisener, H. J. (1957). Naturwiss. 44, 120. Reisener, H. J. (1958). Z. Botan. 46, 474-505. Reisener, H. J., and Simon, H. (1960). Z. Botan. 48, 66-70. Schrank, A. R. (1950). Ann. Rev. Plant Physiol. 1, 59-74. Scott, T. K., and Briggs, W. R. (1960). Am. J. Botany 47, 492-499. Shibaoka, H., and Yamaki, T. (1959). Botan. Mag. Tokyo 72, 152-159. Shibata, K., Benson, A. A., and Calvin, M. (1954). Biochim. et Biophys. Acta

15, 461-470. Shropshire, W., Jr. (1963). Physiol. Revs. 43, 38-67. Shropshire, W., Jr., and Withrow, R. B. (1958). Plant Physiol. 33, 360-365. Sierp, H., and Seybold, A. (1926). Jahrb. wiss. Botan. 65, 592-610. Steeves, T. A., and Briggs, W. R. (1960). J. Exptl. Botany 11, 45-67. Tetley, U., and Priestley, J. H. (1927). New Phytologist 26, 171-186. Thimann, K. V., and Curry, G. M. (1960). In "Comparative Biochemistry" (M.

Florkin and H. S. Mason, eds.), Vol. I, pp. 243-306. Academic Press, New York. Thimann, K. V., and Curry, G. M. (1961). In "Light and Life" (W. D. McElroy

and B. Glass, eds.), pp. 646-670. Johns Hopkins Press, Baltimore, Maryland. Van Overbeek, J. (1933). Rec. trav. botan. néerl 30, 537-626. Van Overbeek, J. (1936). Rec. trav. botan. néerl 33, 333-340. Van Overbeek, J. (1939). Botan. Rev. 5, 655-681. Virgin, H. I. (1957). Physiol. Plantarum 10, 170-186.


Voerkel, S. H. (1933). Planta 21, 156-205. von Guttenberg, H. (1959). Planta 53, 412-433. Wald, G., and du Buy, H. G. (1936). Science 84, 247. Wallace, R. H., and Haberman, H. M. (1958). Plant Physiol. 33, 252-254. Wallace, R. H., and Schwarting, A. E. (1954). Plant Physiol. 29, 431-436. Went, F. W. (1926). Koninkl. Akad. Wetenschap., Proc. 30, 10-19. Went, F. W. (1928). Rec. trav. botan. néerl. 25, 1-116. Went, F. W. (1956). In "Radiation Biology" (A. Hollaender, ed.), Vol. III, pp.

463-478. McGraw-Hill, New York. Went, F. W., and Thimann, K. V. (1937). "Phytohormones," pp. 1-294. Macmillan,

New York. Wilden, M. (1939). Planta 30, 286-288. Wildman, S. G., and Bonner, J. (1948). Am. J. Botany 35, 740-746. Zimmerman, B. K. (1962). "An Analysis of Phototropic Curvature in Oat

Coleoptiles." Ph.D. Dissertation, Stanford University, Stanford, California. Zimmerman, B. K., and Briggs, W. R ; (1963a). Plant Physiol 38, 248-253. Zimmerman, B. K., and Briggs, W. R. (1963b). Plant Physiol. 38, 253-261.

Chapter 9

SOME EFFECTS OF LIGHT ON CHLOROPLASTS AND PLANT PROTOPLASM

Hemming I. Virgin

Department of Plant Physiology, University of Göteborg Göteborg, Sweden

1. General Introduction The predominant role of light in the life of the green plant is mani-

fested in photosynthesis. Besides this process, however, there are other important light-induced reactions, which play certain roles in order for photosynthesis to proceed at an optimal rate. In this chapter three such responses will be described.

As the chlorophyll pigments are the main prerequisites for the light absorption in photosynthesis—and their formation is a light-dependent process—this reaction will be considered first. Second, the peculiar move-ments of the chloroplasts will be discussed. The real significance of these movements is not yet fully understood but they are no doubt of impor-tance for the maintainance of a high photosynthetic rate. Finally, the light-induced protoplasmic streaming will be described. Here we are dealing with a puzzling phenomenon which caught the interest of plant physiologists a long time ago, but is still an unsolved question. Sup-posedly protoplasmic streaming contributes to the translocation of the varied metabolic products within the cell, particularly the photosyn-thetic products.

2. Chlorophyll Formation

2.1 Introduction

Except for a few cases, e.g., seedlings of gymnosperms and cotyledons of some angiosperms, higher plants kept in darkness do not develop any chlorophyll. Under these conditions they turn yellowish green owing to the presence of xanthophylls and other carotenoids. If the plants are exposed to continuous light, they slowly turn green owing to the forma-tion of the chlorophylls a and b. This light-dependent chlorophyll forma-

273

274 HEMMING I. VIRGIN

tion consists of several different light-induced reactions which all together form what is called the "greening process" (Smith, 1961).

2.2 Pigment Changes in Briefly Irradiated, Dark-Grown Plants

One of the first stages—if not the first—in the series of reactions resulting in greening is a rapid formation of small amounts of chloro-phyll a.

This stage can easily be studied on dark-cultivated seedlings of, for example, beans, barley, wheat, or corn. In such material it is possible to detect spectrophotometrically (very dim light) an absorption peak at around 650 τημ, whereas chlorophyll a has an absorption around 677 ιημ (Shibata, 1957). A substance having this absorption can be extracted from the seedlings in the dark with such various organic solvents as methanol, acetone, ethyl ether, etc. The peak shifts a little depending on the solvent used (Smith and Benitez, 1955). If the seed-lings have been irradiated prior to the extraction, only chlorophyll a can be found in the extract. An exposure of only a few minutes in ordinary daylight is required to induce formation of chlorophyll a. Owing to the strong fluorescence of the substance present in the dark as well as of chlorophyll a it is also possible to follow this shift in the peaks by measuring the continuous change of the fluorescence spectrum of the irradiated leaves (Fig. 1). The peaks of the fluorescence spectra are shifted a little toward longer wavelengths as compared to the position of the peaks in the absorption spectra of the two substances.

It is, however, not possible to notice any change of leaf color by the naked eye after an impulse of light short enough to cause this shift in the spectrum.

The disappearance of the substance with the absorption peak at around 640-650 ιημ is quantitatively correlated to the chlorophyll a simultaneously appearing (Koski et al., 1951). From this the conclusion may be drawn that the substance present in the dark is the precursor to chlorophyll a. It has therefore been named protochlorophyll.

In the light it is not possible to find any trace of protochlorophyll in normal, green leaves. This is due to the photosensitivity of the reaction: protochlorophyll -» chlorophyll a. But if a green leaf is placed in the dark new protochlorophyll is formed until a certain level is reached, the height of which depends on, among other factors, the age, the temper-ature, and the chlorophyll a concentration of the leaf (Virgin, 1961a). In leaves where just the protochlorophyll originally present has been transformed to chlorophyll a by a short impulse of light the level of protochlorophyll will become particularly high.

The transformation takes place even at temperatures far below the

9. CHLOROPLASTS AND PLANT PROTOPLASM 275

650

3

LU U

22 8 U £T O 13 _ l LL

1

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Λ/Υ Vi 1 /Λ / ι / l I y γ y \\ 1 \ΛΛ V 1 \ Y \ \\ / W \ \\ i L / ^ \ V 1 7>\V\ v 1 / \ \ \ v 1 / ■ * ^ . \ \ . ν 1

^ 5 ^ ^ S ^ ^C^^w^^VH

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FIG. 1. Fluorescence spectra of dark-grown barley leaves after irradiation with red light (660 ταμ; 33 ergs/cm2/sec) for different periods of time indicated. One unit for the fluorescence equals about 0.0005% of protochlorophyll, calculated on a fresh-weight basis. (After Virgin, 1955a.)

+ 20 WC

FIG. 2. Percentage transformation of protochlorophyll to chlorophyll a in dark-grown barley leaves when irradiated for 10 min with 100 ft-candles incandescent light at different temperatures. (After Smith and Benitez, 1954.)

276 HEMMING I. VIHGIN

freezing point. In Fig. 2 it can be seen that even at as low a temperature as —77°C the transformation is as high as 33%. The irregularities seen in the figure between —10°C and — 20°C are probably caused by freezing damages to the tissue owing to the formation of ice crystals within this temperature range. The progressive lowering of the trans-formation percentage with a lowering of temperature also suggests that the reaction, which is of a second-order type (Smith and Benitez, 1954; Virgin, 1955a), is not strictly a photochemical intramolecular process, but involves intermolecular interactions as well.

2.3 Action Spectrum for the Protochlorophyll Transformation

The action spectrum for the transformation of protochlorophyll into chlorophyll a corresponds closely to the absorption spectrum for proto-chlorophyll (Fig. 3). I t is therefore evident that by absorbing light this pigment is converted to chlorophyll a.

300

(Λ (Λ LU Z ÜJ >200 i -o ÜJ L_ U. LU

100

400 500 600 m/±

FIG. 3. Action spectrum for the conversion of protochlorophyll to chlorophyll a in leaves of dark-grown albino corn and absorption spectrum for protochlorophyll dissolved in methanol. (After Koski et al, 1951.)

2.4 Chemistry of Protochlorophyll

The chemical composition of protochlorophyll was elucidated during 1939-1940 by Fischer and Oesterreicher. Its molecule differs from chloro-phyll a only in the lack of the two hydrogen atoms in positions 7 and 8,

CMHK» ABSORPTION SPECTRUM ACTION SPECTRUM

150

o 100 P

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which in protochlorophyll are replaced by a double bond. This structural analysis was performed on a special form of inactive protochlorophyll present in the inner seed coats of certain members of the pumpkin family.

Most of the protochlorophyll in leaves is present as chlorophyllide— i.e., it lacks the phytol group—as is also the chlorophyll a formed after a few minutes of irradiation. Following storage in the dark for about 30 min after irradiation the phytolization increases to about 70% (Wolff and Price, 1957; Virgin, 1960).

2.5 The Protochlorophyll Holochrome

The transformation of protochlorophyll to chlorophyll a only takes place in vivo or in specially prepared extracts (see below). Protochloro-phyll extracted by means of organic solvents has lost its ability to transform, probably because the light-dependent transformation consists of a combination of photochemical and enzymatic reactions. The pig-ment-protein complex has been called the protochlorophyll holochrome (Smith, 1952).

A holochrome preparation which retains its ability in vitro to trans-form in light can be isolated from disintegrated bean leaves by extraction with a glycine buffer followed by purification using fractional precipita-tion with ammonium sulfate and repeated dialysis against distilled water. The molecular weight of this holochrome, determined with the ultra-centrifuge method, has a value of 0.7 X 106. To every protein molecule one or possibly two pigment molecules are attached. The holochrome so prepared retains its ability to transform into chlorophyll a holochrome in light for several months (Smith, 1961).

2.6 The Light-Response of the Holochrome

If the holochrome, prepared as mentioned above, is irradiated with UV light a transformation also takes place. Complete transformation can be obtained with light from about 366 τημ to 436 τημ (absorption by the pigment molecule itself). Only about 20-30% transformation is obtained at wavelengths shorter than 366 πΐμ, even if the irradiation period is tripled. This indicates that light absorption by protochlorophyll itself is not a limiting factor since the percentage transformation should increase with the time of irradiation. Some mechanism other than absorp-tion must be involved in the utilization of the activating energy (McLeod and Coomber, 1960).

Studies of the effect of pH on the rate and extent of inhibition of the transformability of protochlorophyll holochrome indicate that residues of tyrosine, cysteine, and lysine are probably attached to the proto-chlorophyll molecule (Smith and Coomber, 1960). The effect of UV light on the transformation shows that the light absorbed by tyrosine


residues is transferred to and activates as many of the pigment molecules as are associated with tyrosine. The protochlorophyll molecules attached to nonabsorbing amino acid residues are not transformed.

2.7 Quantum Yield

The quantum yield for the tranformation of the holochrome in visible light has been determined by Smith (1958) using an aqueous buffered preparation of protochlorophyll holochrome irradiated by mono-chromatic light of 642 m/A and 644 m/x. The quantum yield obtained gave an average of 0.60, a value close enough to 0.5 to suggest that two quanta are required for the conversion of one molecule of protochlorophyll to chlorophyll. The fact that two hydrogen atoms are involved in the con-version speaks in favor of one quantum being required for the transfer of each hydrogen atom.

2.8 Later Stages of the Greening Process

If the chlorophyll a concentration during continuous irradiation of a previously dark-grown leaf is plotted against time (Fig. 4, curve I)

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0.10

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£0.06 o —I X o

0.04

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FIG. 4. The different phases in chlorophyll formation in dark-grown wheat leaves, when continuously irradiated. I, Not pretreated; II, prior to continuous irradiation the leaves were given a short light impulse of 5 min, followed by darkness for 6 hours. (After Virgin, 1955b and 1958.)

\l*1>< 2 > < 3

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one can clearly distinguish three phases in the formation of chloro-phyll a.

First, there is a very rapid formation of chlorophyll a which is com-pleted after about two minutes in ordinary daylight. This formation of chlorophyll a is the result of the aforementioned transformation of the protochlorophyll already present in the dark-grown leaf. During this period the concentration of chlorophyll a rises from zero up to about 0.009 mg/gm of fresh weight.

Second, there is a rather slow phase in the chlorophyll a formation (2) which lasts for 1 to 3 hours, depending on the age of the leaf and on the degree of starvation.

Third, an acceleration in the rate of formation sets in (3) so that after about 4 hours the rate at room temperature is kept at about 0.03 mg of chlorophyll a per gram of fresh weight for several hours.

While, during the first phase, the rate of formation of chlorophyll a is determined mainly by the speed of the transformation of proto-chlorophyll to chlorophyll a, the rate during the other two phases is determined by the speed of formation of new protochlorophyll, as it has been shown that all chlorophyll a derives from protochlorophyll. This means that the changes in the rate of formation just described must be reckoned back to changes in the rate of the formation of protochloro-phyll.

Quite another course of the chlorophyll a formation is obtained if, instead of exposing the dark-grown leaf to continuous light, one gives it just a short light impulse and thereafter keeps it in complete darkness for 5 to 6 hours. If the leaves are now exposed to continuous light, the rate of chlorophyll a formation is high from the very start of irradiation (Fig. 4, curve I I ) . An action spectrum for the effect of such a short light impulse (Fig. 5) reveals a maximum response at 660 ηΐμ, and a comparatively low response to blue light. Thus it is quite different from the aforementioned action spectrum for the transformation of proto-chlorophyll by continuous light, which has peaks in both red and blue light. The action spectrum for a flash of light instead resembles that for a whole group of so-called photomorphogenetic responses including photo-periodism and seed germination. Characteristic for these responses is a reversibility between the action of red and far-red light (see Chapter 10). Such reversibility can also be demonstrated for light effects on chlorophyll formation (Price and Klein, 1961).

The high rate of chlorophyll a formation which sets in after con-tinuous irradiation of a dark-grown leaf for 1-3 hours thus depends on the effect of the light given during the first few minutes of the irradia-tion. This light is acting on a mechanism which governs the formation of the protochlorophyll in the leaves, whereby it takes about 1 to 3


FIG. 5. Action spectrum for the effect of light on the lag phase in chlorophyll a formation. (After Virgin, 1961b.)

hours at room temperature for the whole sequence of reactions to reach this final step, namely the maximal acceleration of the protochlorophyll formation. The reactions lying between the primary light absorption and the acceleration of the protochlorophyll formation are not known.

2.9 Formation of Chlorophyll b

The leaves of higher plants also contain, besides chlorophyll a, chlorophyll b in the proportion a:b = 3 : l . The formation of chloro-phyll b normally also requires light. In previously dark-grown seedlings the first traces of chlorophyll b can be found after a period of irradia-tion of about 1 hour, whereafter the proportion a : b is kept more or less constant. I t has repeatedly been proposed that chlorophyll b derives from chlorophyll a (cf. Egle, 1960) but definite proof for this has not yet been presented.

2.10 Structural Changes in the Chloroplasts during Irradiation

Proplastids in dark-grown seedlings are quite different in structure from normal chloroplasts developed in light. Very rapid changes take place during the first few minutes of irradiation and as both red and blue light gives rise to such changes, it might be assumed that these changes are connected with the transformation of the protochlorophyll into chlorophyll a (Eriksson et al.y 1961). The slower structural changes following the rapid phase are probably connected with phytolization and a building up of the chlorophyll-containing lamellae.


3. Chloroplast Movements

3.1 The Phenomenon and Terminology

In the photosynthesizing green plant the chlorophyll pigments are with few exceptions localized in the chloroplasts. In higher plants these bodies usually have the shape of small disks with an average diameter of 4-6 τημ and a thickness of about half the diameter. In algae the shape can vary from disks to threadlike formations with great variations in size. Viewed under a microscope the cells of a green leaf seem more or less filled with these dark green bodies. A closer look reveals that the chloroplasts are embedded in the cytoplasm and line the cell walls, but are absent from the cell sap-filled vacuole.

The chloroplasts seem to be quite motionless in terrestrial plant cells whereas they often show a rapid motion in cells of water plants. Leaves of the common water weed Elodea are a particularly good mate-rial for studying this. In the cells of the middle rib and in those close to it the chloroplasts are often circulating along the cell walls and through the vacuole, following the fine strands of cytoplasm which often divide the vacuole in smaller compartments like a network. This move-ment of the plastids often starts some minutes after the onset of the strong microscope illumination and is thus an effect of the light. The movement of the chloroplasts so induced is passive, however, the plastids being carried by the moving protoplasm, the streaming of which has been induced by the light.

If plant cells have been subjected to light of various intensities for longer periods of time—up to several hours—one will find that the chloroplasts have attained different positions dependent upon the light intensities. This displacement is an active one, i.e., it is performed by the single chloroplasts alone and independently of the main movements of the rest of the protoplasm and is thus to be clearly distinguished from the movement mentioned above. I t is due to some kind of active trans-fer of the single chloroplasts in the cytoplasm and appears to be a kind of phototaxis.

I t is most practical to distinguish chloroplast movements resulting from high-energy reactions and low energy reactions, corresponding roughly to the response to full sunlight and to overcast sky. The chloro-plasts often assume very characteristic positions in these two types of light intensities. I t has also been shown (see below) that there are probably several reactions involved in the response, i.e., reactions re-sulting from light absorption in different pigment systems. I t is difficult to distinguish between primary effects on the chloroplasts and on the


surrounding cytoplasm when a whole cell is irradiated. The fundamental knowledge about chloroplast movements derives from the classical works by Senn, who in his monograph of 1908 has presented the basic facts about this phenomenon.

Investigators have distinguished between several general types of chloroplast distributions in the cell. The more common types are listed below:

1. Diastrophy. The chloroplasts are separated in two groups lining the two cell walls nearest to and further-most from the light source. This is a common type of distribution in weak light. Sometimes it is also seen in cells kept in darkness.

2. Apostrophy. The chloroplasts line the radial walls of the border-line cells. This type of distribution can also be seen in some species in cells kept in darkness.

3. Parastrophy. The chloroplasts line the cell walls which because of internal reflections and refractions are most shaded. This is a common type of response in strong light intensities.

4. Epistrophy. The chloroplasts line the outside bordering cell walls. This response is common in medium light intensities.

5. Systrophy. The chloroplasts are assembled in a cluster around the nucleus. This is a common transient type of distribution, obtained by a transfer of cells from darkness to very strong light.

Other positions of chloroplasts are described, but they can be con-sidered special cases. I t should be emphasized that the above-mentioned cases may be looked upon more as morphological rather than physio-logical types, since different species do not behave in the same way when treated under similar conditions. Since not all chloroplasts in a cell respond in the same way, it is possible at certain light intensities to obtain patterns of distribution which can be considered intermediate.

Among the chloroplast positions mentioned above the first four repre-sent arrangements which are interpreted as true adaptations to the prevailing light intensities, taking into account the light falling on the chloroplasts themselves. The adaptation is coupled to the photosyn-thetic process carried on in the plastids (Zurzycki, 1955). The positions are here comparatively stable at constant light intensities. In the case of the systrophy, where the chloroplasts are assembled around the nucleus, the position is transient and after some hours the plastids dis-perse. During systrophy one can also notice a small decrease in the rate of photosynthesis, but only at high light intensities (Stâlfelt, 1945). The fact that the plastids are assembled around the nucleus indicates that this movement is a kind of protective response.


3.2 Methods of Determination

To obtain an objective measure of the course of the change of the distribution of the chloroplasts several methods have been employed. One can directly count the number of plastids which can be found at a certain position, or one can determine photometrically the transmission of the cells which is affected by the position of the chloroplasts. Recent studies have shown that cinematographic methods are a great help in such determinations (Zurzycki and Zurzycka, 1953; Zurzycka and Zurzycki, 1957).

3.3 Some Typical Cases of Chloroplast Movements

Special attention has been given to chloroplast movements in plants with large cells where it has been possible to follow more closely differ-ent phases of the rearrangement of the plastids.

Classical experimental objects for such studies are the aforemen-tioned water plant Elodea, the duck weed Lemna the moss Funana and the fern Selaginella. Leaves from these plants have only a few layers of cells, easily seen under even a low-power microscope. The chloroplasts are rather large and respond readily to light without any disturbing protoplasmic streaming, at least in Lemna and Funana. Other much-studied objects are some algae that have only one large disk-like chloro-plast which, according to the prevailing light intensities, aligns itself at varying angles to the plane of the incident light beam. Examples of varying chloroplast positions in some of these objects are shown in Fig. 6.

When a change in displacement takes place between dark position (apostrophy) and low-light position (epistrophy) or between low-light (epistrophy) and strong-light position (parastrophy), the movements occur in a purely statistical manner, i.e., the percentage of chloroplasts in the low-light position gradually increases or decreases {Funana and Lemna). The same is the case when the conditions are changed from darkness to strong light. If the cells with chloroplasts in the strong-light position (parastrophy) are suddenly darkened, however, the chloro-plasts place themselves in the complete weak-light position (epistrophy) before they move into the dark position (apostrophy). However, the course of the parastrophy-apostrophy reaction develops in two steps. In the first step the chloroplasts reach epistrophy and in the second they assemble in the proper apostrophic arrangement. At first this double process develops regularly if light and darkness are presented inter-mittently but after some time only epistrophy is reached very quickly,


FIG. 6. Examples of epistrophy and parastrophy in some plants. (After Zurzycki, 1953.)


much quicker than before, while the second stage is continued less often. Finally, apostrophy is never reached and epistrophy is maintained in darkness (Zurzycka and Zyrzycki, 1954).

3.4 Speed of Movement

The speed of the moving chloroplasts differs strongly from one object to another (Table I ) . Also the sensitivity to a light impulse varies.

TABLE I SOME EXAMPLES OF THE SPEED OF CHLOROPLAST MOVEMENT0

Species

Arabis arenosa Funaria hygrometrica Lemna trisulca Elodea densa

Mean

0.417 0.245 5.41

25.02

Velocity in μ/min

Maximum

1.6 1.5

26.4 336.0

° Zurzycka and Zurzycki, 1957.

Within the region 408-510 πΐμ where the moss Funana has its maximum response, 3 ergs/cm2 during 5 hours is enough to elicit a movement of the plastids (Voerkel, 1934), while in other material much higher energies have to be administered in order to get an effect.

3.5 Movements of the Single Chloroplast

If one follows the movement of a single chloroplast one can see that it does not always go by the shortest path to a new position and the speed of the rearrangement from one position to another is often differ-ent as compared to the reverse. Examples of paths traced by chloroplasts in leaves of Lemna during change from parastrophy to epistrophy and the reverse are seen in Fig. 7.

The cinematographic techniques and the analysis of the variability in the chloroplast movements during the course of the epistrophy-para-strophy reaction (I) and the reverse (II) have made it possible to dis-tinguish between two morphological types of chloroplast movements— at least in Lemna trisulca (Zurzycka and Zurzycki, 1957). Character-istic for (I) is an intricate movement, usually involving meandering and frequent changes in direction. The velocity is variable and there are intervals without movement. External factors have great influence on this displacement. During the other reaction (II) the displacement is less complicated, slightly wavy, and the chloroplasts show no changes in direction. The velocity is more constant and external factors have


FIG. 7. The course of the epistrophy—> parastrophy reaction of chloroplasts of Lemna trisulca L. Top: chloroplast locations at 5-min intervals. Bottom: distance-time curves. (After Zurzycka and Zurzycki, 1957.)

much less influence on this reaction than on (I). These differences have been verified statistically.

