PHOTOSYNTHESIS

Teacher's Guide

This teacher's guide is designed for use with thePhotosynthesis series of programs produced byTVOntario, the television service of The OntarioEducational Communications Authority. Theseries is available on videotape to educationalinstitutions and nonprofit organizations.

The GuideAuthor: Bob WhitneyEditor: Carol SevittDesigner: Roswita BusskampReviewer: Murray Lang

Contents

Introduction.........................................................1Seeing the Light ................................................. 2Absorbing the Light ............................................ 6The Light Reaction ........................................... 10The Dark Reaction ........................................... 13C3 and C4 Plants ............................................. 16The Fluid-Transport System ............................. 19Bibliography......................................................23Ordering Information ........................................ 24

The SeriesProducer: David ChamberlainProject Officer: John AmadioAnimation: Cinescan

Canadian Cataloguing in Publication Data

Whitney, Bob

Photosynthesis. Teacher's guide

To be used with the television program,Photosynthesis.Bibliography: p.ISBN 0-88944-124-3

1. Photosynthesis (Television program)2. Photosynthesis. I. TVOntario. II. Title.

QK882.W54 1987 581.1'3342 C87-099661-4

® Copyright 1988 by The Ontario EducationalCommunications Authority.All rights reserved. Printed in Canada.

Introduction

The Photosynthesis series of six 10-minuteprograms is intended to convey a basic under-standing of biological principles to seniorhighschool students.

various pigments involved in photosynthesis. Itencourages students to study the absorptionspectra of plant pigments and devise means ofseparating and identifying them.

This teacher's guide gives a more detailedaccount of the material in each program. It alsosuggests related before-viewing activities, whichare directed at teachers. After-viewing activitiesare directed at students, and can be photo-copied and distributed. These activities includelaboratory exercises, model building, researchassignments, and even the dramatic simulationof the events of photosynthesis. A bibliographyat the end provides highschool and college-levelreferences for further study.

The series begins with a historical survey ofearly ideas and experiments in the area ofphotosynthesis; many of the latter can be dupli-cated in a highschool lab. The first program,"Seeing the Light," develops the chemical equa-tion that represents photosynthesis, and intro-duces the light and dark reactions and themorphological features of photosynthetic organs.

"The Light Reaction" traces the pathways ofelectrons and protons through the thylakoids,introducing students to the major electroncarriers. In "The Dark Reaction," students gainan understanding of the Calvin cycle's complexseries of reactions at the molecular level.

The two final programs follow up some interest-ing related concepts. "C3 and C4 Plants" investi-gates the C4 plants to discover why they haveevolved in some tropical areas, but have failedto displace C3 plants anywhere. "The Fluid-Transport System" explains the position of plantsin the ecosystem. The problem of getting waterto the photosynthetic machinery at the tops oftall trees is considered.

The series should convey the complexity andsophistication of plants, and spark students'curiosity to pursue further studies.

"Absorbing the Light," builds on the morphologi-cal theme at the molecular level, exploring the

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Objectives

After viewing this program, students should beable to do the following:

1. Identify the reactants and products of photo-synthesis.

2. Write a balanced chemical equation for photo-synthesis.

3. Describe, in a general way, the contributionsof the light-dependent and light-independent(dark) reactions.

4. Name the site of photosynthesis in plants.5. Describe a use of isotopes of elements in the

study of biology.

Program Description

The earliest experiments in photosynthesisestablished the identities of reactants andproducts and the need for light to drive theprocess. In 1771, Joseph Priestley found that"something" in air supported the burning ofcandles and the breathing of animals wasrestored by plants. A few years later, Jan Ingen-Housz learned that this restoration of airoccurred only in light and that only the greenparts of the plant were able to do it. Later JeanSenebier found that carbon dioxide was used upin the process and assumed that this was thesource of the oxygen released. In later years,water was recognized as both a reactant andproduct of photosynthesis, and chlorophyll as anecessary participant.

Photosynthesis is represented by this chemicalequation:

The overall reaction of photosynthesis has beenbroken up into two complex series of reactions:the light-dependent reactions use the "old" waterand give off oxygen; the light-independent(dark) reactions combine products of light-dependent reactions with carbon dioxide to formorganic matter and release "new" water as awaste product.

Anatomically, the reactions of photosynthesisoccur in the parts of a plant that contain chloro-plasts. These are most abundant in the leaves,particularly in the palisade cells located near theupper surface of the leaf. Within the chloroplast,elaborate systems of membranes, the lamellae,organize chlorophyll molecules so they presentan immense surface area to the light.

Before-Viewing Activities

1. Discuss the general nature of photosynthesisand its importance in gathering and storingenergy in a form that can be used by othermembers of the biological community.

2. Look at photosynthesis from a human point ofview and discuss its importance in concen-trating energy as biomass and fossil fuels forspace heating, transportation, and poweringindustry, as well as its role as feedstock forthe organic chemicals industry.

After-Viewing Activities

ACTIVITY 1. Examining aChloroplast

The distinction between "old" water (a reactant)and "new" water (a product) awaited the devel-opment of means to produce and detect theheavy isotope of oxygen, 0-18. When reactantwater molecules are tagged with such an iso-tope, the oxygen in the water appears in theoxygen molecules released as a waste product.

Locate an electron micrograph of a chloroplastand trace or photocopy it. Identify the outermembrane, stroma, lamellae, thylakoids, andgrana. While it is a two-dimensional structure,calculate the approximate surface area tovolume ratio (ratio of total length of membranesvs. cross-sectional area) of the organelle as itappears and as it would, if there were no internalmembranes.

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PROGRAM 1 / Seeing the Light

Method (see Fig. 1.1)

sodium bicarbonatesolution

Fig. 1.1: Apparatus for Activity 2.

Elodea onwire support

container

1. Fill the jar about 3/4 full with sodium bicarbon-ate solution.

2. Invert the funnel (stem upwards) with the wiremesh screen inside.

3. Place the Elodea plants around the wiremesh, but inside the funnel wall. The base ofthe Elodea stems should point upwards.

4. Place the funnel, wire mesh, and Elodea inthe container.

5. Fill the test tube with sodium bicarbonatesolution and invert it over the stem of thefunnel.

6. Place the apparatus in the light for one or twodays, or until enough gas has collected in thetube to be tested.

7. Test the gas collected in the test tube byplunging a glowing splint into the gas.

8. Record all observations and draw appropriateconclusions.

Discussion Questions

ACTIVITY 2. Testing of GasReleased by a PhotosynthesizingPlant

The purpose of this experiment is to collect thegas released by an aquatic plant and test itsability to support combustion.

Apparatus

Large beaker or battery jarFunnel to fit within above (a short stem is advan-

tageous)Cone of hardware cloth or window screening

that fits loosely into the funnelTest tubeWood splint and match or Bunsen burnerLight source (or unshaded window)0.1% sodium bicarbonate solution, well aerated6-8 shoots of Elodea canadensis

1. Why was sodium bicarbonate supplied to theplants? Why was the solution aerated?

2. What gases might have collected in the testtube? For each gas, explain where it mayhave come from.

3. How is this experiment similar to Priestley'sexperiment?

ACTIVITY 3. Are Light, CarbonDioxide, and Chlorophyll Necessaryfor Photosynthesis?

The following supplies and procedures arecommon to Parts A, B, and C of this activity.

Apparatus for testing leaves

BeakerLarge test tubeForcepsWatch glass or Petri dishLight source or sunny windowSafety goggles

test tube

invertedfunnel

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Wet towel to act as emergency fire extinguisherBoiling water (use electric kettle or coffee urn if

possible)Denatured ethanol (CAUTION: INFLAMMABLE)Lugol's iodine solution

Blank microscope slideCover slip for microscope slideGlycerine (in dropper bottle)

Method

Method for Testing Leaves for the Presenceof Starch

Caution: Wear safety goggles whileworking with chemicals and flames andwork only from a standing position!

Do not use an open flame to boil ethanolnor to boil water while ethanol is beingheated in the room.

1. Remove the test leaf from the plant.2. Using forceps, immerse the leaf for 30 sec-

onds in boiling water in the beaker.3. Add about 30 mL of ethanol to the test tube

and immerse the leaf in it.4. Put the test tube into the beaker of boiling

water.5. Leave the leaf in boiling ethanol for four min-

utes, replacing the water in the beaker asnecessary to maintain boiling.

6. Remove the leaf from the ethanol with forcepsand dip it in boiling water for 30 seconds.

7. Flatten the leaf in a watch glass or Petri dishand cover it with Lugol's iodine solution. Aftertwo minutes, examine the leaf for blackpatches that indicate the presence of starch inthe treated leaf.

1. Remove the geranium plant from storage andtest one of its leaves for starch. Immediatelyfasten the cross to the upper surface of one ofits leaves.

2. Place the plant in the light for a day.3. Remove the treated leaf and test it for the

presence of starch.4. Tear a small piece of leaf from an area that

tested positive for starch and mount it (bottomsurface upwards) in glycerine on a micro-scope slide. After two minutes, examine thesection with medium and high magnificationsand describe the distribution of black color inthe leaf.

Discussion

1. Account for the distribution of starch in the leafas seen with the naked eye and with themicroscope.

2. Why was the plant kept in the dark for 24hours prior to the experiment?

3. Was there a "control" in this experiment?Explain.

PART B: Is Carbon Dioxide Necessary forPhotosynthesis?

Apparatus

PART A: Is Light Needed forPhotosynthesis?

Apparatus

Supplies for testing leaves (outlined previously)Potted geranium plant (kept in the dark for 24

hours)PaperclipCross (about 2 cm x 2 cm) cut from black card-

boardCompound microscope

Supplies to test leaves for starch (described pre-viously)

Potted geranium kept in the dark for 24 hours250 mL Erlenmeyer flaskOne-hole stopper for above, slit into the hole

along one sideVaselineSolid sodium hydroxide (CAUTION:

EXTREMELY CAUSTIC)Scoopula or plastic spoonRetort standAdjustable burette clamp or extension clamp

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Method

Caution: Wear safety goggles whilehandling sodium hydroxide. If you getany chemical on your hands, washthem thoroughly.

1. Remove the geranium plant from storage.2. Place about 10 pellets of sodium hydroxide

i nto the flask.3. Place the rubber stopper over the petiole of

one leaf, with the broad end toward the stemof the plant.

4. Seal the blade of the leaf in the flask, usingvaseline to complete the seal.

5. Support the flask so the leaf will not bedamaged.

6. Expose the plant to light for a day.7. Disassemble the apparatus and test the leaf

from the flask and one other leaf from theplant for the presence of starch (method fortesting for the presence of starch describedpreviously).

8. Explain your observations.

ACTIVITY 4. Review

1. Write a balanced chemical equation for theoverall reaction of photosynthesis.

2. Explain why early conclusions about photo-synthesis were drawn by physicians andclergymen rather than by scientists.

3. If the hydrogen of water supplied to a plantduring photosynthesis were labelled in someway, where would it be found following photo-synthesis? Explain.

4. Sketch the appearance of a chloroplast as itwould be seen in cross-section with the trans-mission electron microscope and label itsparts.

PART C. Is Chlorophyll Necessary forPhotosynthesis?

Apparatus

Supplies to test leaves for starch (describedpreviously)

Potted variegated coleus plant (white/green)

Method

1. Place a variegated coleus plant in the light fora day.

2. Perform a starch test on one of its leaves(method for testing for the presence of starchdescribed previously).

3. Explain your observations.

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PROGRAM 2 / Absorbing the Light

ObjectivesAfter viewing this program, students should beable to do the following:

1. Describe the internal organization of achloroplast.

2. Associate the light reactions with thylakoids.3. Explain the concept of "spectrum" and apply it

to absorption, reflection, and transmission oflight by leaves.

4. Identify the pigments found in chloroplasts.5. Distinguish between the roles of antenna

. pigments and reaction centre pigments.6. Explain how organic molecules absorb light in

the visible range.7. Identify the two photosystems and account for

their names.

Program Description

tional wavelengths of light and may increase theefficiency of photosynthesis. They are alsoresponsible for the brilliant colors of foliageevery autumn. It is common to refer to thesepigments as "antenna" pigments, since theygather energy and direct it to a reaction centrewhere it is utilized. The reaction centre alwayscontains chlorophyll a.

Although the various pigments have differentroles and chemical structures, they share onefeature. All possess a series of alternating singleand double bonds within the molecule. Theseconfer the ability to absorb light in the visiblerange, but each molecule absorbs only a fewwell-defined wavelengths. Thus, the existence ofmany different pigments in slightly differentchemical environments extends the range ofwavelengths (photons) that can be absorbed bythe chloroplast and increases the efficiency ofphotosynthesis.

Chlorophyll is located in membranes within chlo-roplasts. The membranes are called lamellaeand, at points along their length, form disk-likeexpansions, the thylakoids. Thylakoids formstacks within the chloroplast. These stacks arethe grana. The lamellae, and especially thethylakoids, are the location of the light-depend-ent reactions of photosynthesis.

The groupings of antenna pigments and theirassociated reaction centres constitute a photo-system. There are two photosystems spreadthrough the lamellae. These are named on thebasis of the wavelength absorbed by theirreaction centres as P 680 and P 700 Each plays adifferent role in the light reactions and both arevital to photosynthesis.