3.0 Influence of the Protoplasmic State on the Movement

I t has repeatedly been postulated that the light-induced movements of the chloroplasts depend on the physical state of the surrounding protoplasm. The relationships are rather complicated, however. Factors such as temperature, ions, and light which act on the protoplasmic viscosity also affect the chloroplast movements but in different ways depending on the chloroplast positions. As regards the influence of temperature, the epistrophy —» parastrophy reaction is strongly affected, the effect consisting in variations in the speed of the reaction, strictly related with the viscosity of the protoplasm (Fig. 8) (Zurzycka and


Zurzycki, 1950, 1951). The reverse reaction seems, on the other hand, to be more or less unaffected by these factors (Fig. 9).

FIG. 8. The light-induced change of epistrophy into apostrophy in Lemna trisulca L. at different temperatures. Ordinate: percentage of chloroplasts in epistrophy. (After Zurzycka and Zurzycki, 1950.)

20h

,o-rf£=*?»-

i/Sw#c

± ± ± ± I I 10 20 30 40 50 min 60

FIG. 9. The change of apostrophy into epistrophy in Lemna trisulca L. at different temperatures. Ordinate: percentage of chloroplasts in epistrophy. (After Zurzycka and Zurzycki, 1950.)

3.7 Action Spectra

Accounts in the literature on the action spectrum for eliciting changes in chloroplasts distribution are rather contradictory. I t has


been established that within the visible region of the spectrum a change from dark position to weak light position can only be elicited by blue light, even if red light in some instances has been shown to have a small effect here also. It is difficult to distinguish between the purely phototactic movements and the oft-times simultaneously elicited proto-plasmic streaming with a possible different action spectrum. This can explain conflicting results. In recent investigations on Lemna trisulca, Zurzycka (1951) found that the changes from apostrophy to parastrophy take place in both red and blue light. The curves for the two types of reactions are seen in Fig. 10. Of interest in this connection are the find-

·/. 100

80

60

A0

20

0 400 500 600 mu 700

FIG. 10. Action spectra for the epistrophy —» parastrophy reaction and the re-verse in Funaria hygrometnca. (After Zurzycka, 1951.)

ings by Voerkel (1934) that far-red light in some instances can elicit a movement (see below). As to the movements of the chloroplasts in cells of higher plants containing a great number of plastids, it must be stated that detailed action spectra for the phenomena have not yet been presented.

Recent studies on the response of cells containing only one large chloroplast, e.g., the algae Mesocarpus, Mougeotia, and Mesotaenium, may shed more light on this problem. Haupt (1959b) has presented a


rather detailed action spectrum for the behavior of the Mougeotia chloro-plast (Fig. 11). When irradiating with medium intensities of light

look

6ol·

INDUCTION REVERSAL

■—rU-

500 JJ 800

FIG. 11. Action spectrum for induction and reversal of the chloroplast move-ment in Mougeotia. (After Haupt, 1959b.)

(1000 ergs/cm2 sec) only red light within the region 550-700 m/x is active with a maximum at 679 ηΐμ. And, in addition, an impulse of far-red light with maximum at around 720-730 ηΐμ,, administered after the red light impulse, nullifies the effect of the red light. The spectrum reveals that at least parts of the photoinduced chloroplast movements are elicited by light absorbed in the phytochrome system (see Chapter 10). The reversal by far-red light can account for many contradictory results found in the older literature.

According to Zurzycka (1951) the high-energy reaction in Lemna is elicited by both blue and red light. In Mougeotia blue light has in some cases been shown to have a small effect, but this question is not yet settled. I t might be possible that the high-energy reaction of the chloroplasts also involves a reaction where blue and far-red light is active, and which is counteracted by red light in accordance with find-ings by Mohr and Wehrung (1960) for some other photomorphogenetic responses. Just recently Haupt and Schönbohm (1962) have presented a detailed action spectrum for a blue-light induced reaction.

The low-energy reaction in Mougeotia can be elicited by polarized red light. This is most effective when the plane of polarization is vibrating perpendicular to the cell axis, and less effective when it is vibrating parallel to it. As far as the antagonizing effect of far-red light is concerned, however, there is no difference between light polarized in the two directions. From these findings it might be possible to draw conclusions as to the orientations of the pigment molecules responsible for the light absorption (Haupt, 1960).

3.8 Mechanism

The driving forcé behind the movement of the chloroplasts is inti-


mately connected with the localization of the light-absorbing agent. As early as 1880 Stahl put forward the hypothesis that light worked via absorption in the cytoplasm, i.e., not by absorption in the chloroplasts themselves. This is possible for movement elicited by blue light, as light-induced protoplasmic streaming and changes in the consistency of the protoplasm also depend on blue light. Recent experiments by Bock and Haupt (1961) indicate that the movement is very likely induced via light absorption in the surrounding cytoplasm. But light hitting the single chloroplasts also has an effect. Senn (1908) sup-posed that the chloroplasts are surrounded by a thin layer of viscous protoplasm—the peristromium—performing amoeboid movements by sending out small proturberances. And, as a matter of fact, Strugger (1956) was able by electron microscopy to distinguish such a plasmatic envelope which was later also identified by other workers at least for some chloroplasts. Just recently by means of a combination of cinema-tographic technique and phase microscopy it has been shown that al-though the chloroplasts remain stationary, the envelope of optically dense cytoplasm surrounding the individual chloroplasts is in constant motion, giving rise to protuberances which may extend for several chloroplast diameters (Honda et al., 1961; Hongladarom et al., 1961). The protuberances may segment into particles which appear to be iden-tical with mitochondria and the mitochondria sometimes coalesce with the chloroplast envelope, following which they no longer appear as mitochondria. If it can be proven that such formations are common to all chloroplasts showing phototactic movements in the cell, we shall have come one small step nearer the explanation of the mechanism of chloroplast movement, although the real cause for their movements re-mains an enigma. No doubt, however, chloroplast movement is a phenomenon essentially distinguishable from the protoplasmic stream-ing and other properties of the protoplasm such as viscosity and permea-bility induced by light although the effect of light upon these may secondarily affect the speed of rearrangement of chloroplasts in cells. As the cytoplasmic layer also has a consistency generally decreasing in direction inwards against the vacuole, even a very short movement of the chloroplast toward or away from the sticky outer layers of the protoplasm can have a great influence on its rate of the movement. Zurzycka and Zurzycki (1957) claim that some types of phototactic chloroplast movement, particularly for example, the epistrophy-para-strophy reactions, are associated with streaming of the cytoplasm whereas other types, e.g., the reverse reactions, are associated with the equalizing of stresses arising in the cytoplasm under the influence of strong light and the subsequent contraction of the cytoplasmic fibres.


The last conclusion was derived from studies of chloroplast movements in centrifuged cells.

4. Protoplasmic Streaming

4.1 The Phenomenon and Types of Streaming

The cytoplasm in the living cell is never quite at rest. The cells always contain small particles which are moving along in more or less defined paths, probably following nonvisible strands of the cytoplasm. This phenomenon of protoplasmic streaming or "cyclosis" is an energy-consuming process and a sign of metabolic processes going on in the cell.

The mode of the streaming shows considerable differences in differ-ent cell materials. I t also varies depending upon age, vacuolization, and other factors. I t is beyond the scope of this article to describe in detail the variability of this phenomenon (see Kamiya, 1959), but some main features will be mentioned.

For a long time plant physiologists have distinguished between three different types of streaming which may occur in the living cytoplasm.

First we have "agitation" or turbulent motion. I t can be character-ized as a movement of small cytoplasmic particles in the interphase be-tween cytoplasm cell sap, and can often be observed in many Conjugatae, in Spirogyra, and other algae. The motion is erratic and haphazard. By statistical analysis it can be shown not to be the result of Brownian move-ment. Agitation movement can probably be observed by a close study of all cytoplasmic interphases.

A type of streaming called "circulation" is found in plants with large cells having transvacuolar strands, such as stinging hairs of Urtica, cells of Spirogyra, Allium, and others. It is manifested as a more or less rapid movement of a rather constant speed of small par-ticles along the cytoplasmic strands. In one and the same strand particles can go in opposite directions.

If the cytoplasm is only lining the periphery of a cell, and if it streams like a rotating belt in the optical plane, the streaming is called "rotation." Rotation is the most regular of the various types of proto-plasmic streaming; it is the main type used for quantitative observa-tions, and, practically the only one upon which the influence of light has been studied. I t is a common type in many aquatic plants such as Elodea, Vallisneria, Chara, and Nitella, as well as in root hairs, pollen tubes, and cambial cells of terrestrial plants.

Besides these more common types of streaming other special inter-mediate types have been described. Two of these are worth mentioning: The "tidal" type and the "shuttle" type. The first type, found in hyphae


of Phycomycetes, is characterized by a movement in either acropetal or basipetal direction. The direction can be changed by altering the water content by transpiration or by the action of osmotically active sub-stances. The shuttle streaming is found in Myxomycètes. In these organisms the rate of flow as well as the amount of cytoplasm carried along with the streaming is exceedingly great as compared with the ordinary protoplasmic streaming in plant cells. The characteristic feature in this kind of streaming is an alternation which follows a rhythmic pattern. The streaming has much in common with the amaeboid move-ment. Most of our present knowledge of the influence of different agents on protoplasmic streaming derives from studies on Myxomycete plas-modia. Whether conclusions from this material can be directly adopted to the streaming in the green plant cell can be questioned, however. On the one hand the "cells" are quite differently organized and on the other hand the type of streaming is of a completely different type.

4.2 Methods of Measurement

Measurements of the absolute speed of protoplasmic streaming can most easily be made by timing the movement of particles between two selected points. A prerequisite for this method together with most others is, however, that all the particles passively following the streaming protoplasm have about the same speed, which is not always the case. In most measurements photographic methods of different kinds have been employed, whereby the streaks on the film given by dark-field illu-minated protoplasmic granules have been measured. Also comparison methods have been used, whereby the rotating cytoplasm is compared with a running belt, set up outside the microscope (for literature, see Kamiya, 1959). A similar principle was used by Zurzycki (1958).

4.3 Factors Affecting the Streaming

The different types of streaming described here can be elicited by a multiplicity of agents from chemicals to mechanical shocks. As already mentioned the rotation type of streaming is most frequently studied in higher plants upon which the major part of the following account de-pends. Among the factors affecting the streaming, light plays an im-portant role. As the light energy acts via its absorption in a pigment system which in its turn is coupled to chemical systems, the effect of chemicals on the streaming is not without interest when probable mechanisms for the light action are being discussed. But here our knowledge is practically nil.

The protoplasm of cells shows a particularly strong response to certain amino acids, usually of the a-types. Thus L-histidine in as low a


concentration as 1:650,000,000 can elicit streaming in cells of Vallisneria kept in darkness (Fitting, 1929).

4.4 Effect of Light

That light elicits protoplasmic streaming can easily be seen on living green cells under a microscope. The light from the illuminator sooner or later increases streaming during the course of the observation. One can distinguish two different kinds of light influence. On one hand, the light can affect the rate of an already existing streaming, and, on the other, a light impulse can elicit a streaming in an inactive protoplasm which then continues for a certain period. However, the data in the literature differ in many respects owing to the variety of material used (Haupt, 1959a). Some plants, for example, Vallisnena, show a very high sensitivity to light (Schweikerdt, 1928). Intensities as low as 0.05 ft-candles can produce streaming, provided the period of irradiation is long enough. At high light intensities shorter irradiation times suffice to elicit a response than at low light intensities, but the reciprocity law [intensity (/) X exposure time (t)] does not hold true for Vallisneria. The stimulating effect of light in this case is greater at low intensities than at high light intensities for the same exposure (It). The rate of the streaming, at least in cells of Elodea and Vallisneria, is thus to a certain extent proportional to the intensity of the light. At very high intensities the induced streaming starts to decrease after 5-15 min and may eventually stop completely, probably due to irreversible injuries.

The reaction time also depends on the light intensity although an exposure of at least 5 min seems to be required even at higher intensities. The reaction time is therefore much longer than the duration of the exposure to light. In this respect there are similarities between this response and the phototropic response. When subthreshold stimuli are given intermittently, one obtains a summation if the ratio of light and dark period is between 1:1 and 1:2 (Schweikerdt, 1928).

4.5 Effects of Salts on the Light-Reaction

The light-induced streaming is strongly influenced by salts of differ-ent kinds. In subepidermal cells of Vallisneria Jager (1958) found that no streaming could be induced in leaves which after a dark period of three days were treated for 1 hour in 0.001 M solution of KC1, RbCl2 or CaCl2. This inhibitory effect on the light-reaction was shown to disappear during a subsequent water treatment. This would suggest that the presence of certain amounts of K+ and Ca2+ in the protoplasm in-hibits the light-reaction. During a subsequent treatment of the leaves in water the cations present in the plasma—absorbed by negative


groups—are removed in such a way that the light-reaction is again possible.

In respect to different salts, KC1, KN03, KHC03, KH2P04, and K2S04 show identical effects which must therefore be attributed to the potassium ion. Other cations, administered as chlorides with the excep-tion of aluminum, show an influence specific for that ion. Some of the ions also have positive effects on the light-reaction (Fig. 12). This figure

80

6OI-

2oU ol

HoO HoOm H2u H20

1 L ti ίΐι RbCI KCl NaCl LiCl SrC!2 CaCl2 MgCI2 LaCU

CaS04 Co(NH3)6Cl3 AI2(S04)3

FIG. 12. The effect of different cations on the light-induced initiation of proto-plasmic streaming in subepidermal cells of Vallisneria leaves. Black blocks: effect in darkness. Open blocks: effect after exposure to light. (After Jager, 1958.)

also shows that some salts initiate streaming in the dark. I t was found that cations which do not inhibit the light-reaction to an appreciable extent initiate streaming in the dark. This means that the streaming percentages found after exposure are only partly the result of the action of the light for these cations (among others: Na+, Al3+, Li+, Sr2+, and Mg2+). Ions normally occurring in the protoplasm, i.e., Ca2+ and K+, strongly inhibit the light-reaction. The series of ions arranged in ac-cordance with their physiological effectiveness resemble the lyotropic series, with the exception of Ca2+. This suggests that an effect on the permeability of the cell is of minor importance here.

I t is of interest to note that the potassium ion does not inhibit the streaming as such but prevents the initiation of streaming by exposure to light or by the treatment with chemicals such as amino acids.

A clear understanding of the ultimate nature of ionic influence on light-induced streaming cannot be reached until we know the mechanism inducing streaming. As will be shown later, a strong correlation exists between the protoplasmic viscosity and the rate of streaming, changes in viscosity often being correlated with changes in rate of streaming (Seifriz, 1952). Even if the viscosity has not been measured along with the rate of streaming in this special case, when the cells are placed in


different ionic environments the possibility of an ionic influence on rate of streaming via viscosity changes cannot be excluded.

4.0 Streaming in the Avena Coleoptile

A short light-impulse elicits changes in the rate of the streaming in dark-adapted coleoptile cells. The type of reaction in the coleoptile cells differs from that in the other cells hitherto mentioned in that the light causes a short lasting decrease in the rate of the movement. In cells of water plants, the response usually consists of an increase in the rate of streaming. But also here the effect of two consecutive light impulses can be added to each other provided the time elapsed between them does not exceed a certain maximum value (Fig. 13).

0 A 8 12 16 0 C 8 12 16 20 24 28min

FIG. 13. The effect of two consecutive light-impulses on the protoplasmic streaming in the Avena coleoptile. The arrows indicate a light impulse of 190 ergs/cm2 for 8 sec. (After Bottelier, 1934.)

From Fig. 13 one can also see that there is a short latent period of about 3 to 4 min after the onset of the light-impulse, during which time the response of the cell to a new light impulse is decreased.

The light-induced protoplasmic streaming of the Avena coleoptile cells has long been a center of interest because it was thought to have some bearing on auxin transport preceding phototropic movements.

On the whole, the course of streaming responses to light of the protoplasm in the Avena coleoptile show great similarities with other light-responses of the coleoptile, such as photoelectrical phenomena, light-growth reactions, and phototropic bendings. Whether these similari-


ties are due to a real relation between the different responses or are only coincidental cannot yet be stated (for literature, see Galston, 1959).

4.7 Relationship between Streaming Rate and Protoplasmic Viscosity

The dependence of protoplasmic streaming on the viscosity has been repeatedly shown by many authors (see Seifriz, 1952). The viscosity of the protoplasm is a term used to express a complicated property of the living substance. As the cytoplasm does not behave as a Newtonian liquid its properties cannot be expressed by simple mathematical for-mulas. What is meant nowadays by protoplasmic viscosity is the set of properties of this semifluid mass which can be semiquantitatively ex-pressed in terms of movability or fluidity when the cell is subjected to such physical influences as tend to rearrange its contents.

The velocity of protoplasmic streaming depends to a great extent on temperature (Lambers, 1925; Zurzycki, 1951), mainly because of the high dependence of protoplasmic viscosity on temperature. The rela-tionship between viscosity and temperature is almost linear (Hayashi, 1960). As a consequence of this, the motive force of the streaming seems to be almost constant, at least between 5°C and 25°C (Fig. 14). This would suggest that the difference of the velocity of protoplasmic stream-ing at various temperatures depends mainly upon the change of the viscosity of protoplasm with temperature.

% I 1 200 l· °

i ° I

I ° I

100 l· · · · · I o · · · i Γ · o · · L o 8 ·

I ° I Γ o o I

L ° I o l I I I I l _ J 0 5 10 15 20 25°C

FIG. 14. The effect of temperature on the motive force and protoplasmic viscosity in internodal cells of Chara. Open circles: protoplasmic viscosity. Black circles: motive force. Ordinate: relative values of motive force and viscosity in per cent of the value at 10°C. (After Hayashi, 1960.)


The viscosity of protoplasm as measured by means of centrifugation is very sensitive to light (Stâlfelt, 1946; Virgin, 1951) as shown in the typical response curves of Fig. 15. Generally a rather rapid primary

FIG. 15. Changes in the protoplasmic viscosity in Elodea densa after short-lasting irradiations with 22,000 meter candles of white incandescent light. Irradia-tion (time indicated in the figure) started at zero time. (After Virgin, 1951.)

change in viscosity follows illumination—an increase or decrease de-pending on the intensity of the light-impulse. The primary reaction is followed by oscillating changes in viscosity which may go on for many hours after the first light-impulse. In constant light the viscosity is also never constant but shows continuous fluctuations with oscillations of varying amplitudes and lengths.

The changes in the protoplasmic viscosity induced by illumination resemble light-induced changes in protoplasmic streaming in the Avena coleoptile (Bottelier, 1934). This points to a close connection between viscosity and streaming, although there are indications that a decrease in the viscosity of the cytoplasm can take place long before a corre-sponding increase in the protoplasmic streaming sets in, but the observa-tions are scarce (Virgin, 1951). In most cases the observations on light-

298 H E M M I N G I . VIRGIN

induced viscosity changes and changes in the rate of protoplasmic streaming induced by light have been performed on different plant materials.

Light-induced viscosity changes are local; only the illuminated part of the tissue or even an illuminated part of a single cell being affected (Fig. 16). This means that if the stimulus is transmitted from irradiated

FIG. 16. Leaf cells of Elodea densa partly irradiated with white incandescent light. After the irradiation the leaves were centrifuged.

to nonirradiated areas it moves slowly. For example, only 15 to 30 min after local irradiation of a cell does streaming become general in the cell.

4.8 Action Spectra

Information about action spectra for the light-induced streaming is scarce and in many respects conflicting. In most cases a strong re-


sponse to blue light is reported, while the opinions about the effect of red light vary. According to Ritter (1899) blue as well as red light is active in eliciting streaming in quiescent cells and for enhancement of an already existing streaming in Elodea and Vallisnena. Still stronger responses in red light have been reported by Nothmann-Zuckerkandl (1915) who also found far-red to be active for induction of streaming. Schweikerdt (1928) determined the threshold value for the effect of red, green, and blue light of the same energy content on Vallisneria, and found for the three light qualities a response of 100:20:25, respec-tively, in relative units, suggesting chlorophyll as the light-absorbing agent.

Because the accuracy of the measurements of energy and the purity of the monochromatic light used in these older studies is not known, it is difficult to draw decisive conclusions from them. I t is quite evident, however, that plant cells containing chloroplasts do respond to red light to a certain extent, suggesting that chlorophyll might be involved in the absorption of the active-light energy. But as oxygen also affects the streaming, the effect of the red light may be only a secondary phenomenon, i.e., the streaming might be induced secondarily by the increase of oxygen concentration resulting from photosynthesis. Sup-porting the latter suggestion is the fact that cells lacking normal amounts of chlorophylls, e.g. the Avena coleoptile, show little if any response to red light.

The most accurate action spectrum for the influence on the stream-ing is given by Bottelier (1934) for epidermal cells of the Avena coleoptile, light causing a decrease in the rate of streaming (in contrast to the accelerating effect of light on streaming in Elodea cells). Blue light (Hg-line 436 πΐμ) retards streaming most; the 366 πΐμ line (long UV) has a lesser effect, while green, yellow, and red light are without effect (Fig. 17). Although the response curve given by Bottelier con-sists of only six points it is evident that we are dealing here with a blue sensitive process—with an action spectrum very similar to that for phototropic phenomena. According to Virgin (1954) light-induced changes in plasma viscosity (Elodea) occur only in the range of 400 πΐμ to 510 m/A with possible peaks about 430,470, and 490 m/x (Fig. 18). The general similarity between the action spectra for the two light-de-pendent processes—the change in viscosity and the change in rate of streaming—is strong evidence for a close relationship between them. Since the details in the spectrum hitherto published are poor it is not possible to state whether the absorbing agent is a carotenoid or riboflavin (cf. Galston, 1959).


PH0T0TR0PISM STREAMING

>-> z UJ

- I w Σ

„ 3

<

580 mjj

FIG. 17. Action spectrum for the light-induced protoplasmic streaming in the Avena coleoptile compared with the response curve for the phototropic reaction (tip response). The divergence in the blue part of the spectrum can be brought back to the difference between absolute energy response and quantum response. (After Bottelier, 1934, and Shropshire and Withrow, 1958.)

500 π\μ

FIG. 18. Action spectrum for the light-induced decrease in the protoplasmic viscosity of Elodea densa. (After Virgin, 1954.)

4.9 Mechanism

The mechanism of protoplasmic streaming is not known, although numerous hypotheses have been presented. In the older literature are


descriptions of model systems with movements seemingly like those in living protoplasm. By local changes in the surface tension—e.g., by mix-ing polar and nonpolar reagents or by the induction of surface electrical potentials—movements like protoplasmic streaming may be induced. Since knowledge of cell structure and metabolic processes has deepened, the inadequacy of the models has become evident. Most recent work on protoplasmic streaming has been performed on slime mold (Myxomy-cete) plasmodia (Kamiya, 1953) which, being colorless, have little direct bearing on the action of light on streaming in cells of green plants.

In recent discussions on the mechanism of protoplasmic streaming the contractility of individual protein fibers and sol-gel transformations taking place on the submicroscopical level have become centers of in-terest. From myxomycete plasmodia a protein system has been isolated which responds to adenosine triphosphate (ATP) much as muscle myosin B (actomyosin) does, namely, by a great increase in viscosity. I t is therefore plausible to consider the presence in the myxomycete plas-modium (and possibly in all plant protoplasm) of a contractile protein similar to myosin B of muscle. For a more detailed account of these theories, see Kamiya (1959).

Because protoplasmic streaming in cells of higher plants is dependent upon oxygen and is inhibited by respiratory poisons it is considered likely that the driving force comes from energy released by metabolic activity. ATP is found to accelerate streaming. Removal of 0 2 and treatments with KCN and CO, oddly enough, have no particular effect on streaming of myxomycètes and may sometimes result in an increase of streaming, possibly because of increased glycolysis. On the other hand, the driving force of streaming is extremely sensitive to agents inhibitory to the glycolysis, such as NaF and iodoacetate. From this the conclusion has been drawn that perhaps it is only the ATP pro-duced in the undifferentiated cytoplasm which can be used for stream-ing, while that produced in the mitochondrial system through the oxida-tive phosphorylation is useless for this purpose.