If visible light is separated into its componentcolors, a spectrum results. Different spectra canbe obtained for leaves, depending on whetherthe light examined has been reflected, absorbed,or transmitted through the leaf.

The wavelengths of the absorption spectrum aremost important because they represent theportion of the sun's light that may be availablefor the synthesis of carbohydrates.

The major pigments of photosynthesis are thechlorophylls. Chlorophyll a and chlorophyll b arevery similar chemically, with a methyl group onchlorophyll a being replaced by a carbonyl groupon chlorophyll b. Accessory pigments, such ascarotenoids, enable the plant to absorb addi-

Before-Viewing Activities

1. Discuss how a prism or diffraction gratingseparates different wavelengths or colors oflight; follow up with an examination of whychemicals such as dyes and food coloringappear colored when examined in "white"light.

2. Introduce the units (nanometres) used tomeasure wavelengths of light and develop instudents some feeling for the values, innanometres, of visible light ranging in colorfrom violet to red.

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After-Viewing Activities Discussion

ACTIVITY 1. Light Sources

Apparatus

Hand spectroscope, student grade (e.g.,Science Kit Inc.)

Fluorescent light source (e.g., classroom lights)I ncandescent light source (or several with

different wattages)Fresh leaf

Plant growth chambers often use a combinationof fluorescent and incandescent light bulbs togrow plants artificially. The incandescent bulbsare of low power output (e.g., 40 watts). Explainwhy using this combination of lights is a goodpractice.

ACTIVITY 2. Absorption Spectrumof Spinach

Apparatus

Method

1. If using a "Science Kit" hand spectroscope,note the square hole at the narrow end. Thisis the viewing port. At the wide end, there is anarrow slit through which light to be analyzedenters the instrument. There is also a longrectangular opening that provides backgroundillumination for a numerical scale that is part ofthe instrument. Each division of the scale cor-responds to 10 nm. The numbers on the scaleshould be multiplied by 100 to convert themto nm.

2. Aim the instrument so that light from a fluores-cent light fixture passes through the slit andlook through the viewing port. You will seemultiple colored images of the slit superim-posed over the scale. If you wear glasses fornear-sightedness, removing them may enableyou to read the scale with greater ease. Notethe bright bands in the spectrum. These corre-spond to the major emission bands for thefluorescent bulb. Sketch the appearance ofthe spectrum, including only the part that isover the scale.

3. Repeat the observations and sketches, usingeach of the following as light sources:a) incandescent bulbb) sunlightc) sunlight reflected from the surface of a leafd) sunlight passed through a leaf

4. Compare the four spectra and comment onthe significance of any differences.

Hand spectroscopeFocused light source (e.g., fibre-optics micro-

scope illuminator or slide projector with holecut by a paper punch in the middle of a card-board square, sized to fit the projector)

Two 30 mL plastic disposable tissue cultureflasks with caps

Mortar and pestleSmall funnelBeakerLarge test tubeForcepsSafety gogglesWet towel (for emergency fire extinguisher)Fresh spinach leavesClean sandSource of hot water (e.g., electric kettle or

coffee urn)Denatured ethanolSucrose solution, 0.25 mol/L

Method

1. Noting the safety precautions, follow theprocedures of Activity 3 in the previous chap-ter (see page 4) to extract chlorophyll fromone or two spinach leaves, but do not dip theleaves in boiling water a second time. Theleaves may be discarded and need not betested for the presence of starch. Collect theextract of cholorphyll into one of the tissueculture flasks and cap the flask.

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2. Prepare a suspension of spinach chloroplastsby grinding a spinach leaf (with a pinch ofsand) with 30 mL of sucrose solution in themortar. When the suspension is as fine as youcan get it, decant it into the second tissueculture flask and cap the flask.

3. For each flask, observe the spectrum of a lightbeam passed through the contents from top tobottom of the flask (shortest dimension) andobserve, with the unaided eye, any light scat-tered at right angles to the beam. Record allobservations.

4. Save the chlorophyll extract in ethanol for usein Activity 3. (Your teacher may wish you touse the chloroplast suspension in sucrose forthe Hill reaction in the next chapter, if this is tobe performed during the same laboratoryperiod. Check with the teacher beforediscarding it.)

Discussion

1. Compare the absorption of light wavelengthsof each preparation with that of the leaf as awhole (Activity 1).

2. The scattered light you observed with theunaided eye is a demonstration of "fluores-cence". Discuss the significance of this lightand comment on the different fluorescentproperties (if any) between extracted chloro-phyll and extracted chloroplasts.

ACTIVITY 3. Separation of Pigments

Apparatus

100 mL graduated cylinderRubber stopper, #9Small beakerPair of scissorsRulerThumbtackHair dryer (optional)Filter paper (Whatman #1) strip, 25 cm x 2.5 cmExtract of chlorophyll in ethanol (see Activity 2)Chromatography solvent consisting of 12 mL of

90% acetone mixed with 100 mL of petroleumether (100-120 degrees)

Method

Caution: Perform this step in a fumehood. Acetone fumes can cause liverdamage and/or cancers

1. Place 10 mL of chromatography solvent intothe cylinder and insert the stopper firmly in thetop of the cylinder. This must be left standingfor at least half an hour to allow the solventvapors to saturate the air inside, before it isused in step 6.

2. Transfer the chlorophyll extract into thebeaker.

3. Fold the filter paper transversely at a pointabout 5 cm from one end.

4. Build up a dark band of chlorophyll extractalong the fold by dipping the fold into theextract in the beaker until it just wets thepaper, withdrawing the paper, and drying theextract. Repeat the process until there is adark band of chlorophyll extract along the fold.This requires great patience, since eachapplication must be dried before the next canbe applied. However, the darker the chloro-phyll band, the easier it will be to carry out thelast step.

5. Trim the paper so that the chlorophyll band isabout 3 cm from the end. Cut the width of thestrip to 1.5 cm, choosing that portion of thepaper having the most uniform band of chloro-phyll extract. Trim the last 2.5 cm off the endnear the extract of chlorophyll to a uniform,symmetrical point.

6. Working in the fume hood, set up the filterpaper strip in the apparatus so that the point,but not the chlorophyll extract, is immersed inthe solvent at the bottom. The strip will haveto be trimmed to fit your cylinder (about 22 cmlong) and may be gently folded so that it willhang straight in the cylinder. Use the thumb-tack to fasten the filter paper to the rubberstopper. See Fig. 2.1 for help in setting up theapparatus.

7. Allow the solvent to be absorbed and move upthe paper. It will take about an hour for thesolvent to reach the top gradation on the

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8.

cylinder. At that time, remove the paper and,working in the fume hood, mark the position ofthe solvent front and circle each colored spoton the paper. This is best done with pencilbefore the paper is completely dry.After the paper has dried, take it to your deskand, for each spot, calculate the ratio of thedistance travelled by the colored compound(to the centre of density of its spot) to thedistance travelled by the solvent. This shouldbe a constant (the R F = ratio of fronts) foreach substance.

5. How did you distinguish between chlorophyll aand chlorophyll b in this experiment?

ACTIVITY 4. Research

Association of a particular function with a spe-cific pigment often requires study of an actionspectrum in which the dependence of the func-tion on light wavelengths is compared with theabsorption spectrum of the pigment suspected ofmediating the function. A classic study of theaction spectrum of photosynthesis was per-formed by T.W. Engelmann. In the library, doresearch on Engelmann's experiment anddescribe it in a short written report (about 300words).

Fig. 2.1: Chromatography Apparatus

Discussion

1. How many pigments were in your leaf extract?2. What is the value of your "ratio of fronts" for

the pigments you think are probably chloro-phyll a, chlorophyll b, and carotenoids?

3. Compare your "constants" with those obtainedby other students for the same pigments.Account for any differences.

4. Why do pigments travel different distances?What property distinguishes a pigment thattravels a long distance from one that travels ashort distance?

rubber stopper

filter paper

graduatedcylinder

folded area withdried pigmentextract

excess solvent

ACTIVITY 5. Review

1. Name three major pigments involved in photo-synthesis and discuss the role played byeach.

2. Explain why leaves turn yellow, orange, or redin autumn, or when they are diseased.

3. Evaluate chromatography as a means ofidentifying pigments in a complex mixture ofpigments.

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Objectives

After viewing this program, students should beable to do the following:

tron from the chlorophyll molecule. Any electronlost by the chlorophyll must be replaced, if theprocess is to continue and perform useful work.For P 680, replacement electrons are drawn fromwater in a process known as photolysis.

1. Cite the importance of double bonds in deter-mining a molecule's ability to absorb light.

2. Explain the concepts of oxidation andreduction.

3. Identify water as the source of electrons, andoxygen gas as a waste product.

4. Define photolysis.5. Identify and explain the role of the two photo-

systems in the light reaction.6. Explain the concept of a proton pump with

specific reference to plastoquinone.7. Relate phosphorylation of ADP to a flow of

protons down a proton gradient.

Program Description

Within the chloroplasts of green plants areabundant thylakoids. Here reside the essentialcomponents of the light reactions of photosyn-thesis. Within every stack of thylakoids are about200 photosynthetic units, each containing about300 pigment molecules.

The electron transport system, a series of mole-cules in the thylakoid membrane, carries elec-trons to P 680 and P 700 and from P 700 to NADP.NADP is the coenzyme that supplies electronsand hydrogen nuclei to reduce carbon com-pounds in the dark reactions of photosynthesis.

Near the beginning of the electron transportsystem is a special carrier, plastoquinone (PQ),which is fairly mobile within the thylakoid mem-brane. Oxidized PQ is at the outer edge of themembrane. As it receives electrons from P 680 itcombines them with protons from the stroma(fluid surrounding the thylakoid) to complete itsreduction. The reduced plastoquinone, PQH 2,migrates to the inner edge of the membranebefore becoming oxidized again. There it passesits electrons to the next carrier in the sequencebut the protons are released into the thylakoidinterior. Then the oxidized plastoquinone returnsto the outer edge of the membrane to repeat theprocess.

These chlorophyll and carotenoid molecules acttogether as an "antenna," absorbing photonsand channelling their energy to a single reactioncentre. The reaction centre contains a singlemolecule of chlorophyll a near an electronacceptor molecule. The reaction centres P 680 andP 700 are named on the basis of the longestphoton wavelength (measured in nanometres)that they can utilize.

The existence of conjugated double bonds (i.e.,alternating single and double bonds) within eachmolecule determines the wavelengths of lightabsorbed by that molecule. In fact, when achlorophyll molecule absorbs a photon, themolecule becomes "excited" and places anelectron from a double bond into one of its outerorbitals. An electron acceptor molecule near thereaction centre can collect such a "loose" elec-

During the process of photolysis, protons arealso released into the thylakoid interior. Thisleads to an increased concentration of protonswithin the thylakoid (lower pH) and a decreasedconcentration in the stroma (higher pH). Theresult is a proton gradient across the thylakoidmembrane. A channel through the thylakoidmembrane permits the spontaneous flow ofprotons back into the stroma. This flow of posi-tive charge constitutes an electrical currentwhich can be made to do work.

This is accomplished by a complex of enzymes,the CF1 particle, which traps the protons andharnesses their energy for the phosphorylationof ADP to form ATP, used in the dark reactionsof photosynthesis.

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PROGRAM 3 / The Light Reaction

Before-Viewing Activities After-Viewing Activities1. Review the overall equation for photo-

synthesis, the ultrastructure of the chloroplast,and the identification of pigments. As stu-dents' background knowledge may vary, itmay also be necessary to review (or teach)the concepts of oxidation, reduction, andoxidation/reduction potential.

2. It may be helpful to compare the behavior ofbiological systems to that of an electrochemi-cal cell.

Fig. 3.1 Electrochemical cell

ACTIVITY 1. Discussion of CyclicPhotophosphorylation

The electron transport system carries electronsfrom water to NADP. Additional ATP can begenerated by "cyclic photophosphorylation," andclass discussion of the process would be useful.Electrons are pumped to a high-energy state byP 700 then shunted back to the beginning of theelectron transport system, generating morephosphorylating capacity.

ACTIVITY 2. The Hill Reaction

This exercise mimics experiments done in thelate 1930s and early 1940s by Robert Hill andhis associates. They attempted to find outwhether isolated chloroplasts, exposed to ironions as electron acceptors, would give off oxy-gen in the presence of light. Since oxygen isdifficult to analyze, you will attempt the experi-ment using DCPIP (2,6 dichlorophenolindophe-nol) as the electron acceptor. The E 0 value ofDCPIP places it at about the midpoint of theelectron transport system. Reduction of theDCPIP in the presence of chloroplasts willsuggest that electron flow is taking place. Noteany color changes that occur as DCPIP oscil-lates between oxidized and reduced states.

I n biological systems, a series of electron carri-ers substitutes for the wires of the electrochemi-cal cell. Point out that the electrochemical celldoes not operate without a salt bridge, that theflow of positive ions through it constitutes acurrent, and that it should be possible to harnessa positive current to do work.

3. Students should be aware of the alternativeformulation of reduced NADP, which is -chemically speaking - more accurate:

Apparatus

Fresh spinachTwo 30-mL disposable plastic culture flasksMortar and pestleTwo tubes suitable for centrifugingCentrifugeMicroscope slide and cover slipCompound microscopeOverhead projector

* Spinach from a cellophane package may have been-stored too long for this experiment to achieve its desiredresults.