In higher plants the conditions favorable to streaming are somewhat different from those favoring streaming in myxomycètes. In most cases streaming is only possible when oxygen is present. And as a matter of fact the response to oxygen deficiency can be so great that at normal 0 2 concentrations the diffusion of the oxygen into the cells can be the limiting factor for the streaming as Eymers and Bottelier (1937) have shown for etiolated Avena coleoptiles. In green plants like Elodea (Zurzycki, 1951) streaming can be induced by light through the mediation of the oxygen liberated during photosynthesis (Zurzycki, 1951). In this case the action spectrum for the streaming is similar to that for photo-synthesis.


The fact that the myxomycete plasmodium but not cells of higher green plants can show streaming for a long time after deprivations of free oxygen can be explained by the assumption in the former of a greater pool of ATP which can continue to supply energy for a certain period.

The site for the motive force for protoplasmic streaming has been studied in cells of Chara (Hayashi, 1960). The main locus for generation of motile force for streaming seems to be in the interfacial layer between the sol endoplasm and the gel cortical layer containing embedded chloro-plasts. Most determinations of the magnitude of the driving force in protoplasmic streaming have been made on plasmodia of Myxomycètes (Kamiya, 1953). A few data can be found from other materials. Using the centrifugal microscope, Virgin (1949) found a force of 200-360 times gravity necessary to counteract streaming movement in the protoplasm of Elodea cells. In Myxomycete plasmodia a force about 200 times gravity balanced the streaming (Hayashi, 1960).

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(M. B. Allen, ed.), pp. 257-277. Academic Press, New York. Smith, J. H. C , and Benitez, A. (1954). Plant Physiol. 29, 135-143. Smith, J. H. C, and Benitez, A. (1955). In "Moderne Methoden der Pflanzen-

analyse" (K. Paech and M. V. Tracey, eds.), Vol. 4, pp. 142-196. Springer, Berlin.

Smith, J. H. C , and Coomber, J. (1960). Carnegie Inst. Wash. Yearbk. 59, 325-330. Stâlfelt, M. G. (1945). Svensk. Botan. Tidskr. 39, 365-395. Stâlfelt, M. G. (1946). Arkiv Botan. 33A:4, 1-17. Stahl, E. (1880). Botan. Ztg. 38, 298-304. Strugger, S. (1956). Ber. deut. botan. Ges. 69, 177-178. Virgin, H. I. (1949). Physiol. Plantarum 2, 157-163. Virgin, H. I. (1951). Physiol. Phntarum 4, 255-357. Virgin, H. I. (1954). Physiol. Plantarum 7, 343-353. Virgin, H. I. (1955a). Physiol. Plantarum 8, 389-403. Virgin, H. I. (1955b). Physiol. Plantarum 8, 630-643. Virgin, H. I. (1958). Physiol. Plantarum 11, 347-362. Virgin, H. I. (1960). Physiol. Plantarum 13, 155-164. Virgin, H. I. (1961a). Physiol. Plantarum 14, 384-392. Virgin, H. I. (1961b). Physiol. Plantarum 14, 439-452. Voerkel, S. H. (1934). Planta 21, 156-205. Wolff, J. B., and Price, L. (1957). Arch. Biochem. Biophys. 72, 293-301. Zurzycka, A. (1951). Ada Soc. Botan. Polon. 21, 17-37. Zurzycka, A., and Zurzycki, J. (1950). Acta Soc. Botan. Polon. 20, 665-650. Zurzycka, A., and Zurzycki, J. (1951). Acta Soc. Botan. Polon. 21, 113-124. Zurzycka, A., and Zurzycki, J. (1954). Acta Soc. Botan. Polon. 23, 279-288. Zurzycka, A., and Zurzycki, J. (1957). Acta Soc. Botan. Polon. 26, 177-206. Zurzycki, J. (1951). Acta Soc. Botan. Polon. 21, 241-264. Zurzycki, J. (1953). Acta Soc. Botan. Polon. 22, 299-320. Zurzycki, J. (1955). Acta Soc. Botan. Polon. 24, 27-63. Zurzycki, J. (1958). Acta Biol. Cracov. 1, 123-129. Zurzycki, J., and Zurzycka, A. (1953). Acta Soc. Botan. Polon. 22, 679-687.

Chapter 10

PHOTOCHEMICAL ASPECTS OF PLANT PHOTOPERIODICITY

Sterling B. Hendricks

Mineral Nutntion Laboratory, Agricultural Research Service U. S. Department of Agriculture, Beltsville, Maryland

1. Introduction A reversible photoreaction

660 πιμ D a r k n e s s

Ρβ60 < P730 >

^660 ( 1 ) 730 m/x

in which 660 and 730 m/A are the respective absorption maxima of two forms of a blue chromoprotein, phytochrome (P), controls many aspects of growth and development of higher plants (Borthwick et al., 1952). The responses include flowering, stem elongation (etiolation), leaf move-ment and expansion, seed germination, anthocyanin production, plastid formation, and bud dormancies. P730 changes to P66o in darkness (Borth-wick et al., 1954). Many seasonal responses of plants, including growth, flowering, and the autumnal color changes, depend primarily upon the rate of this reversion. These time-dependent responses, which are termed "photoperiodic," are important for the preservation of the species in un-favorable seasons by control of reproduction and dormancy.

This pigment change was discovered from action spectra for the various responses. Pertinent results are presented by H. A. Borthwick and his associates (Hendricks and Borthwick, 1955; Borthwick and Hendricks, 1960), chiefly in journals devoted to the plant sciences. The original papers referred to in the review articles describe the important experimental conditions and variations.

Knowledge of the physiological responses eventually led to spectro-metric methods for the detection of phytochrome in living etiolated seedlings. These methods permitted the development of an assay neces-sary for isolation of the pigment. Measurements on the isolated pigment could then, in turn, be correlated with the physiological findings.

The historical account of research on phytochrome which follows is 305

306 STERLING B. HENDRICKS

interesting because it states an attempt to explain physiological re-sponses on a molecular basis. Since knowledge of plant photoperiodicity is currently undergoing such marked advances, the topic is best treated as a review of research developments. A large part of the observations on causation have been made by a single group of workers so, perforce, their work is the basis for this presentation. In the discoveries leading up to present concepts, empirical observations are interwoven with the re-sults of physiological and physical experiments. This work eventually led to a biochemical understanding of the problem and finally to a physio-logical explanation as well. The first step toward understanding of causation came from experiments dealing with light as a stimulus rather than with the detailed display of flowering or some other response to the stimulus.

2. Discovery of Photoperiodism Etiolation, the typical growth of plants in darkness, was perhaps the

first of the phenomena controlled by phytochrome to be related to light. This unusual stem growth was evident in the great elongation of plants growing in shade or under opaque objects. Early experiments on the growth of plants in light of various colors obtained from sunlight by the use of colored solutions as filters (in Senebrier flasks) usually led to pronounced etiolation because of low light intensity. This work indi-cated an effectiveness of red light but did not rule out the possibility that the responses arose solely from photosynthesis. The first action spectra, measured by Vogt in 1915, indicated that red light is most effec-tive in the suppression of etiolation. The ineffectiveness of blue light indicated a type of control other than photosynthesis.

A century ago Caspary (1861) noted that seed of Bulliarda aquatica (Tillia aquatica) required light for germination. During the next 50 years seeds of many other plants were found to have similar require-ments (Kinzel, 1913-1926). The first experiments on the nature of the light response were made by Flint and MacAlister (1935) who found that the germination of lettuce seed was enhanced by red radiation and suppressed by radiation near the red limit of the visible spectrum.

The recognition that the flowering of many kinds of plants de-pended on the season long preceded the experiments on the causes of the dependence. It was first supposed that the responses were due to variations of temperature. The discovery by Garner and Allard (1920) that flowering depended on the relative length of day and night was very surprising. These investigators found the phenomenon which they called "photoperiodism" to be widely displayed but not universal among seed plants. Some plants were found to flower when days were short and

10. PHOTOCHEMISTRY OF PLANT PHOTOPERIODICITY 307

others on long days; thus, the terms short- and long-day plants. A most important aspect of the work of Garner and Allard was the evidence that duration of time can be measured by plants.

Because a possible relationship between the individual phenomena was unsuspected, a separate literature developed around each phe-nomenon.

3. Action Spectra in Living Material The stimuli for the individual responses are light and darkness.

Light has two or three properties of interest here: (1) energy = intensity X time and (2) wavelength; darkness has only one—time—which is in-itiated by the absence of light and terminated by its presence.

An action spectrum expresses the energies in the various wavelength bands required to produce a given response. It is best to know the radiant energy at the site of action or absorption, but this may not be possible owing to uncertain corrections for absorption and scattering between the light source and the site in living material. Lack of this knowledge limits the expression of results to dependence or response on incident energy per unit wavelength region per unit area.

A most important requirement of an action spectrum is that irradia-tion should be of as short duration as possible in order that the stimulus should not be confounded by the change that it produces. This energy requirement must be attained with a spectrum of high purity, par-ticularly with regard to scattered radiation, and with a dispersion ade-quate for irradiation of the object in a narrow wave band of the order of 5 to 10 τημ. These conditions can now be met by the use of inter-ference filters.

Parker et al. (1946) used a large, two-prism (glass) spectrograph having a dispersion of 1.5 m^/cm at 500 m/*. The light path of the in-strument is illustrated in Fig. 1. The resolution is slightly reduced by placing the prisms in the convergent beam. The slit width at the object

Thermopile Monitor

UViolet Focal plane

Plants placed here

iont surfaced flat mirror

6 0 ° glass prisms

Slit 2M. focal length IO"concave mirror front surfaced

FIG. 1. Optical path in a spectrograph used for action spectra measurements on seed plants. See Section 3 for details.


was usually 3.0 cm (corresponding to an actual width of 0.5 cm) and was as broad as allowed by possible fine structure in action. The positive crater of a 12-KW carbon arc was used as a radiation source. Irradia-tion was of the order of 0.30 mw/cm2 at 700 mju, and 0.10 mw/cm2 at 450 τοαμ, with scattered light in the 400 to 700 mju, part of the spectrum of the order of 0.001 of these values, A grating spectrograph giving somewhat higher irradiation and having the advantages of linear dis-persion is in use at the Argonne National Laboratory (Monk and Ehret, 1956).

Results obtained for inhibition of flowering of Biloxi variety soybean plants (Parker et al., 1946) are shown in Fig. 2. All leaves except a recently expanded one were removed from each plant of a group that had been growing on 18-hour light- and 6-hour dark-periods. The plants, which are vegetative under these conditions, were placed on 10-hour light-periods and 14-hour dark-periods for six cycles which is adequate to induce some flowers. Groups of plants were arranged near the middle of each dark period. With their leaves along the focal plant of the spectrograph, the plants were then irradiated for various times and returned to darkness for the remainder of the dark portion of the cycle. After six cycles they were returned to 18-hour light and 6-hour darkness to permit development of any flowers induced by the long nights. The numbers of flowers developing on four replicate plants given various exposure times are indicated in Fig. 2. A solid curve, the portion of the

5 4 0 0 5800 6200 6600 7 0 0 0 7400 7 8 0 0 Wavelength in angstrom units

FIG. 2. An action spectrum for suppression of the flowering response of Biloxi soybeans in the region of 580 to 730 ταμ (Parker et al., 1946). The number of flowers initiated on four plants are shown. See Section 3 for details.

10. PHOTOCHEMISTRY OF PLANT PHOTOPERIODICITY 3 0 9

action spectrum for incident light, is drawn through the array for a stage of one flower induced on four plants and a dotted curve is drawn for half suppression.

Composite action spectra for induction and inhibition of flowering, seed germination, and etiolation are shown in Fig. 3a and 3b. The action spectra in Fig. 3b were obtained after irradiation with high energy in the red region near 650/A. The spectra represent the reverse phenomenon of Fig. 3a; for example, inhibition instead of promotion of germination as in Fig. 3a.

Effectiveness of radiation has been measured for control of flower-

1 i i i i i i i I I l i i i i 1 560 600 640 680 720 760 800

WAVE LENGTH IN MILLI -MICRONS

(a) (b) FIG. 3. (a) Action spectra for germination of lettuce seed, suppression of

flowering of cocklebur, enhancement of elongation of pea leaves by 45%, C; and initiation of flowering of barley, D. (b) Action spectra for reversal of the poten-tiated germination of lettuce seed and suppression of cocklebur flowering.


ing, anthocyanin formation, etiolation, and germination throughout the visible and near-infrared regions. A subsidiary maximum for action is observed in the region of 400 τημ where about ten times more energy than in the red part of the spectrum is required for prevention of flower-ing of cocklebur and soybean plants. The action is minimal in the region of 480 τϊΐμ and none is observed for wavelengths greater than 820 τημ.

A simple deduction from the action spectra is that the initial stimulus for the control of flowering of both long- (barley) and short-day plants (cocklebur, soybeans), etiolation, and germination must be the same, although these developmental characteristics have no other apparent features in common. This fact illustrates the usefulness of action spectra in finding a point of secure knowledge without postulating what lies be-fore or after. Light is a unique kind of probe, entering the plant without disturbance other than at the place of action.

4. Physiological Evidence of a Photoreversible Reaction

The various responses potentiated by red light with maximum effectiveness near 660 τημ are reversed by suitable irradiation in the region of 700 to 750 τημ as indicated by the action spectra of Fig. 3. Radiation sources exposing considerable areas to radiation in these regions are convenient for physiological work. Fluorescent tubes giving "white" radiation with short wavelength-limiting filters of red cello-phane or other red plastics can serve as sources in the region of 600 to 680 m/A, the region of greatest effectiveness for "red" action (Fig. 3a). Incandescent filament lamps with combined red and blue cellophane filters serve as sources for far-red radiation with wavelengths greater than 700 ηΐμ. A water filter can also be used with the latter source. Energy fluxes of the order of 1.0 mw/cm2 in the effective regions are readily attained

Germination responses of lettuce seed to a number of exposures to red and far-red radiation in succession are shown in Fig. 4. In this experiment, seed of the Grand Rapids variety of lettuce were allowed to imbibe water in darkness for 16 hours and then exposed to the red and far-red sources in succession for 4 min. After each exposure the various lots of seed were returned to darkness. Germination of the lettuce seed depended only upon the last exposure of the sequence. More than 15 types of seed, representative of various varieties and species of plants, have been shown to respond in this reversible way to radiation (Toole et al., 1957). A similar response has been found for the germina-tion of spores of a fern (Mohr, 1956).

Reversibility of the potentiated etiolation response of the pinto bean

FIG.

4.

Rev

ersi

bilit

y of

po

tent

iate

d le

ttuce

see

d ge

rmin

atio

n by

red

and

far

-red

rad

iatio

ns i

n su

cces

sion.

Se

e Se

ctio

n 4

for

deta

ils.

» o

O

a W

S »-H

CO

O

•3 « O

H

O

M

O

Ö

«1

00


FIG. 5. Changes induced in internode length of pinto beans at the end of an 8-hour day. The plant on the left received no supplementary radiation, the one in the center far-red radiation for 5 min, and the one on the right far-red radiation for 5 min followed by red radiation for 5 min.

is shown in Fig. 5. Many other varieties of beans as well as varieties of other species of plants respond similarly. A reversible potentiated etiolation response was also found for the first internode of a fern (Laetsch and Briggs, 1962).

Reversibilities of potentiated flowering of plants requiring long nights (soybeans and cocklebur) or short nights (barley and hyoscyamus) for flowering have also been shown. In these responses, however, care must be used in restricting the total irradiation time to a period of less than 30 min because of the dark reversal and biological action of the pigment.

The reversibility is a readily observed response to red and far-red radiation and indicates a growth response to radiation dependent upon the reversible reaction provided the irradiation is of the order of a few millijoules per square centimeter.

5. Measurement of Photoeffectiveness [<*?) and Dependence of Response upon the Degree of

Pigment Conversion The product of the molar absorption coefficient (a) and the quantum

efficiency (<p) for conversion can be measured for the first-order change


of a reversible pigment without the pigment being visibly evident or of a known concentration. This was first shown by Warburg and Negelein (1929) for the photoreversibility of the carbon monoxide inhibition of cytochrome oxidase-linked respiration.

The solution of the differential rate equation for conversion of phytochrome (Butler, 1961)

— — = —E$e\<p$ 660 660 + ^λ(€λ<^λ)73θ[ί>73θ]

is

ι Λ „ /r> r> \ — 0.43ϋ/λ(€λ<Ρλ)660 / , „ lOg ( 660 — Î W ) = 5 t + C

* 730oo

where Pm = mole fraction at time t = 1 — P730 P660oo = mole fraction of Pm at t = <*>

E\ = incident energy (Einsteins per square centimeter per second) Em\ = extinction coefficient of Pm at λ (square centimeter per mole,

base e) 660 = quantum yield of reaction P6eo —► P730 (moles per Einstein)

c = a constant of integration If a wave band is selected where absorption is essentially that of Ρββο the solution is simply

log P66o = -0.43.EX(€X<PXW + c and a similar equation holds for P730 in its absorption region where overlap with that of P660 is negligible.

The energies {E\t) required for two degrees of physiological response, say germination percentage (a) and (6), are measured for λ = 660 τημ and for the reverse reaction at λ — 735 ΐΆμ after P660 is fully converted to P735 by irradiation in the region of 660 m/x. These lead directly to values of the mole fractions P660 for germinations (a) and (6) and to values Of (Εχφχ)ββο and (Εχφχ)

730· Values of (α )6βο for lettuce seed germination found in the indicated way are 2 χ 104 liter/mole/cm and for (<χφ)730 from Lepidium virginicum seed germination 0.1 X 104 liter/mole/cm. These high values, approach-ing a possible maximum of a near 105, indicate φ is the order of magni-tude of 1.0 or that the sum of y>660 and 730 approaches 1.0, which would be in agreement with photoconversion through a triplet spectroscopic state.

Variation of internode length of pinto beans with degree of phyto-chrome conversion is shown in Fig. 6a and 6b. Similar types of curves have been measured for flowering and seed germination (Hendricks et al, 1956).


(a)

100,

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Fraction of pigment in far-red absorbing form

(b)

FIG. 6. (a) Variation of intemode length of pinto bean following the indi-cated irradiations at the beginning of each dark period, (b) Variation of intemode length of pinto beans with the mole fraction of PT3o. Circles correspond to re-sponses to red radiation with the pigment initially in the Ρββο form and crosses to far-red radiation with the pigment initially in the P™ form.


6. Physical Detection of Phytochrome I t was long apparent that some type of assay was needed for phyto-

chrome. A physiologically active compound of unknown biochemical action is usually detected by the physiological response upon its réin-troduction into a living system. A classic example for plants is the assay for auxin by means of the phototropic response of an Avena coleoptile or the curvature of a split stem section. A similar test for phytochrome was unlikely because of its probable protein nature.

Change in absorption of radiation with change in form of phyto-chrome offered a possible, but demanding, approach to an assay. If the concentration [C] of phytochrome is 10~7 molar, a reasonable level in a tissue, then the change in optical density at 660 mp upon change in form is

Δ OD = a[C] = 2 X 104 X 10~7 = 0.002

per centimeter length. This is near the noise level of most spectrograph detecting systems. Moreover, the absorbancy at 660 ταμ of a centimeter thickness of green material is > 106 and the order of 103 for dark-grown plants. The material also scatters the radiation.

An improvement leading to the successful method (Butler et al., 1959) is offered by differential spectrophotometry, in which the differ-ence in the change in absorbancy at 660 and 735 ηΐμ, Δ(ΔθΌ), is measured as phytochrome is changed in form, that is

Δ(Δθϋ) = [OD660 - OD735] with P660 present — [OD66o - OD735] with P735 present

A further spectrographic requirement is for the detector to subtend a solid angle at the sample of the order of π radians or better to collect scattered light. The noise level of the detecting system should be less than 0.001 OD at an OD of 3.0. A simple spectrophotometer meeting these requirements can be constructed with the appropriate interference filters (Birth, 1960).

The plant material to be tested should be as free of chlorophyll as possible. Dark-grown material contains protochlorophyll which is con-verted to chlorophyll upon the irradiation necessary for changing the form of phytochrome. Immediately after conversion the material changes in absorbancy quite rapidly but is stabilized after a few minutes.

In the first attempted assay for phytochrome by differential spectro-photometry, seven-day old tissue of dark-grown chloramphenicol-treated turnip seedlings was placed on a 1-cm thick plaque in the light path. I t was irradiated to convert protochlorophyll and then irradiated for about


30 sec with a strong source in the region 600 to 680 τημ after which ΔΟΌ was measured betwen 660 and 735 τημ. The tissue was next irradi-ated in the region > 700 ταμ and AOD was measured. The difference of Δ(Δθϋ) was found to be of the order of 0.02.

Phytochrome is readily detectable in many plant tissues by means of differential spectrophotometry. Shortly after the development of the method it was detected in dark-grown shoots of maize, barley, rye, oats, sorghum, wheat, and several varieties of beans. It was also observed in florets of cauliflower and in the seed of avocado as obtained from markets.

7. Isolation of Phytochrome Using differential spectrophotometry, efforts could be made to isolate

and purify phytochrome from tissue. Its detection in vitro was entirely successful (Butler et al.} 1959). Phytochrome in solution has the usual properties of proteins. It coagulates at 50°C at room temperature in the presence of acid with loss of reversibility. It can be precipitated by salt and adsorbed on the usual protein adsorbants.

Dark-grown maize seedlings are a convenient material for the extrac-tion of phytochrome. The seedlings can be broken by grinding with sand or, alternatively, frozen at —16°C and then harvested, after cool-ing with solid C02. The material is then placed in twice its volume of dilute pH 7 buffer in the presence of about 0.05 M —SH-containing material. Fiber and cell wall debris are removed by filtering through cheesecloth and the solution is clarified by centrifuging. The optical density per centimeter at this stage is about 0.004, or 0.02 in a 5-cm path length which can be used with solutions of low absorbancies. Thus, the initial scale of detection is only severalfold greater than the noise level of the spectrometer.

The Δ(ΔθΌ) values can be increased by concentration of the original solution from a protein content of a few milligrams per milliliter to the order of 50 mg/ml. In general, this is accomplished by salting out with ammonium sulfate, followed by solution in dilute buffer or by adsorption on diethylaminoethyl cellulose, followed by elution at moderate phos-phate concentrations. In either case, additional water can be removed by dialysis under vacuum or against polyethylene glycol. In this way, solutions can be obtained with a Δ(ΔθΌ) per centimeter of the order of 0.1 and a possible phytochrome content of the order of 0.1% of the protein.

Upon concentration some of the solutions become somewhat cloudy. A bluish-green pellet is obtained upon centrifugation. This pellet is partially soluble in dilute buffers, giving clear blue solutions that re-


versibly change color upon irradiation. The procedure is apparently one of selective denaturation. While the controlling factors are not fully understood, the method of preparation is the most effective one yet developed (Siegelman et al, 1961; Bonner, 1961).

Further purification of phytochrome is proving difficult as is usual with cytoplasmic proteins of leaves. Repetition of adsorption, molecular-sieve and salting-out procedures decrease the absorbancy arising from impurities in the blue and near-ultraviolet parts of the spectrum and give solutions adequate for measurement of the optical properties of the pigment between 300 ταμ and the limit of detectable absorption near 800 τημ for P735.

The occurrence of phytochrome in a number of green plants showing physiological evidence of its action was verified by extraction, which permits its separation from chlorophyll and its concentration prior to assay by differential spectrophotometry (Lane et αΖ,, 1962). Cocklebur, sorghum, and spinach are among the plants giving positive results.

The major problem of identification of the chromophoric group in phytochrome by chemical degradation is still to be accomplished. The absorption spectrum is very similar to that of allophycocyanin (0 h Eocha, 1960) as shown in Fig. 7. The prosthetic group of allophy-cocyanins is a bilidiene which suggests that the group for phytochrome might be a bilidiene or a bilitriene. The formula for a bilitriene and

2.0

1.5

αφ

i.o

Q5

o 550 6 0 0 6 5 0 700 750 8 0 0

Wavelength rn/u

FIG. 7. Action spectra for phytochrome conversions and the absorption spec-trum for allophycocyanin (ordinates, liters per mole per centimeter).


the approximate configurations of ds-ds-cis, cis-ds-trans, and trans-trans-trans isomers are shown in Fig. 8. The spatial features of these types of compounds have not been studied.