1 1

1 2

Eyedropper calibrated at 1.0 mLFine wire screen50-mL calibrated beakerAluminum foilClean sand3.5% sucrose solution0.02% DCPIP solution

Method

1. Place a little sand in the bottom of themortar.

2. Add part of a leaf (about 4 g) of freshspinach.

3. Add about 25 mL of sucrose solution (usebeaker to measure).

4. Grind the spinach thoroughly with the pestle.5. Strain the ground spinach into the beaker

through the screen. If the volume of spinachextract is less than 20 mL, add sucrosesolution to bring the volume to 20 mL.

6. Divide the extract equally between the twotubes.

7. Wrap one culture flask in aluminum foil.8. Centrifuge each tube of the extract for two

minutes at 3000 r/min.9. Into each flask, pour the contents of one

tube.10. Add 1.0 mL of DCPIP solution to each flask

and swirl contents.11. Place the unwrapped flask on the stage of

the lighted overhead projector for 5 to 10minutes.

12. While you are waiting, withdraw a drop ofthe contents from the foil-wrapped flask,prepare a wet mount, and record observations with the compound microscope at highpower.

13. When the time is up, remove the unwrappedflask from the overhead stage, unwrap thefoil-covered flask, and compare the colors ofthe contents of the two flasks.

Discussion

1. DCPIP is often used in titrations to determinethe vitamin C content of fruit and vegetablejuices. How would you counter the suggestion

that the experiment's results were caused byvitamin C in the spinach extract?

2. Do you think that oxygen was being createdduring the experiment? Why or why not?

3. Is it likely that ADP was being phosphorylatedto ATP during the experiment? Explain.

4. Is it likely that NADP was being reducedduring the experiment? Explain.

5. Design a possible procedure to measure therate of color change over time in the ex-

periment.

ACTIVITY 3. Library Research

Recent specialized research has given scientistsa clearer understanding of the photosyntheticprocess. Use your library's most recent booksand articles to find answers to the followingquestions:

1. How do we know there are two photosystems(P 680 and P 700)?

2. How was it decided where, in the sequenceof reactions, to place the various electroncarrier molecules?

3. How do we know that the light reactions ofphotosynthesis proceed much faster than thedark reactions?

4. What evidence supports the idea that aproton gradient drives the phosphorylation ofADP?

PROGRAM 4 I The Dark Reaction

ObjectivesAfter viewing this program, students should beable to do the following:

1. Explain how the availability of radioactiveisotopes was necessary before the dark reac-tions could be understood.

2. Explain the role of ribulose diphosphate as anacceptor of carbon dioxide.

3. Account specifically for the requirement ofATP and reduced NADP in photosynthesis.

4. Explain why both light reactions and darkreactions take place only in light.

5. Account, generally, for the fact that chloro-plasts never run short of ribulose diphosphateas photosynthesis proceeds.

Program DescriptionThe dark reactions of photosynthesis occur inthe stroma of the chloroplasts and use the ATPand reduced NADP generated by the lightreactions to join and reduce carbon dioxidemolecules. The direct products are carbohy-drates such as glucose, cellulose, and starch,but fats, oils, and the carbon skeletons of aminoacids (proteins) are also generated. Since light isnot directly used during these chemical altera-tions of carbon dioxide, the reactions are said tobe light-independent (or dark). However, sinceneither ATP nor reduced NADP is stored to anappreciable extent, the dark reactions take placeonly while light is supplied to the plant.

The complex series of reactions is called theCalvin cycle after Melvin Calvin who discoveredmost of the steps, using pulse-labelling tech-niques with the green alga, chlorella.

carbon compound that is broken immediately intotwo molecules of phosphoglyceric acid. Ribulosediphosphate is formed from ribulose phosphate,using energy and a phosphoryl group contrib-uted by ATP from the light reactions. Its energylost, ADP returns to the light reactions to acquireadditional energy.

Products of the light reaction are required againas each phosphoglyceric acid molecule isphosphorylated (using ATP) and reduced (usingreduced NADP. During the reduction, inorganicphosphate is split off so two molecules ofphosphoglyceraldehyde are produced in thiscentral series of reactions. These can, after oneof these molecules is slightly changed, formglucose phosphate or, ultimately, starch. Theseare usually considered the end products of thedark reactions. Most of the phosphoglyceralde-hyde, however, is used to regenerate ribulosephosphate by way of a complex series of inter-actions that completes the Calvin cycle. It takessix turns of the cycle to lead to a net gain in thecell of one glucose molecule.

Before-Viewing ActivitiesSummarize for the students the light reactions,specifically the output by them of reduced NADPand ATP. Recall the importance of these com-pounds as suppliers of energy to reduce carbonskeletons of molecules and to join carbon skele-tons in non-redox reactions respectively. Notethe large increase in potential energy and de-crease in entropy as carbon dioxide is fixed intosimple sugars, then into polysaccharldes. Alsonote the need of free energy to effect suchreactions.

The first product to appear after radioactivecarbon dioxide has been used is phosphoglyc-eric acid. This contains three carbon atoms.Plants that form this compound are, therefore,named C3 plants. The actual fixing of carbondioxide involves its attachment to a 5-carboncompound, ribulose diphosphate, forming a 6-

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After-Viewin Activities

ACTIVITY 1. Discussion of theCalvin Cycle

Discuss with the students the importance ofcyclical sequences of reactions and compare theCalvin cycle with the citric acid (Krebs) cycle ifthe latter has already been studied. To encour-age some in-depth study of the reactions of theCalvin cycle, encourage students to predictwhich carbon atom(s) of glucose would becometagged with radioactivity following exposure ofleaf cells to radioactive carbon dioxide. Theyshould, of couse, be able to explain how theyarrived at their answers. If styrofoam spheresand paint are available, have small groups ofstudents prepare models of the different com-pounds of the Calvin cycle and link the modelsinto a mobile to hang in the classroom.

ACTIVITY 2. Dramatizing Photosyn-thesis

In groups of six to eight students, write a play todepict the events of photosynthesis. It must bepossible to perform the play in ten minutes orless. Using a designated portion of the class-room for the set, arrange the furniture to repre-sent the interior of a leaf cell or a portion of it. Adrawing or map of your proposed stage set mustaccompany your script.

Prepare a cast of "chemical characters" andassign each member of your group to play oneor more of these characters. A person may beassigned many roles but can only appear onstage in one role at a time. Any chemical that isactively involved in photosynthesis is a validcharacter, but compounds that are essential butpassive should be part of the set. The more valid"characters" you include in your play, the morecredit you will get.