Rl &2 R3 ^4 **5 **6 R7 ^ 8

I I I H H H

OH

H \ R 7

' R .

R8 ΙΙγ

FIG. 8. Formulas of a bilitriene showing three possible geometrical isomers.

8. Properties of Phytochrome In Vivo and In Vitro The monomolecular nature of the photoreaction P660 P73o was first

based on physiological evidence of temperature independence. Lettuce seeds allowed to imbibe water exposed at two temperatures to a suc-cession of red and far-red irradiations (Borthwick et al., 1954). The temperatures were maintained only over a period of 30 min, after which the seeds were exposed to the same temperature for development of germination. The identical responses at the two temperatures shown by the results listed in Table I indicate that the change of phytochrome is not limited by a possible second reactant. This conclusion is verified by the unmodified retention of reversibility after prolonged dialysis, several salting-out procedures, and adsorptions, processes which would remove a second reactant.

A difference absorption spectrum for phytochrome in living maize tissue in the region >500 m/x, is shown in Fig. 9. It is evident that the


TABLE I PHOTOREVERSAL OF PROMOTION AND INHIBITION OF GERMINATION

OF GRAND RAPIDS LETTUCE SEED

Irradiation

Germination (%) at 20° after irradiation

At 26°C At 6-8°C

R R—I R—I—R R—I—R—I R—I—R—I—R R—I—R—I—R—I

70 6

74 6

76 7

72 13 74 I * 75 11

absorbancies of P660 and P735 at their respective maxima are approxi-mately equal. Extracts having this property can also be obtained but after a number of separative procedures or long standing at —17°C the ratios of absorbancies P735:P66o are less than one and values as low as one-tenth have been found after tryptic digestion or manipulation in the absence of —SH compounds, particularly for extracts from barley. An example of a difference spectrum made with a somewhat altered material from barley is shown in Fig. 10. The changes in absorbancy ratios arise chiefly from a decrease in absorbancy of P735. The changes

Δ 0. D.

+ .01

>. 0

- . 0 1

' T - - —■»'■■- — Γ - " » Far-Red irradiated

^—sM^~ vs Red irradiated

\ ^ / ' '

1 i 1 i i 500 600

Wavelength- m μ 700 800

FIG. 9. A difference spectrum for phytochrome in living dark-grown maize seedlings.

indicate a modification in the relationship of the chromophoric group to the neighboring protein, which might indicate denaturation. Upon repeated reversals the apparently unmodified P660 gives the changed P735 and, accordingly, it too must be modified.

An action spectrum leading to αφ values can be obtained for phytochrome after the method described in Section 5 on thin solutions

3 2 0 STERLING B. HENDRICKS

WAVELENGTH-m/*

FIG. 10. A difference spectrum of a solution of phytochrome separated from dark-grown barley. Slight conversion from PT3o to Ρββο in darkness is indicated.

of low total absorbancies. In this case the first-order character with regard to radiant energy can be tested by assay after various irradi-ances and is found holding as was assumed in Section 5. Values of αφ measured on maize extracts at various wavelengths for the two forms are shown in Fig. 7. It is evident φββ0 exceeds <p735 three- to fourfold, with equal absorbancies of the two forms. This ratio of quantum effi-ciencies is also found for phytochrome separated from barley with absorbancy ratios of a73o/a660 of about 0.8/1.0.

Fluorescence was observed for P66o and the action spectrum was measured for its excitation. Best results were obtained by cooling the sample —196°C in liquid nitrogen with measurement of emission for wavelengths >710 τημ. Maize tissue treated in this way gave a maxi-mum of emission near 667 τημ, followed by that of chlorophyll with a maximum near 678 τπμ. Fluorescence was not found for P735 and is seemingly negligible.

Denaturation of the pigment gives a material having an irreversible maximum of absorption near 660 τημ which also is fluorescent.

9. Dark Reversion of P735 and Time-Sensing in Photoperiodism

A natural and early question about photoperiodic control of flower-ing was: How is time measured? The first speculations favored day length as the determining factor. However, flowering controls on dark


and light periods of various lengths, including periods other than those totaling 24 hours, left little doubt that the period in darkness is effec-tive (Hamner and Bonner, 1938; Emsweller et al, 1941). This con-clusion is supported by the effect on flowering control of long dark periods by irradiations near the middle of the period with a few milli-joules per square centimeter energy in the region of 660 τημ. The effect of darkness, while pertinent, did not suggest the nature of the control.

If a substance exists in interconvertible forms, one form will have the lowest energy state and the second form might be expected to revert to the first form after a period of time. In other words, either Pe60 or P735 might show reversal in darkness.

The existence and direction of a possible change was first tested with the germination of lettuce seed (variety Grand Rapids) (Borthwick et al, 1954). Groups of these seeds were allowed to imbibe water in darkness at 20°C and were then exposed to red radiation to potentiate germination by conversion of P66o to P735. The seeds were then placed in darkness at 30°C or 35°C to block germination, and returned after various times to 20°C in darkness. Seeds held at 30°C for 24 or more hours failed to germinate upon return to the favorable temperature. They were induced to germinate by a second irradiation at the lower temperature, showing that the pigment was still effective. Apparently P735 reverted to P660 in darkness.

While the direction of change was evident from the results on lettuce seed germination, it was too slow to be effective for flowering control. Phytochrome from different sources was not necessarily expected to have equal reversion times. A general argument about flowering indi-

χΐο~2

4(

3

^ 2

O

S

0

0 1 2 3 4 5 HOURS

FIG. 11. Reversion of P735 to Ρββο in dark-grown maize seedlings. Ordinates are A(AOD) values measured with a differential spectrophotometer as described in Section 6.

CORN

L \ ^^

\ rV

V P660 1 1

P660 +P735 3 φ

P660 + P735 27°

'>*—

'"""0'"··-···©............0


cated that appreciable conversion of P735 to Ρββο must take place in about 10 min because critical lengths of dark periods can be measured to about this limit.

The development of an assay by differential spectrophotometry afforded a method for following the dark conversion of phytochrome in etiolated plants, The results for maize are shown in Fig. 11. Samples of maize tissue with both P660 and P735 present were assayed after being subjected to periods of darkness at room temperature. It is evident that total Δ(ΔθΌ) values decrease when P735 is the initial form. P735 is also observed to change to P660 with a half decrease in about one hour which does not necessarily indicate that the change is first order.

Physiological measurements on flowering gave further evidence of time-dependent control of the dark reversal of P735. Seven lots of snap-dragon plants were placed on 8-hour day and 16-hour "night" periods, with the respective irradiation of low intensity (50 foot-candles) from an incandescent filament light during the "night." The time periods were as follows: (a) continuous; 10% exposure out of each, (b) 15 min, (c) 30 min, and (d) 60 min; (e) a single interruption for 96 min; and (f) no irradiation (night controls). Representative plants after

FIG. 12. Flowering of snapdragon in response to change of Ρ™$ to Peeo (see Section 9 for details), indicating a detectable shift in darkness in less than 15 min.


six weeks' exposure to these cycles are shown in Fig. 12. The marked difference with an interruption of the night at 15-min and 30-min intervals as compared with continuous irradiation is evident. The P735 which is produced by the irradiations and which is effective for delaying flowering of snapdragon evidently reverts in 15 min of darkness to an extent easily shown by the differences in flowering between plants (a) and (b) and plants (b) and (c). Similar results were obtained with petunia and chrysanthemum varieties.

In resume, the primary timing factor in photoperiodic control of flowering and other growth responses arising from phytochrome action is the dark conversion of P735 to P66o· This reversion is probably related to the time-dependent metabolism during the night resulting from util-ization of photosynthetic products of the preceding day. An interaction is evident in the varying effectiveness of a given irradiation as a "light break" during the night depending upon the time. Such interaction of two or more time-dependent systems can lead to a degree of tempera-ture independence and generate the rhythmic changes evident in the nyctic responses during prolonged dark periods (Bunning, 1935, 1936).

10. Equivalent Control of Unrelated Displays The close similarity of action spectra for the control of flowering,

germination, and etiolation illustrated in Fig. 3a and b, and discussed in Section 3 has a simple explanation; namely, they are the action spectra for the interconversion of phytochrome. Some of the differences are due to the way in which response depends upon conversion, upon the presence or absence of screening pigments, and upon the relative values of αφ6βο and a >73o.

The very fact that the displays are varied and unrelated indicates that the control of the radiation is at some remote but common point. To seek this common point is equivalent to investigating the manner in which phytochrome behaves. The first step in elucidating its mode of action is to find which form of phytochrome is physiologically active.

11. The Active Form of Phytochrome The etiolation response is useful in studies of phytochrome. P660 is

present in dark-grown peas and phytochrome serves to control the size of the leaf. Irradiation in the region of 660 ηΐμ, adequate to convert < 0 . 1 % of P66o to P735 causes an increase of more than 15% of the realizable increase in leaf size accompanying full conversion. I t there-fore follows that P735 is an active form because its amount relative to


that in darkness is greatly increased by the low irradiance, while that of P660 is reduced from 1.000 to 0.999.

Evidence is also afforded by seed germination. Seeds have long been known to remain dormant in soil without germinating. Experiments Tyere started during the last century (Beal, 1905; Darlington and Steinbauer, 1961) on the maintenance of viability of the buried seed. These experi-ments actually investigated the deep dormancy or essential lack of metabolic activity maintained by the persistent seed. Successful testing for viability was probably related to the exposure to light of the seeds during inspection of freshly dug lots. Phytochrome alone was probably changed by light from P660 to P735 and in the latter form "activated" the whole germination process.

On the basis of this evidence, P735 is probably an enzyme or a hormone. The synthesis of anthocyanin gave an unexpected impetus to this aspect of research on phytochrome.

12. Photocontrol of Anthocyanin Synthesis Synthesis of the prominent red and blue anthocyanin pigments of

many fruits and flowers is controlled by radiation. It was early recog-nized by a horticulturist of the last millenium when he exposed apples to sunlight to deepen their color. The autumnal coloration of leaves was also vaguely related to the action of light. However, the first sys-tematic study of the effects of light and darkness on anthocyanin synthesis was published by Senebrier in 1799. Sachs in 1863 and Sorby in 1873 described the development of anthocyanin in plants as a response to light.

The basic work on the relation of light to anthocyanin synthesis will be considered only in its relation to phytochrome activity. By way of illustration the work on the formation of cyanidin in turnip (Siegelman and Hendricks, 1957) and sorghum (Downs and Siegelman, 1963) will be cited. The amount of anthocyanin [4] in moles per square centi-meter of tissue in the linear region is

[A] = koupEt

where k is a constant, and a, <p, E, and t are defined in Section 4. The receiver of radiation apparently does not vary with time and

uses absorbed energy with a rather high efficiency φ to form the product, anthocyanin. The action spectra for this reaction differs for each of more than eight tissues of the various plants that have been studied. Although a complete explanation for the reaction is lacking it is certain that phytochrome is involved.

Alcohol production in apple peels floating on sucrose solutions is


suppressed by irradiation in spectral regions where anthocyanin syn-thesis is prominent (Fig. 13) (Siegelman and Hendricks, 1958a,b). I t seems that cyanidin, the chromophore of the glycoside moiety, and alcohol are alternate products of the same substrate, influenced respec-

OJ

5 4 >>* o X o 3 -J < Id 2 o Σ o S i

9 25° DARK

/ ? 21° DARK

9" ' , ' 4 19° LIGHT A f 15° LIGHT & , 11° DARK

10 20 30 40 50

IRRADIATION IN HOURS

60 70

FIG. 13. Effect of light and darkness on formation of ethanol by apple peels floating on 2% sucrose solutions.

tively by light and darkness. A two- or three-carbon compound such as acetate or pyruvate seems to be involved and it is also known that simple compounds are produced. Apparently a number of different pig-ment systems can serve as photoreceptors for this reaction, suggesting a function in photosensitized oxidation (Blum, 1941). This reaction will be called "high-energy" dependent.

The anthocyanin synthesis induced by the high-energy reaction is completed only after some hours in subsequent darkness. The time for half completion is of the order of 12 hours (Siegelman and Hendricks, 1957). If the irradiation is restricted to a few hours and then followed by transformation of P735 to P66o by far-red radiation, the anthocyanin synthesis in plants responsive to radiation is blocked. This conversion is effective to a decreasing extent if the far-red irradiation is delayed; the half time for the delay to have maximum effectiveness is about 3 hours.

Action spectra for this second photocontrol of anthocyanin produc-tion after potentiation by the high-energy reaction can be run immedi-ately after the high-energy reaction. Results of measurements of the action spectrum for glycoside formation in sorghum (Wheatland variety)


seedlings made in this way is shown in Fig. 14. The spectrum shows phytochrome conversion and gives the expected reversal.

FIG. 14. Action spectra for promotion and inhibition of anthocyanin formation in sorghum seedlings (var. Wheatland).

The time course of anthocyanin synthesis can be expressed as

Cyanidin has the formula

intermediates —* phytochrome photoactivation to P73B —> inter-mediates —> anthocyanin glycoside.

substrates —> pyruvate or acetate —> high energy photoreaction —>

to ethanol in darkness

120REPROMOTION

110 110

100 100

90 90

N: "'e 8OE 80(,) (,)

....... .......b 70 x b 70

)( )(

en 60 en 60at at

~50...

~500

>- >-~40 ~40w IJJZ Zw30 W30

20 20

10 10

580 600 620 640 660 680 700 720 680 700 720 740 760 780 800 820WAVELENGTH (m)J)


It is a "C6—C3" compound. The C9 moiety is believed to be formed from glucose by the shikimic acid pathway (Neisch, 1960). The C6 or phloroglucinol moiety is probably formed through an activation of acetyl radicals, CH3CO, the features of which are yet to be discovered.

The multiplicity of responses controlled by phytochrome suggests that it is associated with acetyl conversion. The many pathways of this conversion are discussed by Lynen and Decker (1957) and Decker (1959) and include the synthesis of steroids, the synthesis and break-down of fats, the synthesis of chlorophyll, and esterification.

The relation of anthocyanin synthesis to phytochrome action is that it probably restricts possible action to a small number of reactions yet to be discovered. A biochemical explanation of this reaction should be possible from a careful study of the products and substrates involved.

13. Stages between the Light Impulse and Its Physiological Effects

As with anthocyanin synthesis, intermediate steps must be present between photoactivation and the effects expressed in stem elongation, flowering, or seed germination. This aspect of the subject is far afield from photochemistry and there are few facts to aid in explaining the phenomenon in both stem elongation and flowering. Something, possibly one or more compounds, moves from the place of excitation to the receptor tissue. The effects of the agent are evident in flowering by a modification of response unless the irradiated leaf remains on the plant for a period up to 24 hours. The supposed regulating material is called "florigen" (Cajlachjan, 1937).

Translocation of a material regulating stem elongation and subject to phytochrome control is shown by the absence of a phototropic response to unilateral red-irradiation. This compound might be a gib-berellin or a compound possessing gibberellin-like activity. I t effects a radial coordination in lengthening of elements of the stem.

The photochemical aspects of the distinction between plants requir-ing long nights for flowering and those flowering in short nights are intriguing even though explanations are lacking. If red radiation is used to interrupt a long night for these two types of plants, it prevents flowering in the one but causes flowering in the other. The action of P735 is very probably unique. I t may be considered as a material effec-tive only in a limited range of concentration, below which range it is inadequate to promote flowering and above which it is inhibitory (Borth-wick et al., 1956).

Knowledge of the enzymatic action of phytochrome will probably shed some light on the mechanism of this reaction.


14. Some Physiological Aspects of Phytochrome Action

Phytochrome appears to be a regulatory agent in the growth of all seed plants and has been observed, through action spectra, in ferns (Pteridophyta) and in Marchantia (Bryophyta) by control of gemma formation. This leads to the question as to why some plants are inde-terminant in flowering, the tomato and zinnia often being used as examples. Development of the tomato, however, is adversely influenced by continuous radiation; it etiolates and the cuticle of the fruit of some varieties develops a yellow pigment in response to the action of P730 during ripening. A probable effect of P730 is to divert intermediates that are involved in flowering, etiolation, and other aspects of growth. Thus, in plants like wheat that flower in continuous light, P730 diverts the stimulus to flower that reaches inhibitory levels as a dark period is increased in length. In plants like soybeans that require a long night to flower, P730 produced by a night interruption diverts the stimulus below a critical level. An indeterminant plant on this basis is one neither attaining inhibitor levels of flowering stimulus on long nights nor sinking to low levels in continuous light. Failure to control does not indicate the absence of the controlling agent but merely indicates that its effects are less obvious.

The plant-environment interrelationship is of interest with regard to P730 action. Temperature, water, and light regimens are dominant factors for plant growth, and although it is misleading to consider one without the other, for the sake of clarity the interactions of P730 with temperature are considered. The controlling action of P730 has both a temperature response and a degree of temperature independence both necessary for a useful timing system. Dark reversion is enhanced by an increase in temperature as discussed in Section 9, but its rate of action on substrates is also increased, which is compensatory. Other tempera-ture dependencies in multiply-connected metabolic pathways can lead to further stabilization or an approximate constancy of physiological response. The balance in this way, however, is limited in range perhaps to a 10-20°C variation and apparently goes askew at high and low temperatures.

The interplay of phytochrome action and temperature regimens is particularly obvious in seed germination (Kinzel, 1913-1926). This inter-relationship is of great ecological value in the persistence of many annual plants. Many seeds exhibit light requirement for germination in a narrow temperature range, above the range germination will not take place and below it light is not required. As the seed ages the light requirement becomes more necessary. Germination is often enhanced


by temperature alternations in 8-hour high-, 16-hour low-cycling. The longer cycling has been replaced by short periods (2 hours) at tempera-tures of 35 °C for seed of wild pepper grass and lettuce, with this period necessarily preceding the phototransformation of phytochrome. The tem-perature cycling, even though it is the way of enhancing a light response in nature, is often not required and can be replaced by imbibition in 0.2% KN03 solution.

15. Comparison of the Photochemical Aspects of Plant Photoperiodism and Vision

Photochemical change of both rhodopsin and phytochrome involve the geometrical isomerization of a chromophoric group of a protein. An intermediate triplet state with the possible change in geometrical form might be involved in both reactions. In the case of rhodopsin the chromophore eis- 11-retinene, through several intermediates, is disso-ciated from the protein opsin and becomes fraris-retinene. The regen-eration of rhodopsin involves a cyclic process in which eis-11-retinene is regenerated by reduction to an alcohol, reoxidation to the aldehyde, and finally combination with opsin (Wald, 1961). Excitation changes phytochrome P735 to P660 with a change in form but without dissociation from the protein. The photochemical change in the opposite direction, P660 -» P735, is from the thermodynamically stable to the unstable P735 form, which reverts in a moderate amount of time to the stable Ρββο form.

There are indications in anthocyanin synthesis that P735 is an en-zyme. The mode of action of rhodopsin is still unknown but the quick nerve impulse response to its excitation strongly suggests that an enzymatic process would not be fast enough for the action. Rather, a response to a change of protein configuration is not unreasonable, allow-ing salt passage through a membrane to give the necessary amplification for action. Phytochrome change is also very sensitive to protein con-figuration as shown by variation of absorptivity of P735 upon manipu-lation and by the eventual lack of reversibility as in flowering control of the morning glory, Pharbitus nil (Borthwick et al., 1961).

In both rhodopsin and phytochrome the chromophoric group in the active form seems to be stretched across the protein with possibly mutual distortions. The great spatial change of geometrical isomeriza-tion releases the chromophore and allows it to change to a more stable form. At the same time it allows the protein to undergo an even greater shift in atomic position. Excitation of rhodopsin is interpreted as leading to unmasking of two —SH groups and a change in the form of opsin in the neighborhood of the chromophore (Wald, 1961).


16. Conclusion In conclusion, study of photochemical action in plant photoperiodism

led to an essential understanding of the phenomenon. A partial connec-tion has been found between a physiological phenomenon and a bio-chemical change.

REFERENCES

Beal, W. J. (1905). Botan. Gaz. 40, 140-143. Birth, G. S. (1960). Agr. Eng. 41, 432-435, 452. Blum, H. F. (1941). "Photodynamic Action and Diseases Caused by Light." Rein-

hold, New York. Bonner, B. A. (1961). Plant Physiol. 36, (Suppl.), xliii. Borthwick, H. A., and Hendricks, S. B. (1960). Science 132, 1223-1228. Borthwick, H. A., Hendricks, S. B., Parker, M. W., Toole, E. H., and Toole, V. K.

(1952). Proc. Natl. Acad. Sei. U. S. 38, 662-666. Borthwick, H. A., Hendricks, S. B., Toole, E. H., and Toole, V. K. (1954). Botan.

Gaz. 115, 205-225. Borthwick, H. A., Hendricks, S. B., and Parker, M. W. (1956). In "Radiation

Biology" (A. Hollaender, ed.), Vol. I l l , pp. 479-517. McGraw-Hill, New York. Borthwick, H. A., Nakayama, S., and Hendricks, S. B. (1961). Proc. 3rd Intern.

Congr. Photobiol., The Netherlands, 1960 pp. 394-398. Bunning, E. (1935). Jahrb. wiss. Botan. 81, 411-418. Bunning, E. (1936). Ber. dent, botan. Ges. 54, 590-607. Butler, W. L. (1961). Proc. 3rd Intern. Congr. Photobiol, The Netherlands, 1960

pp. 569-571. Butler, W. L., Norris, K. H., Siegelman, H. W., and Hendricks, S. B. (1959). Proc.

Natl. Acad. Sei. U. S. 45, 1703-1708. Cajlachjan, M. C. (1937). Compt. rend. acad. sei. U.R.SJS. 4(2), 79-83. Caspary, R. (1861). Schuften Kgl. physik.-Ökonom. Ges. Königsberg 1860 1, 66-91. Darlington, H. T., and Steinbauer, G. P. (1961). Am. J. Botany 48, 321-324. Decker, K. (1959). "Die aktivierte Essigsäure. Das Coenzym A und seine Acyl-

derivate im Stoffwechsel der Zelle." Ferdinand Enke, Stuttgart. Downs, R. J., and Siegelman, H. W. (1963). Plant Physiol. 38, 25-30. Emsweller, S. L., Stuart, M. W., and Byrnes, J. W. (1941). Bull. Chrysanthemum

Soc. Am. 9, 19-20. Flint, L. H., and McAlister, E. D. (1935). Smithsonian Inst. Misc. Collections

94, 1-11. Garner, W. W., and Allard, H. A. (1920). / . Agr. Research 18, 553. Hamner, K. C, and Bonner, J. (1938). Botan. Gaz. 100, 388-431. Hendricks, S. B., and Borthwick, H. A. (1955). In "Aspects of Synthesis and Order

in Growth" (D. Rudnick, ed.), pp. 149-169. Princeton Univ. Press, Princeton, New Jersey.

Hendricks, S. B., Borthwick, H. A., and Downs, R. J. (1956). Proc. Natl. Acad. Sei. U. S. 42, 19-26.

Kinzel, W. (1913-1926). In "Frost und Licht als beeinflussende Kräfte bei der Samenkeimung." E. Ulmer, Ludwigsburg, Germany.

Laetsch, W. M., and Briggs, W. R. (1962). Plant Physiol. 37, 142-148.


Lane, H. C, Siegelman, H. W., Butler, W. L., and Firer, E. M. (1962). PUnt Physiol. 38, 414-416.

Lynen, F., and Decker, K. (1957). In "Ergebnisse der Physiologie, biologischen Chemie und experimentellen Pharmakologie," pp. 328-424. Springer, Berlin.

Mohr, H. (1956). Planta 46, 534-551. Monk, G. S., and Ehret, C. F. (1956). Radiation Research 5, 88-106. Neisch, A. C. (1960). Ann. Rev. Plant Physiol. 10, 55-80. O h Eocha, C. (1960). In "Comparative Biochemistry of Photoreactive Systems"

(M. B. Allen, ed.), pp. 181-203. Academic Press, New York. Parker, M. W., Hendricks, S. B., Borthwick, H. A., and Scully, N. J. (1946).