The script should include each character'sdialogue and directions as to positional changes,body motions, or gestures to accompany theli nes. The more frequently key scientific wordsdenoting processes, structures, or principles(e.g., reduction, thylakoid) appear in your script,the more credit you will receive.

Each group is responsible for submitting a stageset and script to the teacher by a deadline theteacher will announce. You will also be told ifand when you are to perform your play for therest of the class and whether or not to wearspecial costumes.

ACTIVITY 3. Starch Formation inthe Dark

Apparatus

Supplies to test leaves for starch (see Activity 3,Program 1 on page 3)

50 mL or 100 mL beakerWatch glass or other beaker coverMarking pencil50-mL graduated cylinderCork borerPotted geranium plant stored 24 hours in the

dark5% glucose solution

Method

1. Mark the beaker so you can identify if later.2. Place 20 mL of glucose solution into the

beaker.3. Obtain a fresh geranium leaf. Cut eight discs

from the leaf with a cork borer. Avoid areaswhere there are major veins.

4. Float the discs in the glucose solution. Fourshould float right side up and four upsidedown.

5. Cover the beaker and store it in a dark placefor a minimum of 24 hours.

6. After 24 hours, recover the discs, beingcareful to separate those that were floatedupside down from those that were floated rightside up.

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7. Test each disc for the presence of starch,following the instructions and observing theprecautions outlined for Activity 3, Program 1on page 4.

8. Record and interpret all observations.

Discussion

1. What "control" should have been performed?Why?

2. Why are leaf discs a better choice for thisexperiment than intact plants?

3. ATP is required to combine glucose moleculesinto starch molecules. How could leaf cellsgenerate ATP in the absence of light?

ACTIVITY 4. Research

I n the library, research the experiments of Calvinthat established the intermediates of the darkreactions of photosynthesis. Report on theseexperiments in an essay of about 300 words.I nclude sketches of illustrative chromatogramsand a discussion of the variety of techniques andapparatus used by Calvin or others working thesame field of research.

ACTIVITY 5. Review

1. Discuss the roles of ribulose, carbon dioxide,ATP, and reduced NADP in the dark reactionsof photosynthesis.

2. When a molecule changes chemically but itsformula is unchanged, the reaction is calledan isomerization. Discuss the importance ofisomerization in the dark reactions of photo-synthesis.

3. Explain why the determination of events in thedark reactions had to wait until the 1940s.

4. Experiments have revealed that the rate ofincorporation of radioactive carbon dioxideinto glucose by a plant is unchanged if arapidly blinking light source replaces a steadylight source of the same intensity and quality.This substitution reduces the rate at whichenergy is supplied. Explain this observation.

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Objectives

After viewing this program, students should beable to do the following:

1. Explain the low efficiency of photosynthesis inC3 plants.

2. Outline the differences in arrangements ofcells within the leaves of C3 and C4 plants.

3. Explain the biochemical basis for definingplants as C3 or C4.

4. Compare the chloroplasts of mesophyll cellsand bundle-sheath cells in C4 plants.

5. Explain the role of oxaloacetate, malate, andpyruvate in C4 plants.

6. Explain why C4 metabolism is advantageousin tropical, desert, or saline environments.

Program Description

The biochemical difference is accompanied byan anatomical difference in the arrangement ofphotosynthetic cells within the leaves. Theinterior of leaves is called the mesophyll. In mostC3 plants the mesophyll cells are arranged intoan upper palisade layer and a lower spongylayer. Cells in both layers contain similar chloro-plasts, although these are more numerous incells of the palisade layer. Within the spongylayer lie numerous small veins (vascularbundles) surrounded by nonphotosyntheticbundle-sheath cells. The mesophyll cells of C4plants form rings around the bundle-sheath cells,and the bundle-sheath cells contain chloroplasts.However, the chloroplasts of bundle-sheath cellshave few, poorly developed grana and cannotparticipate in the light reactions. The light reac-tions of such plants are carried out, as youwould expect, in the thylakoids of mesophyllcells.

Comparing the energy stored by plants using theCalvin cycle with the quantity of energyabsorbed in the light reactions reveals an im-pressive theoretical efficiency of 38%. In reality,however, the plants operate at an efficiency ofless than 1%. This is because of the nature ofthe enzyme that is supposed to join carbondioxide to ribulose diphosphate. This enzymealso has an affinity for molecular oxygen, and ifoxygen is combined with ribulose diphosphaterather than carbon dioxide, the ribulosediphosphate enters a series of reactions thateffectively removes it from the Calvin cycle.Since oxygen is much more abundant in air thancarbon dioxide (21 % vs. 0.035%), a substantialloss of ribulose diphosphate occurs in plantsrelying on the Calvin cycle to fix carbon dioxide.

The problem has been avoided by some plantsin tropical or desert areas in which radioactivecarbon supplied to the plant in carbon dioxideappears first in four-carbon compounds ratherthan in the three-carbon compound,phosphoglyceraldehyde. Such plants are calledC4 plants.

The difference lies in the dark reactions which, inC4 plants, require the participation of bothmesophyll and bundle-sheath cells. The twotypes of cells are close and have bridges ofcytoplasm between them, so materials can bepassed back and forth easily. Carbon dioxideenters the mesophyll cells and is incorporatedinto oxaloacetate, which is reduced to malateusing reduced NADP. The malate passes intobundle-sheath cells where it is used to reduceNADP and, as it does so, breaks into the three-carbon compound, pyruvate, and carbondioxide. It is this carbon dioxide that enters theCalvin cycle. The Calvin cycle is restricted to thebundle-sheath cells where there is abundantcarbon dioxide (from decomposition of malate)and little oxygen (because light reactions are notoccurring there). Thus, loss of ribulosediphosphate is minimized and efficiency ofphotosynthesis is increased. Such an adaptationis especially useful in hot, dry areas, since plantsin such areas to retain moisture must, for muchof the day, close down the stomata that allowgases to diffuse in and out of the leaves. Undersuch conditions, carbon dioxide concentrationsdecrease rapidly in the mesophyll cells andoxygen concentrations increase. Therefore, the

PROGRAM 5 / C3 and C4 Plants

1 6

C4 strategy of using mesophyll cells to effec-tively "pump" carbon dioxide into the oxygen-deficient chloroplasts of the bundle-sheath cellsis of great adaptive value.

Before-Viewing Activities

1. Discuss enzyme specificity and the possibilityof there being two equivalent substrates forthe same site. Following logically is a discus-sion and predictions about competitive inhibi-tion of enzymes (interaction of 0 2 or C02 withribulose diphosphate carboxylase).

2. Review with students the concepts of theprevious two programs, especially the releaseof molecular oxygen by the light reactions(photolysis) and the entry of carbon dioxideinto the dark reactions via combination withribulose diphosphate.