Botan. Gaz. 108, 1-26. Sachs, J. (1863). "Über den Einfluss des Tageslichtes auf Neubildung und Entfaltung

verschiedener Pflanzenorgane." Beih. Botan. Ztg. Senebrier, J. (1799). Physiol. veget. {Geneva). Siegelman, H. W., and Hendricks, S. B. (1957). Plant Physiol. 32, 393-398. Siegelman, H. W., and Hendricks, S. B. (1958a). Plant Physiol. 33, 185-190. Siegelman, H. W., and Hendricks, S. B. (1958b). Plant Physiol. 33, 409-413. Siegelman, H. W., Firer, E. M., Butler, W. L., and Hendricks, S. B. (1961). Plant

Physiol. 36 (Suppl), xlii. Sorby, H. (1873). Proc. Roy. Soc. (London) 22, 442-483. Toole, E. H., Toole, V. K., Hendricks, S. B., and Borthwick, H. A. (1957). Proc.

Intern. Seed Test Assoc, Copenhagen, 1956 22, 1-9. Vogt, E. (1915). Z. Botan. 7, 193-271. Wald, G. (1961). In "Light and Life" (W. D. McElroy and Bentley Glass, eds.),

pp. 724-753. Johns Hopkins Press, Baltimore, Maryland. Warburg, O., and Negelein, E. (1929). Biochem. Z. 214, 64-100.

Chapter 11

THE ROLE OF LIGHT IN PERSISTENT DAILY RHYTHMS1

J. Woodland Hastings

Biochemistry Division, University of Illinois Urbana, Illinois

1. Introduction. Rhythms and Biological Clocks Over the past decade there has been developed a strong body of

evidence in support of the proposition that persistent daily rhythms are a manifestation of a biological time-measuring system (for review of the literature, see Hastings, 1959, 1962; Pittendrigh, 1961; also Cold Spring Harbor Symposia, Vol. 25, 1960). I t is implicit in our adoption of this viewpoint that organisms and cells—through the mediation of this biological clock mechanism—are able to vary and regulate the capacities of various physiological and biochemical processes with re-spect to time of day.

The light-dark cycle of the environment is quantitatively the most spectacular, and certainly the most regular and reliable periodicity experienced by the organism. However, although daily rhythms are clearly adaptively oriented to this environmental light-dark cycle, and although there are very pronounced light effects in all biological rhythms, the clock-related biological rhythms are not strictly and directly "forced" by the light cycle. Rather, the mechanism itself derives its intimate control from an endogenous cellular mechanism (Pittendrigh, 1958), a "block box" which we refer to as the biological clock.

The physicochemical nature of this rhythmic mechanism has not been elucidated, but in recent studies it has been found that by blocking ribonucleic acid (RNA) synthesis, the clock is specifically inhibited (Karakashian and Hastings, 1962). Actinomycin D, a specific inhibitor of deoxyribonucleic acid (DNA)-dependent RNA synthesis, blocks the occurrence of rhythmicity of both luminescence and photosynthesis in Gonyaulax polyedra at concentrations as low as 2 X 10-8 M.

Supported by a grant from the National Science Foundation. 333

334 J. WOODLAND HASTINGS

The compelling biological evidence for the conclusion that the mechanism functions without environmental signals comes from experi-ments in which rhythmic organisms are placed in constant light and temperature conditions in the laboratory (Fig. 1). Not only is it observed

10 20 30 40 50 60 70 80 TIME -HOURS

FIG. 1. These curves illustrate the nature of the rhythm of flashing luminescence in Gonyaulax. The curve at the top (A) shows the rhythmicity in cultures exposed to alternating light and dark periods of 12 hours each (LD 12:12). When such cells are transferred to conditions of constant light (120 ft-candles) and constant temper-ature (21 °C) the rhythm persists with a period of about, but not exactly 24 hours (curve B). (After Hastings and Sweeney, 1959.)

that the rhythm continues; it does so with a period which approximates the solar day, but which is slightly different from 24 hours. This is in contrast to a rhythm maintained in the 24-hour light-dark cycle of the environment where the period is exactly 24 hours (Figs. 1 and 18). The fact that rhythms generally possess a period which differs slightly from 24 hours (so long as temperature and light intensity are held constant) is of central importance, since this indicates that environmental variables

I L ROLE OF LIGHT IN PERSISTENT DAILY RHYTHMS 335

which have periods of exactly 24 hours are not involved (Pittendrigh and Bruce, 1957).

We are thus confronted with a biological clock mechanism which functions with absolute time as a principal parameter. There is a feature of all persistent daily rhythms which indicates and underlines the fact that the dimension of time is indeed crucial. This is the so-called "temperature-independence" of biological clocks (reviewed in Sweeney and Hastings, 1960). Experimentally this refers to the lack of large differences in the period of a rhythm when the organism is maintained under constant conditions at different temperatures (Fig. 2). Indeed, we

ÜJ o z LU O </) LU Z

Έ

5h 4

3

2

.A.J

klKAAJ/M./1 JM n K

26.8°±0.7°C 26.5

23.6°±I°C 25.7

I650±I°C 22.8

20 40 60 80 100 120 TIME IN HOURS

140 160 180

FIG. 2. Characteristics of the persistent rhythm of luminescence in Gonyaulax at the three different temperatures noted. The cells were grown on conditions of LD 12:12 at 22°C and transferred at the end of a dark period to constant light (100 ft-candles) at zero time on the graph. The luminescence capacity was meas-ured approximately every 2 hours. The average period in hours measured for each is noted on the graph below the temperature. (After Hastings and Sweeney, 1957.)

would certainly not expect to find a functionally useful biological clock mechanism which was accurate at one temperature but grossly inac-curate at another. Although the biochemical and physiological nature of the mechanism is not known, it is logical to assume that some sort of reasonably accurate compensatory system is involved (Hastings, 1959). Evidence supporting this view comes from the observation that although


small Qio values are generally found, some are greater than 1.0 and others are less than 1.0 (Hastings and Sweeney, 1957; Sweeney and Hastings, 1960).

Studies of the effects of light upon persistent daily rhythms reveal many of their important properties and, moreover, constitute an im-portant experimental means for adducing the nature of the mechanisms involved. It is of more than casual interest to note that the responses of various rhythmic organisms ranging from algae to seed plants and protozoa to mammals are strikingly similar. The present article will deal primarily with aspects which are generally comparable in the various systems studied.

2. The Effects of Continuous Light 2.1 Effects upon Natural Period

Figure 3 illustrates the effect of continuous light at three different

60 80 TIME-HOURS

FIG. 3. This experiment illustrates the effect of light intensity upon the natural period. The cells were grown in LD 12:12 conditions (800 ft-candles during the light period). The beginning of the experiment, shown on the graph as 0 time, fell at the end of a normal light period. At this time, some cells were placed in the dark, and others in light of 120 ft-candles (upper curve), 380 ft-candles (middle) and 680 ft-candles (bottom), the average periods measured respectively being: 24.5 hours (in the dark, not shown on graph), 24.5 hours, 22.8 hours and 22.0 hours. (After Hastings and Sweeney, 1958.)

11. ROLE OF LIGHT IN PERSISTENT DAILY RHYTHMS 337

intensities upon the rhythm of luminescence in Gonyaulax. Not illustrated are measurements at even higher intensities where there is observed essentially no residual evidence of rhythmicity; i.e., the system is alto-gether arrhythmic. Several features are evident. First, there is an effect of light intensity upon the natural period of the rhythm. At higher light intensities the period is shorter. Although most organisms studied ex-hibit a period change with intensity, not all exhibit a shortening.

Aschoff (1960) has investigated in considerable detail this aspect of daily rhythms and has assembled the results of numerous studies. These results are shown in Fig. 4. Aschoff has pointed out that a general rule

— Ί j ( ! ! 1 O.I I 10 100 1000 Lux 10000

Intensity of illumination

FIQ. 4. The effect of intensity of continuous illumination upon the natural period of daily rhythms in various organisms, as indicated: (a) activity, Johnson (1938) ; (b) activity, Aschoff (1960) ; (c) luminescence, Hastings and Sweeney (1958); (d) activity, Hoffman (1960b); (e), (f) activity, Aschoff (1960); (g) leaf movements, Pfeffer (1915). Note that the absolute intensity required for the Gony-aulax system is greater than the others studied. (After Aschoff, 1960.)

may be formulated, namely, that with increasing intensity of illumina-tion, light-active organisms (finches, starlings, lizards) increase their spontaneous frequency (shorter natural period) whereas dark-active ones decrease spontaneous frequency. The explanation of the nature of the interaction between light and the period-determining mechanism of the clock must incorporate this feature, although no suggestion has yet been made to account for it.

2.2 The Nature of Arrhythmic Systems. An Unsynchronîzed Population

or True Arrhythmicity?

Another feature illustrated by Fig. 3 is the damping of the rhythm at higher light intensities. The very fact that continuous illumination

338 J . W O O D L A N D H A S T I N G S

inhibits rhythmic systems underlines again the fact that there exists a strong interaction between light and the clock mechanism. With most rhythmic organisms which have been studied there has been observed an inhibitory effect upon the rhythm under conditions of constant light. The degree of this inhibition is dependent upon the intensity and wave-length of the light. With the rhythm of bioluminescence in Gonyaulax, for example, the inhibition requires relatively bright light. As noted above, the system is completely arrhythmic at very high light intensities (1000 ft-candles).

4 6 8 HOURS AFTER DAWN

1 0

FIG. 5. Measurement of the photosynthetic capacity in single isolated cells of Gonyaulax from a culture grown in LD 12:12 conditions before transfer to con-tinuous light. Cells kept in dim light (50 ft-candles) exhibit a rhythm (solid circles) whereas those kept in bright light (800 ft-candles) do not (open circles). The absolute levels of photosynthesis are not comparable under the two conditions. (After Sweeney, 1960a.)


Two alternative explanations for the nature of arrhythmicity were noted by Pittendrigh (1954). First, all cells or organisms might be truly arrhythmic individually. Second, although each organism or cell possessed a rhythm, bright light might result in a loss of synchrony between cells. Being out of phase with one another might therefore result in a net aperiodicity. Pittendrigh at that time favored the second hy-pothesis, namely that of desynchronization, on the basis of experiments concerned with phase shifting by light.

More recently it has been possible to distinguish directly between these two alternatives by studying the rhythms of photosynthetic capacity and cell division in isolated single cells of Gonyaulax. Sweeney (1960a) demonstrated that with individual cells maintained in constant bright light, no rhythm was observed (Fig. 5). A similar conclusion was obtained from studies of the rhythm of cell division (Sweeney and Hastings, 1960). It is thus possible to conclude that individual cells in a light-inhibited population do not possess an overt rhythm.

2.3 Action Spectrum for Inhibition

The action spectrum for photoinhibition of luminescence Gonyaulax was determined by Sweeney et al. (1959), and is reproduced in Fig. 6.

u 0.5

0.4

?. 0.3

/

0.2 l·

0.1

400

\

\ \° \ • \

\

Action spectrum for photoinhibition

Absorption of cell suspension

V \

*—·σ^ / /

f \ ° o \ o o o\ °\

\

500 600 WAVE LENGTH IN MJU

700

FIG. 6. The action spectrum for photoinhibition of luminescence capacity in Gonyaulax polyedra. The absorption spectrum for a cell suspension is included for comparison. (After Sweeney et al., 1959.)


It may be seen that the action spectrum closely parallels the absorption spectrum of the cell suspension, which is due largely to chlorophyll, and to carotenoids such as peridinin. Since light also inhibits rhythmicity in nonphotosynthetic forms, it is presumed that chlorophyll and perhaps other photosynthetic pigments act simply as sensitizers in the region of 400 to 680 m/jt. Because of the lack of absorption by chlorophyll a beyond 710 τημ, it was suggested that an unidentified additional pig-ment must be acting as the sensitizer for the inhibition in the region of 690 to 735 m/x, where inhibition of the rhythm still occurs. Since this unidentified pigment does not appear to contribute significantly to the absorption by intact cells, it must be a minor component.

Since inhibition of rhythms by light occurs in animals also, it can be assumed that in Gonyaulax the photosensitizers prominently involved in inhibition are not directly involved in the rhythmic mechanism. No action spectrum has been reported for this function in animals, but there is good indirect evidence from the studies of Whitaker (1940) that the eye is involved. He reported that although blinded mice possess a period of about 24 hours in their activity rhythm, they were not sensitive to shifting the phase of the light-dark cycle. In conclusion, it is clear that, although the mechanism of light inhibition in both plants and animals is not understood, it is a generalized and important feature of rhythmic systems.

2.4 Restoration of Rhythmicity in Arrhythmic Cultures

Although it is known that inhibition by continuous bright light is truly an inhibition of each cell, it is of importance to note that rhythms may be initiated anew merely by placing light-inhibited cultures in the dark or in dim light (Fig. 7). Two features of this response should be noted:

(1) Zero time on the graph is the time when cultures were moved from constant bright light to constant darkness or dim light, and the phase of the rhythm is established by the time when this is done, irrespective of solar time (Fig. 8). (2) No previous recent history of exposure to day-night light cycles is necessary. Experiments like this have been carried out with cells maintained in constant bright light for as long as three years; still the characteristic natural frequency is exhibited after placing in darkness, showing that the capability for ex-pressing rhythmicity exists all the while even though the rhythm is inhibited. Furthermore, the cells possess the property of a 24-hour period without benefit of previous "conditioning" of any sort. A com-plete tabulation of the results of experiments similar to these, with a


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FIG. 7. The initiation of an endogenous diurnal rhythm of luminescence by means of a one-step change in illumination from bright light to either dim light (bottom curve) or darkness (top curve). Cultures which had been grown in bright light for one year were moved from bright light (800 ft-candles) to dim light (90 ft-candles) at the time indicated on the graph as 0 hours. Luminescence measurements were made approximately every 2 hours thereafter. The top curve shows a similar experiment where cultures grown in bright light were transferred to the dark at the time indicated as 0 hours. The rhythm persists in a similar way but the amplitude exhibits a damping, due to the requirement for light to supply energy via photosynthesis. [After Sweeney and Hastings (1957) and Hastings and Sweeney (1958).]

variety of other organisms, has been provided in the review article by Bruce (1960).

A similar experimental question may be investigated with organisms which exhibit arrhythmicity by virtue of long-term maintenance in dark conditions. Figure 9 illustrates the arrhythmic nature of eclosion in a Drosophila culture which had been kept in the dark. In a similar culture which had been exposed to an unrepeated light signal (see Section 3) the emergence exhibits a 24-hour rhythm. Light signals as short as


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FIG. 8. Eight additional experiments similar to the one illustrated in Fig. 7 (bottom). Cultures were transferred from constant bright light to darkness at the various times of day, as shown. Time (Pacific Daylight) is from 0700 hours of the first day through 1300 hours of the second day. The values for the first determination in each experiment (which were made just at the time when that culture was put in the dark) show that the cells kept in constant light do not exhibit a rhythm. The time at which the maximum occurs is related to the time when the culture was put in the dark, this being the way whereby phase is established. (After fig. 3, p. 121, Sweeney and Hastings, 1957.)

FIG. 9. This illustrates the restoration of rhythmicity in an arrhythmic culture of Drosophüa. The emergence of adults in cultures grown in constant dark and constant temperature is aperiodic (top). However, in a similar culture exposed to a single 4-hour light signal (as indicated by the white rectangle on the abscissa), emergence exhibits a rhythmicity with a period of about 24 hours (bottom). (After Pittendrigh, 1961.)


1/2000 of a second have been shown to be effective in this way. In addition, it should be noted that, as in the previous cases, this signal gives no information on periodicity or its possible frequency.

A question which arises in experiments of this kind concerns whether the phase-determining transition is light-to-dark or dark-to-light, i.e., dusk or dawn. From the experiments illustrated (Fig. 7) it is clear that a light-to-dark transition (dusk) can establish phase; other experiments (Bruce, 1960) show equally clearly that a dark-to-light transition is effective in determining phase. The clock mechanism is thus sensitive to both kinds of transitions in phase determination.

Although Pittendrigh and co-workers had previously held the view that organisms were sensitive only to a dark-to-light transition with regard to phase (Pittendrigh and Bruce, 1959), their more recent experi-ments fully support the view that organisms are sensitive to both types of signals (Pittendrigh, 1960).

3. Effects of Short Exposures to Light Pulses

3.1 Phase Shift via Single Pulses vs. via Shifted Cycles

The phase of a rhythm, that is to say, the time of day when the maximum in some particular function occurs, bears a relatively fixed relationship to the environmental light-dark cycle. Experimentally, an appropriate phase shift has always been found to occur when a rhythmic organism is exposed to a light-dark cycle having a different phase, either by moving to a different longitude or by exposure to a different artificial light-dark cycle (Fig. 10).

Some years ago, however, Pittendrigh and Bruce (1957) discovered a fact which is of major significance to our understanding of the nature of rhythms. They demonstrated that with organisms maintained on constant conditions it is not necessary to subject the organism to a particular new light-dark cycle in order to shift the phase of the rhythm. Rather, an appropriate single step-type or pulse-type change in light intensity is sufficient, after which the organisms are again main-tained in constant conditions.

They pointed out that this response is comparable to a characteristic feature of an electronic or a mechanical oscillator. A pendulum, for example, when freely swinging, requires only a single disturbance in order to experience a phase change. A change in light intensity, there-fore, may be viewed as the kind of signal whereby a biological clock may be reset. The phenomenon of phase shifting by single pulses is illustrated by experiments shown in Fig. 11. I t must be emphasized


FIG. 10. This experiment illustrates the effect of changing the solar time at which the light and dark periods occur. The upper curve shows the pattern of luminescence changes in an LD culture which had been on the schedule indicated for some time. The black bars on the time axis indicate dark periods. The lower two graphs illustrate the effect of placing cultures (which were previously on schedule shown in the top graph) in an LD schedule in which the light and dark periods were at a different time of day. The new schedules were started at zero hours on the graph. Temperature, 26°C, light intensity, 250 ft-candles. (Hastings, unpublished.)

again that any change in light intensity is potentially able to cause a phase shift.

3.2 Variation in Sensitivity to Phase Shifting with Respect

to Time in the Cycle

In experiments with light pulses it is possible to study the variation in sensitivity at different times in the cycle. Detailed studies of the response to single light pulses of rhythmic systems under constant con-ditions have been reported by several workers, including Bruce and Pittendrigh (1958) and Pittendrigh (1960) with various organisms in-cluding Drosophila and Euglena, by Hastings and Sweeney (1958) with Gonyaulax, and by De Coursey (1960, 1961) with the flying squirrel, Glaucomys. All workers have found that the new phase, following a short light perturbation, is not directly related to tihe time when ex-posure occurred. Moreover, there is a remarkable qualitative similarity in the response of the various organisms. During the subjective day


10 20 30 50 60 0 10 20 TIME - HOURS

FIG. 11. These experiments illustrate the way in which the phase of the rhythm of luminescence is shifted following an exposure of the cells to 2}£-hour light pulse and the 1400 ft-candles, 21 °C. Prior to the time shown on the graph all cultures were in LD 12:12 conditions, and at the end of a light period the cells were placed in the dark and the control remained in the dark thereafter. This is there-fore similar to the experiment shown in Fig. 7 (bottom). In the other experiments the time at which a light pulse was administered was varied as indicated by bar on graph so as to effectively scan the cycle. Note the pulses during the early part of the subjective night cause a phase delay and those given during the latter part a phase advance. (Hastings, unpublished.)

phase organisms generally do not respond to light signals; signals given during the first part of the subjective night cause phase delay (the subsequent maximum occurs later than usual) ; signals given during


the last half of the subjective night resulted in a phase advance (max-imum occurs earlier than usual). These features are illustrated for Gonyaulax (Figs. 11 and 12) but are qualitatively the same for other organisms.

4 8 12 16 20 2 4 28 TIME IN CYCLE OF EXPOSURE TO

LIGHT PULSE

FIG. 12. The relationship between time in the cycle when a light pulse is ad-ministered and the resulting phase change. Phase shift is plotted on the ordinate as either a delay or an advance. The abscissa is measured in hours subsequent to the end of the last light period, as in the experiment shown in Fig. 11. Solid circles, data from experiment shown in Fig. 11; open circles, data from an experi-ment with a different rhythm in Gonyaulax, that of the luminescent glow, Fig. 16. (Hastings, unpublished.)


It is evident that if an organism were moved in longitude from east to west that the later dusk would result in the required phase delay. Conversely a movement from west to east would involve a light signal before dawn and result in a phase advance.

3.3 Effects of Duration and Intensity of Light Pulses

Studies on the effects of the duration and intensity of single light signals have been reported with Gonyaulax. Increasing both the duration and the intensity of the light signal increased the amount of phase shift, as shown in Figs. 13 and 14. These experiments were carried out with

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FIG. 13. The effect of the duration of a light pulse upon the phase shift. The experiment was similar to the one shown in Fig. 11 except that the time when the light exposure was begun was fixed, and its duration was varied. The in-tensity of the light was 1400 ft-candles, temperature 21 °C. Different symbols, whose meanings are indicated in Fig. 14, give the phase difference between the control and the expérimentais measured at each of the three maxima in luminescence subsequent to the light exposure. The interpretation of phase shifting light pulses having too long a duration is not profitable, since the sensitivity is different at different times in the cycle (Fig. 12). (Hastings, unpublished.)

light pulses given in the latter half of the subjective night phase, so that a phase advance was measured in all cases. Experiments with other organisms indicate that similar relationships are expected to pertain.

3.4 Action Spectra for Shifting the Phase of the Rhythm by Single Light Pulses

An approach which has often been useful in the study of various light-related biological processes involves the determination of the action spectrum. Measurements of this kind for visible light have been reported for Gonyaulax (Hastings and Sweeney, 1960) and for Para-mecium (Ehret, 1960).


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FIG. 14. The relationship between the intensity of a single 2%-hour light per-turbation and the number of hours by which the phase is shifted. Data are taken from an experiment similar to the one shown in Fig. 11, except that the time at which the pulse was given was the same in all cases. As in Fig. 13, the different symbols marked on the graph give the phase difference between the control and the expérimentais, measured at each of the three maxima in luminescence subse-quent to the perturbation. (After Hastings and Sweeney, 1958.)

The action spectrum for Gonyaulax is shown in Fig. 15. I t is evident that there are two maxima in effectiveness, at 475 m/x and at 650 m/x. Light at the red maximum, however, is considerably less effective than light at the blue maximum.

The action spectrum roughly corresponds with the absorption spec-trum of whole cells of Gonyaulax as well as methanolic extracts. How-ever, the wavelengths of maximum effectiveness do not correspond precisely with the absorption maxima, and the absorption in the blue is much greater and broader than the apparent effectiveness would suggest. This action spectrum might be expected if only chlorophyll c were involved in the reaction, but at the present time the identification cannot be established with certainty.

The possibility that the pigment system involved was similar to that sensitizing the large class of photoreactions induced by red light and reversed by far-red light, as in the case of the germination of lettuce seeds (Borthwick et al, 1952), was investigated. No evidence for

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FIG. 15. The action spectrum for shifting the phase of the rhythm of luminescence in Gonyaulax by single 3-hour exposures to light of different wavelengths, as indi-cated. The relative effectiveness at each wavelength was calculated on the basis of equal incident quanta at each wavelength. The experimental design was very similar to that of the experiment shown in Fig. 11, except that the time and duration of the phase shifting light pulse were held fixed and the wavelength of light was varied. The points plotted are the average of four determinations. (After Hastings and Sweeney, 1960.)

reversal of the red effect (650 m/x) by far-red light (730 ταμ) was ob-tained, and far-red light alone was ineffective in causing a phase shift.

3.5 Phase Shifting by Exposure to Ultraviolet Light

Studies of the effect of UV pulses in phase shifting have been reported by Ehret (1960) with Paramecium and by Sweeney (1960b) with Gonyaulax. Due to damage by UV light only relatively short ex-posures of a few minutes may be used. Such exposures, however, are very effective in causing a persistent phase shift. Unlike visible light, which may cause either a phase advance or a delay, the direction of the shift with UV light is always unidirectional. Moreover, UV light-induced phase shift is photoreactiveable in both species. Like the visible re-sponse, however, there are variations in sensitivity as a function of time in the cycle.


3.0 Transients Prior to the Attainment of the Steady State

In some organisms, notably Droscxphila, the response to a light perturbation involves transients which precede the attainment of a new steady state. This feature has evoked considerable interest with respect to the oscillator model of biological clocks developed by Pittendrigh et al. (1958). These transients are such that the new phase is not at-tained immediately. Instead it may require several days before the final phase is reached.

Although this feature certainly occurs in some organisms, it does not appear to be a general or universal feature of persistent rhythms, as suggested by Pittendrigh (1960). As shown in Fig. 14, there occur no such transients in the attainment of a new phase in the Gonyaulax rhythm. This is also true for some other rhythms (Wilkens, 1960).

4. Effects of Environmental Light Cycles

4.1 Phase Relationships of Rhythmic Processes

Organisms maintained under environmental light-dark cycles exhibit rhythms which, in a particular case, bear a given fixed relationship to the cycle. Considering all organisms, however, particular phase rela-tionships for diurnal rhythms do not appear to be either dictated or excluded. One may cite examples of rhythmic processes with maxima almost any time of day, and the clock system of a given organism is such that rhythms of different processes may be phased differently (Hastings, 1959). Luminescence, cell division, and photosynthesis are clocked so that they occur maximally at different times of day in Gonyaulax (Fig. 16). In mammals, Halberg and his collaborators (1959) have made detailed studies of a variety of rhythmic phenomena and have reported significant phase differences.

Moreover, a particular kind of rhythmic process need not be phased at a particular time of day. In different species of dinoflagellates, a maximum in cell division has been reported to occur at times ranging from 3 A.M. to 10 A.M. (Sweeney and Hastings, 1962) ; and in different species and varieties of Paramecium, the time of maximum mating affin-ity varies (Ehret, 1953). The generalization certainly does not apply in all cases, since we would scarcely expect to find an organism where luminescence was maximum at noon and photosynthetic capacity max-imum at midnight.

Pittendrigh (1961) has recently pointed out that the phase of a rhythm (in an organism maintained on a schedule of alternating light and dark periods) may be viewed as an equilibrium condition, resulting


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FIG. 16. This figure schematically illustrates the several diurnal rhythms in Gonyaulax and their phase relationships to the daily light-dark cycle (LD 12:12). The rhythm of flashing luminescence (induced by mechanical agitation) has a broad maximum in the middle of the night. The rhythm of photosynthesis is maximum during the light period and the rhythms of cell division and steady luminescent glow have maxima at approximately the same time, at about the end of the dark period. Very little light emission actually occurs in the glow in contrast to the flashing emission, which is very bright. The scales are therefore not comparable. The glow rhythm has been used in experiments shown in Figs. 12 and 17.

from the interaction of the resetting signals at dawn and dusk, advancing and delaying effects, respectively (see Section 3.2). As already noted, we earlier termed this "repetitive resetting" (Hastings and Sweeney, 1959; see Section 4.3). Since the precise relationship which the phase will bear to the light-dark cycle may be somewhat different, depending upon the natural period of the rhythm, Pittendrigh has suggested that this effect may constitute the adaptive significance of natural periods which are appreciably longer or shorter than 24 hours. Within narrow limits this phase lability might be achieved, but it is unlikely that this feature could constitute the major explanation for phase determination in the sense discussed in this section.

The phase of rhythm may also be slightly dependent upon photo-period (see Section 4.4). Such an effect has been reported by Pittendrigh (1961) for the rhythm of eclosion in Drosophila, but again the effects are relatively small.

4.2 Changing Phase of the Light-Dark Cycle

Numerous experiments with many organisms have been carried out in which the phase of the rhythm was shifted by changing the time at which the light and dark periods occurred. In such experiments, as was illustrated with the rhythm of luminescence in Gonyaulax (Fig. 10), the


phase (i.e., the solar time at which the maximum in luminescence occurs) may be shifted so that it will bear any desired relationship to the solar day (Pittendrigh, 1954). In cultures which are subsequently transferred to constant conditions of dim light or darkness the phase of the persistent rhythm is related to the most recent light-dark schedule, rather than to solar time, or any other factor.

4.3 Effect of Period of Light-Dark Cycle. Entrainment, Frequency Démultiplication, and Repetitive Resetting

Terminology. Reference to light-dark cycles will be made by using symbols such as LD 10:8. This refers to a light-dark cycle with light for 10 hours alternating with darkness for 8 hours. The period of the cycle is the sum of the two times, namely 18 hours in this case.

The effects of light-dark cycles possessing various periods have not as yet been studied in many laboratories in a very complete way. The studies by Bruce, Pittendrigh, and collaborators are the most extensive, and have been recently summarized by Bruce (1960). Bruce defined entrainment of a rhythm by a light cycle as the phenomenon whereby the imposed cycle causes that rhythm to become periodic with the same period as the entraining cycle. As he indicated, the definition cannot be taken to be applicable to all cases. To interpret properly any particular case, information is necessary concerning the nature of both the rhythmic oscillator as well as the entraining signal. Some of the gen-eralizations and distinctions which now appear possible will be con-sidered.

I t was pointed out by Pittendrigh and Bruce (1957) that biological rhythms exhibit many of features analogous to those of physical oscil-lators. For example, the period of a physical oscillator, such as a pen-dulum, is a characteristic feature of the particular oscillator. The natural period of a biological rhythm is considered to be analogous being, likewise a feature of the cellular mechanism, and not directly dependent upon the external light-cycle.

The phenomenon of entrainment of biological rhythms is also analogous to a similar property of physical oscillators. A given oscillator with a particular natural period may be coupled by appropriate means to a second oscillator possessing a slightly different frequency. Under these conditions the second oscillator may act as an entraining agent, modify the frequency of the first, and establish a phase relationship, even though the energy input from the second to the first oscillator is small. Biological rhythms may be entrained in this fashion by light-dark cycles, but only within limits. When the entraining frequency differs too widely from the natural frequency of the entrained oscillator the coupling

11. ßOLE OF LIGHT IN PERSISTENT DAILY RHYTHMS 353

breaks down and the first oscillator reverts to its natural frequency (Bruce, 1960; Tribukait, 1954). Moreover, with both biological rhythms and physical analogues, when the entraining oscillator is removed the first oscillator reverts to its natural frequency (Fig. 17). This emphasizes

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FIG. 17. This shows the time of day at which the maximum in the luminescent glow occurred on each of 20 successive days in a single culture of Gonyaulax. The values were obtained by measuring the luminescence from the vial approximately every 2 hours. For the first few days the culture was kept on a daily light-dark cycle, and the peak occurred at the same time each day. This has been referred to as "entrainment" or "repetitive resetting" (see text). Subsequently, the light was kept on continuously, and whereas the rhythm continued, its maximum occurred about 45 minutes later each day. Thus the natural period of the rhythm is exhibited. Temperature 24°C; light intensity during LD exposure, 800 ft-candles; during constant dim light, 120 ft-candles. (After Hastings, 1960.)

that the cellular mechanism involved in the rhythmicity is not directly dependent upon the environmental light-dark cycle either for its occur-rence or for its specific natural frequency.

With organisms exposed either to light-dark cycles or to periodic light pulses, having frequencies which differ considerably from the natural frequency of the rhythm, Bruce and Pittendrigh (1956) dis-covered another phenomenon, which they termed "frequency demultipli-

354 J . WOODLAND HASTINGS

cation." Using light cycles which were particular submultiples of 24 hours, they found that the rhythm was entrained to a 24-hour period. An example of this phenomenon in Gcmyaulax is shown in Fig. 18.

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FIG. 18. Effects of exposing cells to light-dark schedules with periods which differ from 24 hours followed by constant dim light. With LD 8:8 (200 ft-candles) and LD 6:6 (800 ft-candles) a repetitive resetting occurred with maxima in luminescence every 16 and 12 hours respectively. With LD 6:6, but (only 200 ft-candles) a type of frequency démultiplication appears to occur, with maxima only every 24 hours. In all cases the rhythms revert to the period of approximately 24 hours when replaced on continuous dim light. (After Hastings and Sweeney, 1959.)

The entrainment of the rhythm in the cases discussed above to the periodic light signals can occur at low light intensities, where the energy involved may be small. However, as was discussed in an earlier section (3.1), light pulses are capable of causing an actual phase shift, but the light energy involved is relatively large (Fig. 15). It was shown with Gonyaulax that light-dark cycles with periods which differ considerably from the natural period (e.g., LD 6:6, LD 7:7 or LD 8:8) could result in an apparent entrainment of the rhythm (Hastings and Sweeney, 1959). However this was correctly interpreted as not being due to true entrainment; rather it was termed repetitive resetting. This interpreta-

I I I 6 HR. DAY (200 ft.c.) - 6 HR. NIGHTl


tion was supported in an experiment where the distinction was drawn. Light cycles with 12-hour periods (LD 6:6) were used; with a dim light frequency démultiplication was observed with maxima occurring every 24 hours; with bright light a repetitive resetting occurred with maxima every 12 hours (Fig. 18).

Repetitive resetting occurs with other rhythms also (Fig. 19). On a

12 14 16

TIME-HOURS

FIG. 19. This experiment illustrates repetitive resetting of the rhythm of cell division (Sweeney und Hastings, 1958). Cultures were grown in bright light (800 ft-candles) with a 16-hour cycle (LD 8:8). Counts were made at various times during the cycle over the course of about 7 days of the number of cells in a particular division stage where daughter cells adhere as pairs. (Hastings, un-published.)

16-hour cycle (LD 8:8), the time at which cell divisions occur is re-stricted to a time just at the end of the light period, every 16 hours.

These experiments again illustrate the generalization stated earlier, namely that the rhythmicity does not derive information concerning period from the environmental light-dark cycle. When the entraining or resetting light-dark cycle is stopped and the cells are placed in constant conditions, the rhythms continue to revert to the natural frequency.

The theory of entrainment of biological rhythms by photoperiod


(Pittendrigh, 1961) proposes that entrainment results from daily repeti-tive resetting as a consequence of the light signals at dawn and dusk. However, since phase shifting by light is strongly dependent upon the light intensity, whereas entrainment is thought to occur with very low light intensities, a different mechanism may be involved.

4.4 Effect of Photoperiod. Biological Clocks and the Problem of Photoperiodism

The nature of the mechanism involved in photoperiodic responses is not known. In considering the general problem, however, it is important to appreciate that the biological measurement in studies concerned with photoperiodism is very often different from the more usual kind of measurement. In biological experiments we frequently measure rates, or at least the measurement is expected to bear a simple relationship to a rate; in photoperiodism we measure a product, an event—or what may be the culmination of many different interdependent processes. The fact that the duration of the light (or dark) period-signaling as it does the annual cycle triggers a complicated response such as flowering, al-ready suggests that a simple rate interpretation, such as the hour-glass model, is not by itself adequate to explain the phenomenon.

Many years ago Biinning (1936) suggested that the mechanism of photoperiodic responses in plants might involve persistent daily rhythms. For a long time this proposal remained with very little experimental support, and it was generally discounted. In recent years, however, it has received increasing support on both experimental and theoretical grounds, particularly since the explicit recognition and development of the relationship between persistent daily rhythms and time measure-ment in organisms. The existence of such a "sense of time" seems necessary to explain a number of experimental observations, including the demonstration that bees have the ability to regularly feed at given times of day (Wahl, 1932; Renner, 1959, 1960), and that numerous animals can adopt a specific orientation with respect to sun or stars, regardless of the time of day (Kramer, 1952; Hoffman, 1960a).

I t would appear probable, therefore, that such an internal clock mechanism could mediate the long-term integration of environmental signals relating information concerning photoperiod. Evidence concerning this possibility has been recently obtained. With Drosophila, as noted earlier (Section 4.1), the phase of the rhythm may be modified by photo-period. In addition, there is a marked effect of photoperiod upon the physiological condition of the flies. Subjected to a long-day photoperiod flies recover more readily from a heat shock than do those maintained on short days (Pittendrigh, 1961).


The more relevant evidence comes, as would be expected, from studies with organisms which exhibit the more classical types of photo-periodic responses. Bünsoe's studies (1960) with Kalanchoë demon-strate a 24-hour rhythm in sensitivity to inhibition of a photoperiodic response by light. Equally impressive are the studies by Hamner (1960), where it has been shown that a photoperiodic response which results from repeated light-dark cycles is favored by using photoperiods close to 24 hours, or multiples thereof.

These results indicate already a relationship between persistent 24-hour rhythms and photoperiodic responses. The pursuit of this area of study may be expected to yield information of an unusually valuable nature.

5. Rhythms of Photosynthetic Capacity There is every indication that a large variety of physiological proc-

esses may exhibit persistent daily rhythmicity in their expression. In animals, for example, processes such as eclosion, color change, body temperature, mating reactivity, luminescent flashing, and various types of locomotory activity have been studied. In different plants biolumines-cence, sporulation, growth rate, leaf movements, and phototaxis exhibit such rhythms.

Diurnal changes in photosynthesis were first reported by Doty and Oguri (1957), who measured the photosynthetic capacity of samples of oceanic phytoplankton. The organisms present were not identified but presumably the samples contained various species of dinoflagellates.

A true persistent daily rhythm of photosynthetic capacity (Fig. 20) was first reported in unialgal cultures of Gonyaulax polyedra (Hastings and Astrachan, 1959), and Sweeney (1960a) demonstrated that this rhythm could be measured in single isolated cells (Figs. 5 and 21).

The rhythm is one of photosynthetic capacity rather than of photo-synthesis. This point is illustrated in both Figs. 20 and 21. Measure-ments of photosynthetic rate as a function of light intensity at different times of day illustrate that the intensity of light needed to saturate the dark steps in the reaction varied with time of day (Fig. 21). As would therefore be expected, when measurements of photosynthesis were made at low light intensities (Fig. 20) the rhythmicity was not evident (Hast-ings et al., 1961).

With cells kept on LD 12:12 the maximum in photosynthetic capacity is at approximately the middle of the light period. Such cells, after being transferred to continuous dim light, continue to exhibit the rhythmicity with a period of about 24 hours (Fig. 20).

Variations in photosynthetic capacity could be caused by changes in

358 3. WOODLAND HASTINGS

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FIG. 20. Measurements of photosynthesis and photosynthetic capacity of Gonyaulax cells maintained in continuous light. Cells were grown in LD 12:12 and transferred to constant light at the end of a dark period, 16 hours prior to zero hours on the graph. Dim light was 110 ft-candles and bright light was 960 ft-candles; temperature, 25 ± 0.3°C. (C14)-Dim-cap refers to the photosynthetic capacity of cells cultured in constant dim light, and records the relative amounts of C1402 incorporation when aliquots were incubated with a tracer in saturating bright light for 15 minutes. (C14)-Dim refers to relative rates of photosynthetic activity in cells cultured in dim light, and records C1402 incorporation when aliquots were incubated with tracer for 60 minutes in dim light. (C14)-Bright refers to relative rates of photosynthesis (or photosynthetic capacity) in cells cultured in bright light, and records the relative amounts of C1402 incorporated when aliquots were incubated with tracer for 15 minutes in bright light. (After Hastings et al, 1961.)

a number of different cell constituents such as, for example, photosyn-thetic pigments. However, no diurnal variations in either chlorophyll or carotenoid content have been found with Gonyaulax. The rhythm might also be due to changes in the concentration or activity of enzymes in-volved in dark reactions of photosynthesis. To date, however, no clue has been found concerning the component which does change.

There has also been reported a rhythm of photosynthesis in Acetabulana major (Sweeney and Haxo, 1961). Moreover, plants from which the nucleus had been removed by severing the basal rhizoids


E

1000 2000

LIGHT INTENSITY IN FOOT CANDLES 3000

FIG. 21. The net oxygen production of a single Gonyaulax polyedra cell at different light intensities measured at four different times in the diurnal cycle using the same cell: curve 1, drawn to 1 hour after dawn; curve 2, 1 to 3 hours after dawn; curve 3, 3 to 4 hours after dawn; curve 4, 10y2 to 11 hours after dawn. (After Sweeney, 1960a.)

retained the rhythm for at least five cycles. It was concluded, that the nucleus is not immediately essential for the maintenance of rhythmicity in Acetabularia. This conclusion might appear to be difficult to reconcile with the recent finding (Karakashian and Hastings, 1962) that actino-mycin D, a specific inhibitor of DNA-dependent RNA synthesis, inhibits the occurrence of rhythmicity—both of photosynthetic capacity and of luminescence. It has been suggested that this might be explained by the possibility that a long-lived RNA may be involved in the rhythm-determining function in Acetabularia.

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Ash off, J. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 11-27. Borthwick, H. A., Hendricks, S. B., Parker, M. W., Toole, E. EL, and Toole, V. K.

(1952). Proc. Natl. Acad. Sei. U. S. 38, 662. Bruce, V. G. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 29-47.


Bruce, V. G., and Pittendrigh, C. S. (1956). Proc. Natl. Acad. Sei. U. S. 42, 676-682. Bruce, V. G., and Pittendrigh, C. S. (1958). Am. Naturalist 92, 295-306. Bünning, E. (1936). Ber. dent, botan. Ges. 54, 590-607. Bünsoe, R. C. (1960). Cold Spnng Harbor Symposia Quant. Biol. 25, 257-260. De Coursey, P. (1960). Cold Spnng Harbor Symposia Quant. Biol. 25, 49 -55. De Coursey, P. (1961). Z. vergleich. Physiol. 44, 331-354. Doty, M. S., and Oguri, M. (1957). Limnol. Oceanog. 2, 37-40. Ehret, C. F. (1953). Physiol. Zoöl. 26, 274-300. Ehret, C. F. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 149-157. Halberg, F., Halberg, E., Barnum, C. P., and Bittner, J. J. (1959). In "Photo-

periodism and Related Phenomena in Plants and Animals" (R. B. Withrow, ed.), pp. 803-878. Publ. No. 55, Am. Soc. Adv. Sei., Washington, D. C.

Hamner, K. C. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 269-277. Hastings, J. W. (1959). Ann. Rev. Microbiol. 13, 297-312. Hastings, J. W. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 131-140. Hastings, J. W. (1962). Proc. Intern. Congr. Physiol., 22nd, Leiden, Holland, 1,

pp. 37-43. Hastings, J. W., and Astrachan, L. (1959). Federation Proc. 18, 65. Hastings, J. W., and Sweeney, B. M. (1957). Proc. Natl. Acad. Sei. U. S. 43,

804-811. Hastings, J. W., and Sweeney, B. M. (1958). Biol. Bull. 115, 440-458. Hastings, J. W., and Sweeney, B. M. (1959). In "Photoperiodism and Related

Phenomena in Plants and Animals" (R. B. Withrow, ed.), pp. 567-584. Publ. No. 55, Am. Soc. Adv. Sei., Washington, D. C.

Hastings, J. W., and Sweeney, B. M. (1960). / . Gen. Physiol. 43, 697-706. Hastings, J. W., Astrachan, L., and Sweeney, B. M. (1961). / . Gen. Physiol. 45,

69-76. Hoffman, K. (1960a). Cold Spring Harbor Symposia Quant. Biol. 25, 379-387. Hoffman, K. (1960b). Z. vergleich. Physiol. 43, 544-566. Johnson, M. (1938). J. Exptl. Zool. 82, 315-328. Karakashian, M., and Hastings, J. W. (1962). Proc. Natl. Acad. Sei. U. S. 48, 2130-

2137. Kramer, G. (1952). Ibis 94, 265-285. Pfeffer, W. (1915). Abhandl. sächs. Akad. Wiss. Leipzig, Math.-naturw. Kl. 34,

1-154. Pittendrigh, C. S. (1954). Proc. Natl. Acad. Sei. U. S. 40, 1018-1029. Pittendrigh, C. S. (1958). In "Perspectives in Marine Biology" (A. Buzatti-Traverso,

ed.), pp. 239-268, Univ. of California Press, Berkeley, California. Pittendrigh, C. S. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 159-182. Pittendrigh, C. S. (1961). Harvey Lectures 56, 93-125. Pittendrigh, C. S., and Bruce, V. G. (1957). In "Rhythmic and Synthetic Processes

in Growth" (D. Rudnick, ed.), pp. 75-109. Princeton Univ. Press, Princeton, New Jersey.

Pittendrigh, C. S., and Bruce, V. G. (1959). In "Photoperiodism and Related Phenomena in Plants and Animals" (R. B. Withrow, ed.), pp. 475-505. Publ. No. 55, Am. Assoc. Adv. Sei., Washington, D. C.

Pittendrigh, C. S., Bruce, V. G., and Kaus, P. (1958). Proc. Natl. Acad. Sei. U. S. 44, 965-973.

Renner, M. (1959). Z. vergleich. Physiol. 42, 449-483. Renner, M. (1960). Cold Spnng Harbor Symposia Quant. Biol. 25, 361-368.


Sweeney, B. M. (1960a). Cold Spring Harbor Symposia Quant. Biol. 25, 145-147. Sweeney, B. M. (1960b). Cold Spnng Harbor Symposia Quant. Biol. 25, 157-158. Sweeney, B. M., and Hastings, J. W. (1957). / . Cellular Comp. PhysioL 49, 115-128. Sweeney, B. M., and Hastings, J. W. (1958). J. Photozool. 5, 217-224. Sweeney, B. M., and Hastings, J. W. (1960). Cold Spring Harbor Symposia Quant.

Biol. 25, 87-103. Sweeney, B. M., and Hastings, J. W. (1962). In "Physiology and Biochemistry of

Algae" (R. A. Lewin, ed.), Part III, pp. 687-700. Academic Press, New York. Sweeney, B. M., and Haxo, F. T. (1961). Science 134, 1361-1363. Sweeney, B. M., Haxo, F. T., and Hastings, J. W. (1959). / . Gen. PhysioL 43,

285-299. Tribukait, B. (1954). Naturwiss. 41, 92-93. Wahl, O. (1932). Z. vergleich. PhysioL 16, 529-589. Whitaker, W. L. (1940). J. Exptl. Zool. 83, 33-60. Wilkens, M. B. (1960). Cold Spring Harbor Symposia Quant. Biol. 25, 115-129.

AUTHOR INDEX

A Ainsworth, S., 29, 83 Airth, R. L., 201, 220 Allard, H. A., 306, 330 Allen, B. T., 52, 62 Allen, M. B., 16, 16, 53, 55, 58, 59, 60,

62, 85, 88, 94, 108, 108, 109, 114, 115, 117, 118, 119, 123, 132, 148, 151, 154, 167, 190, 193

Amann, H., 162, 163, 195 Amesz, J., 85, 108, 139, 152, 162, 163, 165,

194, 220 Anderson, W. F., 75, 81 Androes, G. M., 35, 55, 56, 61, 62 Appel, W., 162, 163, 195 Arisz, W. H., 229, 268 Arnold, W., 60, 62, 129, 151, 159, 160, 168,

173, 182, 185, 186, 188, 190, 191, 198, 194, 202, 212, 220

Arnold, W. A., 191, 196 Arnon, D. I., 58, 62, 63, 113, 114, 115, 117,

118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 143, 144, 145, 147, 151, 151, 152, 153, 154, 157, 158, 162, 163, 193, 196, 218, 220

Asana, R. D., 233, 234, 238, 241, 268 Aschoff, J., 12, 16, 337, 359 Asomaning, E. J. A., 228, 242, 243, 244,

246, 247, 249, 253, 258, 268 Astrachan, L., 357, 358, 860 Augenstine, L. G., 74, 80 Avery, G. S., Jr., 224, 268 Avron, M., 118, 142, 143, 153

B

Bachmann, F., 249, 269 Bailey, G., 90, 93, 108 Baker, R. S., 8, 17, 226, 250, 251, 252,

269, 270 Ball, N. G., 246, 269 Baltscheffsky, H., 142, 143, 152 Baltscheffsky, M., 132, 154, 188, 196 Bamford, C. H., 40, 62

Banbury, G. H., 268, 269 Bandurski, R. S., 252, 269 Bannister, T. T., 183, 187, 193, 195 Barer, R., 85, 108 Barnum, C. P., 350, 860 Bass, A. M., 40, 62 Bassham, J. A., 112, 114, 152 Bawden, F. C., 78, 80 Bayliss, W. M., 9, 16 Beal, W. J., 324, 380 Beaven, G. H., 69, 71, 80 Becker, R. S., 171, 173, 198, 194 Beinert, H., 55, 57, 62, 100, 108, 190, 194 Bellis, D. R., 91, 109 Bendall, F., 139, 153, 162, 163, 196 Bendix, S., 8, 16 Benitez, A., 274, 275, 276, 808 Benson, A. A., 85, 86, 109, 114, 126, 152,

254, 270 Berends, W., 75, 80 Bergann, F., 250, 269 Bergeron, J. A., 161, 186, 194 Berns, K., 75, 81 Beukers, R., 75, 80 Bicking, L., 100, 108 Birth, G. S., 315, 830 Bishop, N. I., 55, 58, 62, 131, 140, 152,

164, 190, 194 Bittner, J. J., 350, 860 Blaauw, A. H., 225, 229, 239, 249, 268,

269 Blaauw, O. H., 160, 196 Blaauw-Jansen, G., 238, 242, 244, 269 Blatt, J., 131, 152 Blinks, L. R., 107, 109, 201 203, 207, 208,

213, 214, 215, 218, 219, 220, 221 Blois, M. S., Jr., 6, 7, 16, 35, 49, 62 Blum, H. F., 5,16, 325, 880 Bock, G., 290, 802 Bonner, B. A., 317, 880 Bonner, J., 10, 17, 226, 239, 243, 248,

270, 271, 321, 880 Bonner, W. D., 131,158 Borthwick, H. A., 10, 17, 249, 269, 305,

363

Numbers in italic show the page on which the complete reference is listed.