After-Viewing Activities

ACTIVITY 1. Further Discussion

Crop plants using C4 metabolism (e.g., corn andrice) are very efficient energy converters. Couldethanol from corn have an increased use as asource of energy for transportation? Why hassuch a high-efficiency process that has beenaround long enough for many plant families toadopt it not displaced C3 metabolism throughevolution? Seek answers by reading further inthe area of photo respiration.

ACTIVITY 2. Microscope Study

Apparatus

Compound microscopePrepared slide: cross-section of Syringia leaf

(lilac)Prepared slide: cross-section of Zea leaf (corn)

Method

1. Draw a strip from the top to the bottom of eachleaf, including a small vessel in each drawing.Be sure each drawing is the same scale andhas a title accompanying it.

2. In each epidermal layer, look for a site wheregases and vapors could be exchanged withthe environment. If you find such a site, in-clude it in the appropriate location of yourdrawing, even if it was not visible in the stripyou originally chose.

3. Look carefully for chloroplasts within each celltype in your drawings. Note that the slides arestained artificially so cell parts will not neces-sarily have the color you intuitively expect. Forexample, chloroplasts are often stained red incommercial preparations. Add chloroplasts ofappropriate size, shape, and location to thecells of your drawing that are found to containchloroplasts.

4. Label the leaf parts with which you are familiaron each drawing.

5. On each drawing, superimpose color-codedarrows to indicate the following pathwayswithin the leaf:a) Carbon dioxide from air to site of incorpora-tion in glucose.b) Oxygen from release point to air.c) Liquid water from plant reserves to photoly-sis site.d) Water vapor being transpired.I nclude a legend of color codes on the samesheet(s) as your drawings.

Discussion

Which plant(s) in the study is/are C3? Which is/are C4? Justify your answers.

ACTIVITY 3. Research

A third type of adaptation to extreme habitats isthat of "Crassulacean metabolism." Researchthis type of adaptation and write a 250-wordreport on it.

1 7

ACTIVITY 4. Dramatizing Photosyn-thesis (Act 2)

Using the same guidelines as in the chapter onProgram 4 (see page 14), write a second act toyour photosynthesis play, entitled "Variations ona Theme." Keep it short - no longer than fiveminutes (excluding time spent in shiftingscenery). Act 2 should address both the C4 andCrassulacean adaptations.

ACTIVITY 5. Review

1. Explain why photosynthesis fails to operate atits theoretical efficiency.

2. In C4 metabolism, why are cytoplasmicbridges (plasmodesmata) between mesophylland bundle-sheath cells important?

3. Why is C4 metabolism more useful in aridthan in moist habitats?

4. If a palaeontologist were to find the fossil leafof an extinct plant, how might he or she deter-mine if the plant had been C3 or C4?

5. Anthropologists can use the ratio betweencarbon-13 (an isotope formed in the atmos-phere by cosmic rays) and carbon-12 (normalisotope) in ancient bones to discover if, whenalive, those people had based their agricultureon C3 or C4 plants. Suggest a reason thatsuch determinations may be possible.

18

PROGRAM 6 / The Fluid-Transport System

Objectives

After viewing this program, students should beable to do the following:

1. Define transpiration and relate it to xylemtransport.

2. Discuss the role of stomata in the exchange ofgases and vapors between the leaf's interiorand the environment.

3. Appreciate the difficulty of raising water to thetop of a tree against the pull of gravity.

4. Relate the structure of a water molecule to thecohesive and adhesive properties of water.

5. Explain how water enters the roots of a plant.6. Outline the active processes upon which

phloem transport depends.

Program Description

As plants evolved, some grew to great heightsand gained a significant advantage in competingwith other plants for light. However, the greatseparation between the leaves and the source ofminerals and water (soil) necessitated theevolution of efficient transport systems.

The exchange of gases between the leaf interiorand the surrounding atmosphere that is neces-sary for photosynthesis is inevitably accompa-nied by loss of water vapor to the air. Thisprocess, called transpiration, is also useful to theplant, as it provides the energy to lift water fromthe roots to the highest branch. Some mineralsaccompany the water and become available formaintenance of leaf tissue. Water and someminerals travel through columns of intercon-nected, dead cells that make up the xylemtissue. This tissue also imparts tensile strengthto the aerial parts of the plant.

Within the xylem tissues, as in all areas of theplant, water molecules interact in importantways. Water molecules are polar - each pos-sesses a permanent, asymmetric separation ofpositive and negative charge. This means thatwater molecules can cling firmly together, a

process called cohesion. Also, they may cling toother polar molecules or the polar hydroxylgroups of macromolecules such as the cellulosethat comprises the cell wall. This process iscalled adhesion. Cohesion and adhesion enablewater to be lifted to great heights through thexylem tissue. Adhesion serves to "anchor" thetop of a water column in the leaf, and cohesionbinds the water column together with a strengthapproaching that of a steel wire of the samediameter. As water evaporates in the leaf, adhe-sion of water to cellulose fibres in the leaf cellserves to pull the column upwards to replace theevaporated water.

I nitially, water enters roots by osmosis. Theconditions for osmosis are established by theactive uptake of mineral ions from the soil. Thesupply of minerals is ensured because the plantis constantly exploring new areas of soil bygrowing and forming new root hairs. The ions areoften associated with soil colloids, but can bedislodged by replacing them, on the colloids, withhydrogen ions generated by the intermediarymetabolism of root cells.

Similar processes are involved in phloem trans-port. Active transport of organic molecules (orcertain inorganic ions) into phloem cells causeswater to enter these phloem cells by osmosis,with a consequent increase in phloem cell turgor.Elsewhere, in the same column of phloem cells,active unloading of solutes leads to a decreasein turgor. The result is a pressure gradient withinthe column of phloem cells from the point ofsolute uptake to the point of solute release. Thiscauses mass flow of phloem cell contents alongthe gradient. The flow is facilitated by perfora-tions in the walls between neighboring cells inthe column. The phloem cells even contain aprotein that causes "clotting," if the columnshould be injured.

1 9

Before-Viewing Activities

1. Review with the students the concepts ofactive transport and osmosis, and the struc-ture of the leaf (Program 5, Activity 2).

2. Discuss the expected rates of diffusion ofcarbon dioxide and water into and out of theleaf. Highlight the fact that water can beexpected to diffuse out of a leaf much morerapidly than carbon dioxide will diffuse into theleaf, under most circumstances (steeperconcentration gradient, less massive mole-cules). As a result, some water loss inevitablyaccompanies photosynthesis.

3. Discuss the importance of the plant's conduct-ing and gas exchange systems in enabling theleaves to photosynthesize effectively.