364 AUTHOR INDEX

307, 308, 310, 313, 318, 321, 327, 329, 830, 881, 348, 359

Bottelier, H. P., 295, 297, 299, 300, 301, 302

Bové, C, 136, 137, 162 Bové, J., 136, 137, 147,152 Bowen, E. J., 106, 108 Boyce, R., 79, 81 Boysen-Jensen, P., 224, 226, 234, 242, 269 Brackett, F. S., 88, 90, 108 Brandt, D. C, 85, 108 Brauner, L., 226, 234, 250, 251, 261, 269 Bremekamp, C. E. B., 229, 269 Briggs, W. R., 8, 17, 226, 227, 228, 229,

230, 231, 232, 234, 236, 238, 240, 241, 242, 243, 244, 245, 246, 247, 248, 259, 261, 262, 263, 264, 265, 266, 269, 270, 271, 312, 830

Brighenti, L., 66, 80 Bril, C , 188, 194 Brim, W., 24, 83 Brode, W. R., 25, 32 Brody, M., 166, 177, 194, 207, 220 Brody, S. S., 166, 177, 185, 187, 194 Broida, H. P., 40, 62 Brown, F. A., Jr., 12, 17 Brown, H. W., 35, 49, 62 Brown, J. R., 94, 108 Brown, T. E., 55, 61, 63 Broyer, T. C, 136, 152 Bruce, V. G., 335, 341, 343, 344, 350, 352,

353, 859, 860 Bubnov, N. N., 54, 62 Buchmann, B., 16, 17 Buder, J., 268, 269 Buller, A. H. R., 268, 269 Bünning, E., 240, 250, 251, 253, 269, 323,

330, 356, 360 Bunsen, R., 229, 269 Biinsoe, R. C, 357, 360 Burkholder, P. R., 224, 239, 268, 269 Busck, G., 13, 14, 17 Butler, W. L., 167, 187, 194, 196, 313, 315,

316, 317, 330, 331 Buzzell, A., 75, 81 Byrnes, J. W., 321, 880

c Cajlachjan, M. C, 327, 880 Caluert, J. G., 24, 82

Calvin, M., 35, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 85, 86, 109, 112, 114, 152, 158, 182, 190, 191, 194, 195, 196, 219, 220, 254, 270

Cannon, C. G., 106, 108 Canterow, A., 13, 17 Capindale, J. B., 114, 115, 151, 154 Carlton, A. B., 136, 152 Carnahan, J. E., 133, 153 Carter, L. A., 53, 60, 63 Caspary, R., 306, 880 Cederstrand, C , 108, 108, 161, 194 Chalmers, R. V., 108, 161, 194, 213, 220 Chance, B., 95, 98, 100, 101, 108, 130,

131, 132, 152, 160, 168, 185, 188, 194, 196

Charney, E., 88, 90, 108 Chessin, M., 6, 17, 79, 81 Ching, T. M., 240, 269 Cholodny, N., 225, 269 Christensen, B. C, 16, 17 Claes, H., 183, 184, 194, 219, 220 Claesson, I. M., 67, 69, 72, 74, 77, 81 Claesson, S., 23, 24, 29, 31, 83 Clare, N. T., 5, 17 Clark, C, 83, 108 Clayton, R. K., 60, 62, 129, 151, 167, 168,

185, 188, 189, 190, 191, 193, 198, 194 Coahran, D. R., 75, 81 Cohen-Bazire, G., 128, 152, 183, 187, 195 Coleman, J. W., 188, 194 Colmano, G., 187, 194 Commoner, B., 35, 52, 53, 55, 59, 60,

62, 68, 190, 194 Conn, E. E., 133, 153 Contopolou, R., 146, 154 Cook, R. M., 91, 109 Coomber, J., 277, 803 Cope, F. W., 52, 68 Crane, F. L., 58, 68, 128, 152, 158 Curry, G. M., 8, 18, 226, 227, 230, 233,

239, 242, 244, 245, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 261, 268, 269, 270

Cynkin, M. A., 114, 153

D

Dam, H., 127, 152 Daniels, F., 4, 17, 23, 88

AUTHOR INDEX 365

Darlington, H. T., 324, 330 Darwin, C, 224, 269 Darwin, F., 224, 269 Das, V. S., 117, 153 Davenport, H. E., 127, 133, 152 Davidson, J. B., 173, 193 Davydov, A. S., 176, 194 Deckes, K., 327, 330, 331 De Coursey, P., 344, 360 Dhar, N. R., 2, 3, 4, 17 Diwar, A. R., 6, 18 Dolk, H. E., 225, 269 Dose, K., 68, 69, 81 Doty, M. S., 357, 360 Doty, P., 69, 75, 81 Doudoroff, M., 146, 153, 154 Downs, R. J., 313, 324, 330 Doyle, B., 70, 73, 81 du Buy, H. G., 226, 227, 250, 270, 271 Duggar, B. M., 15,17, 77, 81, 212, 220 Durham, L. J., 114, 151 Dutton, H. J., 185, 194, 212, 220 Duysens, L. N. M., 85, 88, 95, 97, 98,

108, 108, 130, 132, 139, 152, 160, 162, 163, 165, 166, 168, 185, 194, 196, 208, 220, 256, 270

Dyke, I. J., 246, 269

E

Eden, M., 91, 109 Egle, K , 280, 302 Ehret, C. F., 23, 33, 308, 331, 347, 349,

350, 360 Eib, M., 240, 270 El-Bayoumi, M. A., 177, 178, 195 Elbers, P. F., 186, 196 Eley, D. D., 180, 182, 194 Elsden, S. R., 129, 152 Emerson, R., 60, 63, 107, 108, 108, 159,

160, 161, 166, 185, 194, 203, 207, 209, 213, 220

Emsweller, S. L., 321, 330 Engelmann, T. W., 6, 17, 202, 204, 220 Eriksson, G., 280, 302 Ermolaev, V. L., 179, 196 Estermann, E. F., 69, 74, 81 Evans, H. J., 148, 152 Eymers, J. G., 301, 302 Eyster, C., 55, 61, 63

F

Fair, W. R., 91, 109 Fang, S. C, 240, 269 Fehes, G., 48, 63 Fernandez, J., 173, 194 Ferrari, G., 81 Finsen, N., 12, 17 Firer, E. M., 317, 331 Fischer, H., 276, 302 Fitting, H., 293, 302 Flint, L. H., 306, 330 Fluke, D. J., 23, 33 Fogg, G. E., 148, 152 Forbes, G. S., 24, 33, 106, 109 Forbes, W. F., 68, 69, 71, 81 Fork, D. C , 164, 195, 213, 215, 218, 220 Förster, T., 175, 179, 194 Forti, G., 218, 221 Fox, D. L., 220 Fraenkel, G. S., 8, 17 Franck, J., 158, 166, 170, 171, 185, 186,

195, 256, 270 Freifelder, D., 5, 17 French, C. S., 7, 17, 90, 91, 92, 93, 94,

108, 109, 139, 153, 162, 164, 165, 195, 196, 212, 213, 215, 217, 220, 221, 256, 270, 274, 276, 302

Frenckel, J., 176, 195 Frenkel, A. W., 118, 152, 158, 195 Fröschel, P., 229, 270 Fujimori, E., 191, 196, 201, 221 Fuller, R. C., 147, 152, 186, 190, 194, 195 Futrell, J. H., 29, 33

G

Gaffron, H., 16, 17, 58, 63, 106, 109, 134, 152, 156, 159, 164, 165, 195

Gajewska, E., 75, 81 Galston, A. W., 8, 17, 226, 228, 242, 243,

244, 246, 247, 249, 250, 251, 252, 253, 258, 268, 269, 270, 296, 299, 302

Gandy, H. W., 22, 33 Garner, W. W., 306, 330 Garrett, C. G. B., 180, 195 Geiduschek, E. P., 69, 81 Geldard, F. A., 9, 17 Gest, H., 128, 134, 152, 154 Gewitz, H. S, 142, 154 Ghiron, C. A., 74, 80

366 AUTHOR INDEX

Gibbs, M., 114, 163 Gibson, K. S., 25, S3, 83, 109 Gibson, Q. H., 29, S3 Giese, A. C, 1, 5, 17, 76, 81 Gill, J., 52, 63 Gillespie, B., 240, 270 Ginoza, W., 78, 81 Ginther, R. J., 22, 33 Glass, B., 18, 108, Î00 Goedheer, J. C , 168, 171, 179, 186, 195 Gordon, S. A., 240, 270 Gott, W., 162, 167, 185, 195 Govindjee, 165, 166, 185, 196, 219, 221 Granick, S., 186, 196 Green, G., 58, 63 Griffiths, M., 183, 187, 195, 219, 221 Grossweiner, L. I., 4, 17, 29, S3 Gunn, D. L., 8, 17

H

Haberman, H. M., 252, 271 Haig, C , 229, 258, 270 Halberg, E., 350, 360 Halberg, F., 350, 360 Hall, D. O., 139, 140, 143, 144, 151, 153 Halldal, P., 107, 109 Hammer, K. C, 321, 330, 357, 360 Hand, M. E., 226, 270 Harris, A. Z., 114, 162 Harris, R., 15, 17 Harrison, G. R., 106, 109 Hartline, A. K., 10, 17 Hartridge, H., 100, 109 Harvey, E. N., 14, 17 Hastings, J. W., 333, 334, 335, 336, 337,

339, 341, 342, 344, 347, 348, 349, 350, 351, 353, 354, 357, 358, 359, 360, 361

Hatchard, C. G., 24, 33 Haupt, W., 288, 289, 290, 293, 302 Haurowitz, F., 76, 81 Hawkes, J., 1, 17 Haxo, F. T., 107, 109, 199, 203, 205, 208,

210, 213, 220, 221, 339, 358, 361 Hayashi, T., 296, 302, 302 Hayes, J. E., Jr., 91, 109 Heidt, L. J., 23, 24, S3 Heise, J. J., 53, 55, 59, 62, 63, 190, 194 Hendley, D. D., 133, 153 Hendricks, S. B., 10, 17, 107, 109, 249,

269, 305, 307, 308, 310, 313, 315, 316, 317, 318, 321, 324, 325, 327, 329, 330, 331, 348, 359

Hill, R., 113, 127, 130, 131, 133, 139, 152, 153, 162, 163, 195

Hiskey, C. F., 83, 109 Hjorth, E., 127, 152 Hoch, G., 55, 57, 62, 162, 165, 166, 190,

194, 195, 218, 221 Hoffman, K., 337, 356, 360 Holiday, E. R., 69, 71, 80 Hollaender, A., 12, 13, 15, 16, 17, 68, 77,

81, 82 Holt, A. S., 90, 109, 187, 195 Holt, W. W., 91, 109 Honda, S., 290, 302 Hongladarom, T., 290, 302 Hook, A. E., 79, 82 Horecker, B. L., 116, 154 Horio, T., 118, 129, 153 Horton, A. A., 131, 139, 140, 151, 152, 153 Huiskamp, W. J., 168, 194 Hutchison, C. A., Jr., 42, 63

I Ingram, D. J. E., 35, 40, 52, 62, 63

J Jacobs, E. E., 187, 195 Jagendorf, A. T., 118, 142, 143, 148, 153,

218, 221 Jager, G., 293, 294, 302 James, W. O., 117, 130, 153 Jaycock, M. J., 88, 109 Jenkins, A. D., 40, 62 Jennings, H. S., 8, 17 Jennings, W. H., 187, 196 Johnson, C. M., 136, 162 Johnson, F. H., 16, 17 Johnson, M., 337, 360 Johnston, E. S., 239, 250, 269, 270 Johnston, R. G., 106, 110 Jones, R. F., 201, 221 Jucker, E., 200, 221

K Kahn, A., 280, 302 Kamen, M. D., 118, 129, 132, 134, 162,

153, 154

AUTHOR INDEX 367

Kamf, B. M., 220 Kamiya, N., 291, 292, 301, 302, 302 Kamp, B. M., 139, 162, 162, 163, 165, 194 Kaplan, R. W., 5, 17 Karakashian, M., 333, 359, 360 Karrer, P., 200, 221 Kasha, M., 171, 173, 174, 177, 178, 181,

184, 189, 193,196,196 Katoh, S., 131, 163 Kaus, P., 350, 360 Kautsky, H., 162, 163, 196 Kay, L. D., 114, 162 Kay, W. W., 79, 82 Kearns, D. R., 58, 63, 182, 196 Kegel, L. P., 128, 163 Keiner, A., 13, 17, 107, 109 Kersten, J. A. H., 212, 221 Kessler, E., 63, 141, 148, 163 Kinzel, W., 306, 328, 330 Kistiakowsky, G. B., 3, 4, 18 Kittel, C , 180, 196 Klebe, J. F., 240, 269 Kleczkowski, A., 66, 78, 79, 80, 80, 81 Klein, W. H., 279, 303 Klingenberg, M., 58, 63, 162, 189, 193, 195 Knight, C. A., 74, 78, 81 Kofler, M., 128, 168 Köhnlein, W., 49, 68 Kok, B., 55, 57, 62, 95, 109, 160, 162,

165, 166, 167, 185, 188, 190, 194, 195, 218, 221

Kornberg, H. L., 147, 152 Koski, V. M., 274, 276, 802 Kramer, G., 356, 860 Krasnovskii, A. A., 54, 62 Krippahl, G., 142, 154 Krogmann, D. W., 118, 140, 142, 143, 168 Kruse, I., 127, 152 Kunisawa, R., 128, 146, 152, 164

L Labouriau, L. G., 252, 270 Laetsch, W. M., 312, 880 Lambers, H. L., 296, 808 Lane, D., 75, 81 Lane, H. C, 317, 331 Lang, H. M., 127, 133, 154, 158, 196 Lange, S., 228, 251, 270 Larsen, H., 160, 164, 195 Latimer, P., 87, 88, 90, 109, 187, 195

Lauffer, M. A., 75, 81 Laurens, H., 15, 18 Lavollay, J., 136, 153 Leech, R. M., 130, 158 Lees, A. D., 11, 18 Legallais, V., 95, 96, 100, 108, 110 Leighton, P. A., 4, 18, 25, 88 Leighton, W. G., 24, 25, 88, 106, 109 Lemberg, R., 200, 221 Lemmon, R. M., 35, 62 Lester, R. L., 58, 63 Levine, R. P., 53, 58, 68 Levring, T., 202, 203, 221 Lewis, C. M., 107, 108, 185, 194, 203,

209, 220 Lichtenthaler, H. K., 127, 153 Lindblom, R. O., 35, 62 Lindqvist, L., 24, 29, 31, S3 Linschitz, H., 174, 184, 195 Lippincott, B. B., 52, 53, 55, 59, 62, 68 Liverman, J. L., 243, 248, 270 Livingston, R., 7, 18, 65, 81, 106, 109,

171, 174, 182, 183, 184, 196, 196 Loofbourow, J. R., 106,109 Loos, G. M., 16 Lord, R. C., 106, 109 Losada, M., 58, 62, 63, 115, 122, 123, 124,

125, 126, 128, 136, 138, 139, 140, 147, 151, 161, 152, 158, 154, 157, 158, 162, 163,198,196

Lumry, R., 189, 196 Lundegardh, H., 85, 88, 109, 130, 158 Luse, R. A., 69, 70, 74, 81 Lwoff, A., 151, 168 Lynen, F., 327, 831

M McAlister, E. D., 306, 330 McArdle, J., 171, 196 McClure, D. S., 169, 196 McElroy, W. D., 14,18, 108,109 McGlynn, S. P., 181, 196 Maclachlan, G. A., 153 McLaren, A. D., 25, 27, 83, 66, 67, 68,

69, 70, 72, 74, 77, 78, 79, 80, 81 Maclay, H. K., 182, 191, 193 McLean, I. W., 79, 82 McLeod, G. C., 106, 109, 277, 808 McRae, E. G., 177, 196 Mahan, B. H., 21, 88

368 AUTHOR INDEX

Maling, J. E., 49, 58, 62, 63 Mandai, R., 21, SS Mandl, I., 67, 81 Mangum, B. W., 42, 63 Mann, I., 9, 18 Mann, W. B., 28, 33 Manning, W. M., 185, 194, 212, 220 Marcus, A., 147, 153 Margerum, J. D., 24, 33 Marmur, J., 75, 81 Martin, G., 136, 163 Mast, S. 0., 8, 18 Matthews, L., 75, 81 Mayer, E., 12, 18 Mayor, H. D., 6, 18 Meek, E. S., 186, 193 Menzel, D. H., 1, 2, 18 Merrick, J. M., 146, 153 Mie, C , 85, 109 Minnaert, K , 186, 196 Mitsui, A., 135, 152, 153 Mohr, H., 267, 270, 289, 303, 310, 331 Monk, G. S., 23, 33, 308, 331 Montfort, C , 202, 221 Moraw, R., 32, 33, 63, 100, 103, 110, 162,

168, 188, 189, 197 Moring-Claesson, I., 25, 27, 33 Morita, S., 129, 153 Mortensen, L. E., 133, 153 Mulac, W. A., 29, 33 Müller, A., 32, 33, 49, 58, 63, 100, 103, 109,

110, 162, 167, 188, 189, 190, 193, 195, 197, 221

Murchio, J. C, 53, 55, 58, 59, 60, 62, 85, 88, 109, 190, 193

Myers, J., 139, 153, 162, 196, 217, 221

N

Nakamoto, T., 118, 153 Nakamura, H., 131, 152, 153 Nakayama, S., 329, 330 Nakayama, T. O. M., 183, 184, 194 Nason, A., 148, 152 Negelein, E., 148, 154, 313, SSI Neisch, A. C, 327, SSI Nelson, R. C , 180, 196 Newton, G. A., 128, 129, 153 Newton, J. W., 128, 129, 153 Nieman, R. H., 131, 153

Nishimura, M., 130, 143, 152, 153, 160, 168, 185, 194, 256, 270

Noble, F. W., 91, 109 Norberg, R. E., 53, 55, 59, 63 Norman, A., 78, 79, 81 Norris, K. H., 315, 316, 330 Norrish, R. W. G., 4, 18, 20, 28, 29, 33,

100, 109 Nothmann-Zuckerkandl, H., 299, 303 Noyés, W. A., 4, 18 Nozaki, M., 58, 62, 118, 119, 122, 123, 132,

135, 147, 152,164, 157, 158,193 Nuernbergk, E., 227, 270 Nyman, B., 23, 33

O

Ochoa, S., 116, 117, 133, 164, 158, 197 Oesterreicher, A., 276, 302 Ogata, S., 122, 132, 135, 147, 163, 154 Oguri, M., 357, 360 O'h Eocha, C., 201, 221, 317, 331 Ohta, S., 128, 164 Olcott, W. T., 1, 18 Olson, J. M., 168, 185, 188, 196 Olson, R. A., 187, 196 Oppenheimer, J. R., 185, 193, 212, 220 Ordin, L., 128, 154 Oster, G., 79, 81, 84, 85, 109

P

Page, R., 268, 270 Pake, G. E., 35, 52, 62 Paneque, A, 135, 152, 154 Pardee, A. B., 74, 79, 80, 81 Parfitt, G. D., 88, 109, 180, 194 Pariser, R., 106, 109 Parker, C. A., 24, S3 Parker, M. W., 10, 17, 305, 307, 308, 327,

330, 331, 348, 359 Passera, C , 81 Passoneau, J. V., 53, 55, 59, 63 Pearson, S., 27, S3 Peterson, D. L., 176, 196 Pfeffer, W, 337, 360 Piette, L. H., 53, 55, 58, 59, 60, 62, 63,

190,193 Pincussen, L., 18 Pirie, A., 9, 18, 141, 164

AUTHOR INDEX 369

Pittendrigh, C. S., 12, 18, 333, 335, 339, 342, 343, 344, 350, 351, 352, 353, 356, 360

Pitts, J. N., Jr., 24, 33 Platt, J. R., 171, 196, 219, 221 Polis, B. D., 52, 63 Pon, N. G., 53, 54, 63, 190, 196 Porter, G., 4, 18, 20, 28, 29, 33, 100, 109 Porter, H. K., 153 Porter, K , 24, 25, 33 Price, L., 277, 279, 303 Priestley, J. H., 224, 270 Pringsheim, E. G., 147, 154 Puck, T. T. 256, 270 Pugh, A. C., 184, 196

R Raacke, I. D., 147, 152 Rabideau, G. S., 90, 109 Rabinowitch, E., 87, 109, 161, 165, 166,

170, 171, 173, 185, 187, 188, 194, 195, 196, 219, 221

Racker, E., 116, 154 Rajewsky, B., 68, 69, 81 Ramirez, J., 132, 154 Rand, S. J., 32, 33 Ray, P. M., 239, 248, 269 Rechen, H. J. L., 24, 32 Reinert, J., 226, 239, 251, 252, 255, 270 Reisener, H. J., 240, 269, 270 Renner, M., 356, 360 Rhodes, W., 177, 178, 195 Rice, F. 0., 172, 196 Rideal, E. K., 74, 81 Rieke, F. F., 164, 196 Rigopoulos, N., 190, 195 Ritter, G., 299, 303 Rivett, D. E., 69, 81 Roberts, R., 74, 81 Robinson, J. C , 16 Roquitte, B. C, 29, 33 Roscoe, H., 229, 269 Rosenberg, B., 181, 196 Rosenberg, J. L., 166, 189, 195, 196 Rosenberg, L. L., 115, 128, 154 Roughton, F. J. W., 100, 109 Rubin, J., 134, 152 Rumberg, B., 63, 103, 110, 162, 167, 188,

189, 190, 197, 221 Rupert, C. S., 76, 81

Rushizky, G. W., 74, 78, 81 Rushton, W. A. H., 9, 18 Ryan, F. J., 151, 153

S Sachs, J., 324, 331 Sager, R., 131, 152, 221 Sands, R. H., 100, 108 San Pietro, A., 127, 133, 154, 158, 196 Santamaria, L., 52, 63 Sargent, M. C, 220 Sarkanen, K , 174, 184, 195 Savige, W. E., 68, 69, 71, 81 Scarisbrick, R., 127, 130, 153 Schepartz, B., 13, 17 Schmidt, G., 202, 221 Schmidt-Mende, P., 58, 63, 162, 189,

193, 195 Schönbohm, E., 289, 302 Schrank, A. R., 226, 270 Schucken, V., 106, 110 Scully, N. J., 307, 308, 331 Schwarting, A. E., 252, 271 Schweikerdt, H., 293, 299, 303 Scott, J. F., 23, 33, 83, 109 Scott, T. K., 240, 270 Seifriz, W., 294, 296, 303 Seliger, H. H., 14, 18 Senebreir, J., 324, 331 Senn, G., 282, 290, 303 Setlow, J. K , 75, 79, 81 Setlow, R. B., 23, 33, 70, 73, 75, 79, 81 Sever, R. J., 52, 63 Seybold, A., 202, 221, 228, 270 Sherwood, H. K., 191, 193 Shibaoka, H., 239, 270 Shibata, K., 85, 86, 109, 254, 270, 274, 303 Shiliapintokh, V. I., 54, 62 Shore, V. G., 79, 80, 81 Shropshire, W., Jr., 250, 256, 268, 270, 300,

303 Shugar, D., 68, 69, 74, 75, 80, 81, 82 Siegel, A., 78, 81 Siegelman, H. W., 315, 316, 317, 324,

325, 330, 331 Sierp, H., 228, 270 Simon, H., 240, 269, 270 Simpson, W. T., 176, 196 Singleton, M. F., 55, 56, 61, 62 Sinsheimer, R. L., 23, 33, 75, 82

370 AUTHOR INDEX

Sisler, E. C, 147, 162 Sistrom, W. R., 183, 187, 195, 219, 221 Skow, R. K., 203, 220 Smaller, B., 52, 54, 55, 57, 59, 63 Smillie, R. M., 58, 63, 147, 162, 190, 196 Smith, C, 189, 194 Smith, D. E., 52, 63 Smith, J. H. C , 274, 275, 276, 277, 278,

302, 303 Smith, L., 132, 162, 164, 188, 194, 196 Smith, R. A., 4, 18, 22, 33 Smith, R. N., 25, 33 Söderborg, B., 86, 109 Sogo, P. B., 53, 54, 55, 59, 60, 62, 63,

190, 196 Sorby, H., 324, 331 Spoehr, H. A., 6, 18 Spruit, C. J. P., 189, 196 Stadler, L. J., 76, 82 Stahl, E., 290, 303 Stair, R., 106, 110 Stâlfelt, M. G., 282, 297, 303 Stanier, R. Y., 5, 18, 146, JÄfc 156, 183,

187, 195, 196, 219, 221 Steeves, T. A., 240, 270 Steinbauer, G. P., 324, 330 Stern, B. K., 142, 154 Stout, P. R., 136, 152 Strain, H. H., 199, 221 Strehler, B. L., 14, 18, 191, 196 Strong, J., 106, 110 Strong, R. L., 32, 33 Strugger, S., 290, 303 Stuart, M. W., 321, 330 Stuy, J. H., 75, 82 Suzuki, I., 158, 196 Sweeney, B. M., 334, 335, 336, 337, 338,

339, 341, 342, 344, 347, 348, 349, 350, 351, 354, 357, 358, 359, 360, 361

Symons, M. C. R., 40, 62

T Tagawa, K., 58, 62, 118, 119, 122, 123, 127,

133, 134, 141, 144, 145, 147, 152, 154, 157, 158,193

Takahashi, W. N., 67, 68, 81 Takamatsu, K., 256, 270 Takashima, S., 189, 196 Tanada, T., 203, 221 Tanner, H. A., 55, 61, 63 Tatum, E. L., 151, 153

Taylor, A. R., 79, 80, 82 Taylor, R. P., 24, 33 Teller, E., 172, 185, 195, 196 Terenin, A. N., 179, 196 Tetley, IL, 224, 270 Than-Tun, 148, 162 Thimann, K. V., 8, 18, 226, 227, 230,

236, 239, 240, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 268, 269, 270, 271

Thomas, J. B., 160, 186, 196, 221 Thompson, J., 202, 212, 220 Thrush, B. A., 29, 33 Tichy, C , 141, 154 Timm, E. A., 79, 82 Tocher, R. D., 234, 236, 269 Tollin, G., 55, 58, 63, 180, 181, 182, 190,

191, 192, 195, 196 Tonnelat, J., 28, 33 Toole, E. H., 305, 310, 318, 321, 330, 331,

348, 359 Toole, V. K., 305, 310, 318, 321, 330, 331,

348, 359 Towner, G. H., 91, 109 Townsend, J., 35, 52, 53, 55, 59, 62, 63,

190, 194 Trebst, A. V., 114, 115, 122, 126, 147,

153, 154 Trehame, R. W., 55, 61, 63 Tribukait, B., 353, 361 Tsepalov, V. F., 54, 62 Tsujimoto, H. Y., 115, 139, 140, 161, 154 Turner, A., 76, 81 Tuttle, A. L., 128, 154

U Über, F. M., 76, 82 Umrikhina, A. V., 54, 62 Uretz, R. B., 5, 17, 77, 82

V

Valentine, R. C, 133, 153 van der Hart, J. M., 168, 194 van Niel, C. B., 113, 148, 151, 163, 154,

156, 157, 160, 164, 195, 196, 197, 219, 220

Van Overbeek, J., 226, 238, 241, 270 Vatter, A. E., 187, 195 Vennesland, B., 118, 131, 142, 162, 153,

154 Vernon, L. P., 124, 129, 132, 152, 153, 154

AUTHOR INDEX 371

Virgin, H. I., 252, 270, 274, 275, 277, 278, 280, 297, 299, 300, 302, 803

Vishniac, W., 116, 117, 133, 154, 158, 197 Voerkel, S. H., 250, 271, 285, 288, 80S Vogt, E., 306, 831 Volker, W., 142, 154 Volman, D. H., 24, 25, 83 von Guttenberg, H., 243, 271 von Wettstein, D., 280, 802 Vos, J. J., 168, 194

W Wahl, O., 356, 861 Wald, G., 4, 9, 18, 250, 271, 329, 831 Wallace, R. H., 252, 271 Walles, B., 280, 802 Wang, S. Y., 75, 82 Warburg, O., 106, 110, 135, 142, 148, 154,

313, 881 Wassink, E. C , 212, 221 Watson, W. F., 171, 196 Weaver, E. C , 54, 55, 57, 58, 60, 63 Weaver, H. E., 54, 55, 58, 63 Wehrung, M., 289, 803 Weissbluth, M., 35, 52, 62, 68 Went, F. W., 224, 225, 226, 227, 233, 236,

238, 240, 243, 252, 271 Werkman, C. H., 158, 196 Wessels, J. S. C , 118, 154 West, W., 83, 110 Wettermark, G., 23, 29, 88 Weygand, F., 240, 269 Whatley, F. R., 58, 63, 114, 115, 117, 118,

119, 123, 124, 125, 128, 131, 133, 136, 137, 138, 139, 140, 141, 145, 151, 152, 158,154, 157, 158, 162, 163,196

Whiffen, D. H., 42, 68 Whitaker, W. L., 340, 861 Wierszchowski, K L., 74, 75, 81, 82 Wiessner, W., 147, 154

Wüden, M., 233, 234, 238, 241, 271 Wildman, S. G., 78, 81, 239, 271, 290, 802 Wilhelmi, G., 141, 154 Wilkens, M. B., 861 Wilson, A. T., 114, 152 Wilson, J. F., 234, 236, 269 Withrow, A. P., 24, 33 Withrow, R. B., 16, 18, 24, 83, 250, 256,

270, 300, 803 Witt, H. T., 32, 88, 58, 68, 100, 103, 109,

110, 162, 167, 168, 188, 189, 190, 193, 195,197, 221

Wohl, K., 159, 195 Wolff, J. B., 277, 803 Wood, R. W., 85, 86, 110 Wright, B. E., 148, 154 Wurmser, R., 202, 221

Y Yamaki, T., 239, 270 Yang, C., 95, 96, 110 Yocum, C. S., 160, 164, 195, 207, 221 Young, V. K., 212, 220 Yount, V., 190, 195

Z Zalokar, M., 221 Zaugg, W. S., 124, 154 Zavoisky, E., 35, 63 Zelle, M. R., 68, 82 Zieger, G., 32, 83, 103, 110, 189, 197 Zimmerman, B. K., 228, 230, 231, 232,

242, 244, 245, 246, 248, 259, 261, 262, 263, 264, 265, 271

Zirkle, R. E., 77, 82 Zurzycka, A., 283, 285, 286, 287, 288, 289,

290, 808 Zurzycki, J., 282, 283, 284, 285, 286, 287,

290, 292, 296, 301, 803

SUBJECT INDEX

A Absorbance index, 26-28 Absorption spectra, see also action

spectra bacteriochlorophyll, 187-188 chlorophyll, 171-172, 174 chlorophyll a, 87-88, 89, 94, 156 derivative spectroscopy, 91-94 differential spectroscopy, 94-103 instrumentation for, 89-90 light-induced, 98-103 measurement of

mixtures, 90-94 scattering materials, 83-89

Acetabularia major, 358, 359 Acridine dyes, 5 Actinometers, 24, 31 Actinomycin D, 333 Action spectra, see also absorption

spectra carotenoids, 202-210 chlorophyll, 56-57, 107, 108 interpretation of, 107-108 measurement of, 103-106 photoinhibition of luminescence, 339-

340 photoperiodism, 307-310 photosynthesis, 202-210 phototropism, 249-259 phycobilins, 208-210 Prorocentrum, 107 protochlorophyll, 276 rhythm phase shift, 348-349

Actomyosin, 301 Adenosine diphosphate, 118 Adenosine triphosphate, see also photo-

phosphorylation Adenosine triphosphate, 113, 119, 135, 301 ADP, see adenosine diphosphate Allophycocyanin, 317 Aluminum, 294 Amino acids, see also individual amino

acids photochemistry, 68-69

Ammonium, 135, 142 372

Anabaena cylindnca, 148 Anacystis, 166, 219 Ankistrodesmus, 141 Ankisroclesmus braunii, 148 Anthocyanin synthesis, photocontrol,

324-327 Antibody, photoinactivation, 66 Aphids, 11 Apostrophy, 282, 283, 288 Arsenate, 142 Ascorbate, 118, 119, 135, 136 Ascorbic acid, 128 Astaxanthin, 200 Auxin,

geotropic response, 240 inactivation, 239-240 phototropism, 233-242 red-light effect, 238-239 transport, 225-226, 234-236, 239-249

Avena, 225 A vena coleoptile, protoplasmic stream-

ing, 295-296, 299 Avena sativa, 224

B

Bacterial photophosphorylation, 118 Bacteriochlorophyll, 128, 130, 168, 185,

186 absorption bands in vivo, 187-188 absorption maxima, 156 aggregation in vivo, 186-188 electron transport, 156-157

BacteriochlorophylL, 168, 189 Bacteriophages, 79, 80 Barley coleoptiles, 242 Bean, 224 Bilioprotein, 107, 108 Biological clocks, see biological rhythms Biological rhythm,

arrhythmic systems, 337-339, 340-343 continuous light, 336-343 entrainment, 352-356 phase relationships, 350-351 phase shifting, 343-350, 351-352 photoinhibition, 339-340

SUBJECT INDEX 373

photoperiod effect, 356-357 photosynthetic capacity, 357-359 short light exposure, 343-350

Bioluminescence, 14 Bi-radicals, 40-41 Blepharisma, 5

C Carbon dioxide,

assimilation, 112, 113, 114-116, 148-149 fixation, 122 Hill reaction, 142 photoassimilation, 148-149 photoreduction, 164

Carotene, 126 ß-Carotene, 251 Carotenoids, 107, 108, 166, 182, 273, 299

action spectra, 202-210 chemical structures, 199-200 protective action, 219-220 photosynthesis, 182, 188-189 phototropism, 243, 250-255, 257

Chemosynthesis, 116, 149-151 Chlamydomonas, 57, 58, 60 Chlamydomonas reinhardi, 53 Chlorella, 58, 59, 60, 149, 159, 219 Chlorella pyrenoidosa, 87, 88, 148 Chloride, 294

in photosynthesis, 135-136, 137 Chlor obium, 118, 168 Chlorophyll, 113, 119

absorption bands, 171-172, 174 action spectra, 107, 108 action spectra for R-signal, 56-57 aggregation in vivo, 186-188 chromatic transients, 214r-219 electron resonance signals, 53-61 electron transport, 180-184 energy migration, 174r-179 energy transfer, 211-212

in vivo, 185-186 formation, 273-280 light-induced signals, 53-61 photochemistry in solution, 183-184 photosensitizer, 5 quantum for formation, 288 singlet states, 169-172 triplet states, 172-174, 184, 189

Chlorophyll a, 126, 128, 166, 186, 207, 214, 217, 218, 340

absorption maxima, 156 absorption spectra, 87-88, 89, 94 formation, 273 -274, 277, 278-280 reaction with cytochrome f, 162, 167,

189 Chlorophyll b, 59, 70, 74, 126, 128, 166,

218 formation, 280

Chlorophyllide, 277 Chloroplasts, 143

carbon dioxide assimilation, 114r-116 constituents, 126-128 movement, 281-291 photosynthetic unit, 113-116 protoplasmic streaming, 299 structural changes, 280

Chloroplast extract, 115, 116 Chromatium, 118, 122, 135, 147, 149, 150,

185 chromatophores, 161

Chromatophores, 118, 143, 161 constituents, 128-129 primary light reaction, 129-130

Chroococcus, 209 Chymotrypsin, 70, 74 Coenzyme Q, 128, 189-190 Coilodesme, 206 Copper, 128 Corn, 224 Corn coleoptile, phototropism, 228-230,

234-235, 240-241, 248, 249 Cypridina, 14 Cysteine, 135 Cysteine sulfuric acid, 68 Cystine, 68 Cytochromes, 120, 122, 123, 139, 157

participation in photosynthesis, 130-132

Cytochrome b, 129 Cytochrome b6, 127, 130 Cytochrome c, 131, 132, 168 Cytochrome c2, 129, 130, 132 Cytochrome c-type, 127 Cytochrome f, 127, 130-131, 162, 167 Cytochrome oxidation, 144

D Daily rhythms, see biological rhythms Desoxyribose nucleic acid, photoinactiva-

tion, 75-76

374 SUBJECT INDEX

Diamagnetism, 36-38 Diastrophy, 282 Dinitrophenol, 143 Diphosphopyridine nucleotide, 118, 157 DPN see diphosphopyridine nucleotide Diurnal rhythms, 11-12 Drosophila, 341, 344, 350, 356 DunalieUa, 206, 219

E Electron spin resonance,

application to photobiology, 51-52 application to photophysiology, 35-63 experimental methods, 45-48 methods for biological systems, 48-51 phenomenon, 42-45 studies in photosynthesis, 52-61

Elodea chloroplast movement, 281, 283 Emerson effect, 161-162, 207, 212-213 Enteromorpha, 206 Enzyme, photoinactivation, 66, 69-74 Epistrophy, 282, 283, 285, 286 Etiolation, 309, 310

F Ferredoxin, 126, 127, 133-134, 145 Ferret, 11 Ferromagnetic resonance, 51 Ferromagnetism, 37, 39 Flash photolysis, 4, 20-21

discharge unit, 29-32 monitoring system, 32 photochemistry, 28-32

Fla vine mononucleotide, 118, 120, 140, 142, 144

Flowering, 309, 322 FMN, see flavine mononucleotide Formaldehyde, 112 Free radicals, 21, 40, 69 Fucoxanthin, 200, 203 Funaria, chloroplast movement, 283

G Gene alteration, 76 Geotropic response, 240 Glaucomys, 344 Glucose, photoassimilation, 146 Gonyauhx, 206, 337, 338, 339, 344, 346,

347, 348, 350, 351, 354, 358 Gonyaulax polyedra, 333, 357

H Haematococcus, 219 Helianthus, 225 Hemocyanin, 74 Hill reaction, 123, 140

carbon dioxide requirement, 142 Hydrogen, 150

photoproduction, 134-135, 147 Hydrogenase, 135 Hydrogen sulfide, 112, 113

I Infrared light, 1 Iron, 128

L Lasars, 22-23 Lepidium, 229 Lepidium virginicum, 313 Lemna, chloroplast movement, 283, 285,

289 Lemna trisulca, 285, 288 Lettuce, seed germination, 310, 313, 321 Light-induced absorbance, 98-103 Limulus, 10 Luciferase, 14 Luciferin, 14 Luminescence,

blocking, 333 rhythmicity, 333, 337, 338, 346, 350, 351,

353 Lupus vulgaris, 12

M Magnesium, 118 Manganese, 58, 60, 61, 128,165

photosynthesis, 141-142 Mercury lamps, 21-22 Mitochondria, 117, 290 Monostroma, 206 Mougeotia, chloroplast movement, 289 Mustard, 229 Myers effect, 217

N New Zealand spinach, 114 Nitrate, photoreduction, 148 Nitrogen, 135

photoreduction, 147-148

SUBJECT INDEX 375

O Oat, 224 Oat coleoptiles,

phototropism, 227-230, 233-234, 242, 244-245

pigments, 250-258 Organic acids, photoassimilation, 146-147 Oscilfoloria, 208 Oxygen, 69

role in photosynthesis, 13&-140 Oxygen evolution, 58, 113, 136, 138, 148,

164, 214

P

Paramagnetic species, origin of inorganic systems, 39-42

Paramagnetism, 37 38-39 Paramecium, 347, 350 Parastrophy, 282, 283, 286, 288 Peristromium, 290 Pharbitus nil, 329 Phaseolus, 224, 242 Phenazine methosulfate, 119, 120, 140,

143, 144, 218 Phormidium, 209 Phosphorescence, 173 Phosphorylation,

anaerobic, 117, 118 oxidative, 117 photosynthetic, 116-119 see photophosphorylation

Phosphoglyceric acid, 112 Photoactivation, organic compounds, 147 Photoassimilation,

carbon dioxide, 148-149 glucose, 146 organic acids, 146-147

Photobiology, reference books, 14-16 Photochemistry,

development of, 2-4 filters and monochromators, 23 high intensity, 28-32 light intensity measurement, 23-25 light sources, 21-23 macromolecular inactivation, 65-82 principles of, 19-33 quantum yield calculation, 25-28

Photoinactivation, amino acids, 68-69

desoxyribose nucleic acids, 75-76 enzyme, 66, 6^-74 quantum efficiency

calculation, 65-68 ribonucleic acid, 66, 74-75 virus, 66-68, 77-80

Photon, energy of, 19 -20 Photon absorption, calculation of, 25-28 Photoionization in vivo, 190-192 Photonasty, 223 Photooxidations, 183, 251 Photoperiodism, 304-331

action spectra, 307-310 biological rhythms, 356-357 development of photochemistry, 10-11 discovery of, 306-307 photoeffectiveness, 312-314 photoreversible reaction, 310-312 time-sensing, 320-323 vision, compared to, 329

Photophosphorylation, 218 bacterial, 118 cyclic, 120-121 electron-flow mechanism, 119-126 evidence for electron-flow, 126-145 ferricyanide effect on, 136-137, 138 inhibition, 142-143 light and dark phases, 143-145 noncyclic, 122-126 oxygen participation, 139-140 two light reactions, 137-139

Photoreactivation, 13 Photoreductant, 111 Photoreduction, 183 Photosensitization, 4-6 Photosenitizer, 340 Photosynthesis,

accessory pigments, 199-221 action spectra, 202-210 biochemical outlines, 155-158 biophysical problem, 168-169 blocking, 333 capacity rhythm, 357-359 carotenoids, 211-220 chlorine requirement, 135-136 chromatic transients, 214-219 compared with chemosynthesis, 149-

151 concepts of, 112-113 development of photochemistry, 6-7

376 SUBJECT INDEX

energy transfer, 211-212 examples of, 145-149 inhibitors, 142-143 photochemical reactions of, 111-154 photosynthetic unit, 113-116, 159-161 primary photochemical reactions in

vivo, 188-190 reaction center, 167-168 two light reactions, 137-139 two photochemical system, 161-167

Photosynthetic bacteria, 112-113, 164 Photosynthetic phosphorylation, see pho-

tophosphorylation Photosynthetic pyridine nucleotide re-

ductase, 127, 133 Photosynthetic reaction center, 167-168 Phototactic, 288 Phototaxis, 8 Phototropic, 299 Phototropic induction, 232 Phototropism, 8

action spectra, 249-259 auxin relationships, 233-242 dosage-response curves, 226-233 history of study, 223-226 kinetic studies, 259-266 Phycomyces, 268 pigments, 249-259 Pilobolus, 268 red light influence, 232-233, 242-249

Phycobilins, 126, 128, 166, 186 action spectra, 208-210 chemical structure, 200-201

Phycocyanin, 126, 201, 203, 208 Phycoerytherin, 126, 207, 214, 215 Phycoerytherobilin, 200-201 Phycomyces phototropism, 268 Phytochrome, 24&-249, 313

active form, 323-324 anthocyanin synthesis, 324-327 dark reversion, 320-323 isolation of, 316-318 physical detection, 315-316 physiological aspects of action, 328-329 properties of, 318-320

Phytofaca amencana, 114 Pilobolus phototropism, 268 Plastoquinone, 58, 126, 128

photophosphorylation, 140-141 Pokeweed, 114

Porphyridium cruentum, 130 Porphyra tenera, 131, 139 Potassium, 294 Primary light reaction, electronic nature,

129-130 Proplastids, 280 Prorocentrum, action spectrum, 107 Protochlorophyll, 315

action spectra, 276 chemistry of, 276-277 formation, 274-276, 279

Protochlorophyll holochrome, light-re-sponse, 277-278

Protoplasmic streaming, 291-302 factors affecting, 292-295 light, effect of, 293, 294, 295-296, 297-

300 mechanism, 300-302 salts, effect of, 293 types of, 291-292 viscosity, 296-298

Q Quantasomes, 56, 61 Quantum efficiency, calculation of, 65^68

photoperiodism, 312 photosynthesis, 161, 203, 207 phototropism, 253

Quantum law, 3 Quantum yield, 3, 20

calculation, 25-28 chlorophyll formation, 278 measurement, 23-25

R

Radish, 224 Raphanus, 224, 241-242 Red light,

chlorophyll formation, 279 photoperiodicity, 307-309 phototropic response, 232-233, 242-249,

261 protoplasmic streaming, 299

Rhodopsin, 9, 329 Rhodopspeudomonas sphéroïdes, 219 Rhodospirillum rubrum, 118, 129, 132,

142, 146 chromatophores, 56, 59, 161

Rhodospirillum sphéroïdes, 161, 188

SUBJECT INDEX 377

Rhodoviolascin, 200 Riboflavin, 299

phototropism, 226, 251-252 Ribonuclease, photoinactivation, 66 Ribonucleic acid, 359

photoinactivation, 66, 74-75 synthesis, 333

RNA, see ribonucleic acid

S Scenedesmus, 160 Seed germination, 309, 310 Sefoginelfa, chloroplast movement, 283 Soybean, 308 Spectroscopy, derivative, 91-94

differential, 94-103 Spinach chloroplasts, 114, 118 Spirilloxanthin, 200 Starch, 114 Streptomyces grisetis, photoreactivation,

107 Succinate, 118, 122 Sugar beet, 114 Sunflower, 114 Swiss chard, 114 Systrophy, 282

T Tetragonia expansa, 114 Thiobacillus, 149 Thiosulfate, 122, 135 Tillia aquatica, 306 TMV, see tobacco mosaic virus TPN, see triphosphopyridine nucleotide Tobacco mosaic virus, 5, 72, 74, 77-79 Transforming DNA, 75

Trentepohlia, 206, 219 Triphosphopyridine nucleotide, 123, 128,

131, 134, 138, 139, 140, 148, 149, 162, 163, 218

Triplet states, paramagnetic systems, 41-42

U Ultraviolet light, 1

biological effects, 76-77 medical uses, 12-13 phototropism, 238, 248, 250, 254-255 protochlorophyll conversion, 277 rhythm phase shifting, 349

Ultraviolet radiation, effects upon cells, 13-14

Viva, 206

V , Vanadium, 61

Virus, photoinactivation, 66-68, 77-80 Vision, development of photochemistry,

9-10 Vitamin A, 9 Vitamin K, 120, 127-128 Vitamin K3, 118, 140, 144

w Water photooxidation, 124-126

X Xanthophyll, 126, 273

Z

Zea mays, 224 Zinc, 128

Photophysiology. General Principles; Action of Light on Plants

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Transcript of Photophysiology. General Principles; Action of Light on Plants