After-Viewing Activities

ACTIVITY 1. Investigating WaterTransport in Plants

PART B: Observing Plant VesselsPrepare a dichotomous key that could be usedto classify the cell types found in stewed rhu-barb. A few drops of glycerol should be added toeach preparation, and staining of lignin withphloroglucinol could be attempted.

Note to the teacher: You might want to set up apotometer, using the top portion of the plant de-capitated in Activity 2. However, to demonstratethe lifting power of the aerial portions of plants,mercury has to be used as the working fluid.Mercury fumes are toxic and many jurisdictionsprohibit the use of mercury in schools. If youdecide to demonstrate this activity, be sure themercury is contained in such a way that it cannotvaporize into the air. Also, be certain that thewater used between the plant and the mercuryhas been boiled to free it of dissolved air. Other-wise, air bubbles may develop in the watercolumn under tension and break its cohesive-ness.

ACTIVITY 2. Locating Plant VesselsApparatus

Apparatus

Two microscope slidesMotor oilCover glassStewed rhubarbCompound microscopeGlycerolPhloroglucinol

250-mL beakerFresh celeryWater darkly colored with red ink or eosinScalpel or single-edged razor bladeFine forcepsCompound microscopeTwo microscope slides with cover glasses

MethodMethod

PART A: Comparing the Cohesive andAdhesive Power of Water with that of aNonpolar SubstanceUsing a pair of plain microscope slides, place adrop of water on one slide and press the otheron top. Note how difficult it is to separate theslides unless you slide one over the other. Nowput a drop of motor oil (nonpolar) on one slideinstead of water and note how easily the slidesare separated.

1. Twenty-four hours before the investigation,place a fresh celery stalk into a beaker ofdarkly colored water.

2. Note the distribution of red stain in the stalk.Describe its apparent distribution.

3. Cut a thin section of the stalk (petiole) from anarea to which color has extended. Prepare awet mount of the section and examine it withthe microscope. Sketch the cross-section andindicate the location(s) of vessels transportingwater.

20

4. Dissect from the stalk, a longitudinal section ofthe vascular area. Prepare a wet mount of thevessels, examine it with the microscope, andsketch the arrangement of the cells within avessel carrying water.

ACTIVITY 3. Can Roots Push WaterUpwards?

4. Secure the capillary tubing in an upwardposition, using the burette clamp and placethe entire apparatus aside in a plant-growingarea for several days.

5. Note and account for any changes in the levelof fluid within the capillary tubing. How wouldthe level be affected if the plant were'watered" with 1 percent salt solution?

Apparatus

Healthy potted plant (e.g., tomato)1-metre capillary tubingPetroleum jelly3-cm rubber tubing (approximately)Hose clamp and screwdriverRetort stand with burette clamp

Method

1. Cut aerial portions from plant, leaving one totwo cm of stem projecting above the soil.

2. Coat the stem lightly with petroleum jelly andfasten one end of the rubber tubing over it,using a hose clamp to secure it firmly.

3. Insert the capillary tubing into the other end ofthe rubber tubing.

ACTIVITY 4. How Rapidly Do PlantsTranspire?

Apparatus

Volumeter consisting of side-arm test tube, 1 mLpipette (or graduated capillary tube), andsyringe as shown in Fig. 6-1 (syringe needleshould be sealed in place in the first hole ofthe two-holed stopper, using epoxy cement)

PlasticeneStopwatchPlant cutting, ideally with woody stem (e.g., top

of plant from Activity 2, white pine cutting, orleather fern from florist)

Method

plant cutting

Fig. 6-1:,Transpiration Apparatus

1-mL pipette(or graduated capillary tube)

ubber tubing

side-arm test tube

1. Prepare the plant cutting for the experiment bysnipping the terminal inch or two of stem fromit, while holding the stem end immersed inwater.

2. Secure the cutting in the second hole of therubber stopper using plasticene.

3. Fill the side-arm tube with tap water and insertstopper firmly. Excess water should be forcedout around the stopper and the end of thepipette.

4. The syringe should be filled halfway withwater and attached to the delivery needle.This can be used to adjust the level of water inthe pipette.

5. Use the gradations on the pipette and a stop-watch to calculate the rate of transpiration ofthe cutting. Several environmental conditionscan be tried, such as different light exposures,humidities, temperatures, or wind speeds.

2 1

Duplicate determinations should be madeunder each environmental condition, if timepermits.

6. Account for any variations noted in transpira-tion rates.

ACTIVITY 5. Leaf Impressions

Caution: This experiment should onlye performed In well-ventilated areas

or outdoors. Avoid breathing ethylacetate fumes.

which the stomata are opened by sketching atypical stoma/guard cell complex, as revealedin the replica.

4. Draw conclusions based on your data. De-pending on the nature of your replica collec-tion, you could compare plant species fromexposed habitats with those from shadedhabitats, the same species from exposed andshaded habitats, or the same plant at differenttimes of day.

ACTIVITY 6. Review

Apparatus

Ethyl acetate in drop-dispenser bottlePlastic cover slips or 2-cm squares cut from

overhead transparency acetateSmall paper envelopes e.g., philatelists's enve-

lopes15-cm rulerCompound microscope

Method

Choose various plants growing in differenthabitats or the same plant at different times ofday as your subject of investigation. Examinethe density of stomata and degree of stomatalopening by making replicas of their surfaces.For each of the surfaces of the leaf in turn,place one drop of ethyl acetate on the surfaceand press a plastic square firmly against theleaf surface over the drop for one minute.Then peel away the square and store thesquare in an appropriately labelled envelope.This should be done outdoors, with the leaf re-maining attached to the plant throughout theprocedure.

2. Store each replica in its own envelope andkeep clear records of the plant/leaf surface/habitat/time.

3. In the laboratory, determine the density ofstomata in each replica, using the ruler andcompound microscope. Indicate the degree to

1. Why do horticulturalists recommend that somebranches be pruned from a plant followingtransplantation?

2. Explain how a plant can be killed either byoverwatering or by overfertilizing.

3. Why should you cut 2 cm or so of stalk awayfrom cut flowers, while holding the cut endsimmersed in water, before you place theflowers in a vase for display?

4. Split the cut end of a stem of a white carnationin half longitudinally and place one-half inwater stained with a blue dye and the otherhalf in water stained red. Predict how the blos-som will be colored after a few hours. Justifyyour prediction.

5. Is there any practical limit to how tall a treecan grow? Explain your reasoning.

6. Does xylem transport in a plant stop when theair around the plant has a relative humidity of100 percent? Explain the experimental proce-dure you would use to try to answer this ques-tion.

22

Bibliography

Amon, D.I. "The Role of Light inPhotosynthesis." Scientific American, Novem-ber 1960.

Bassham, J.A. "The Path of Carbon inPhotosynthesis." Scientific American, June1962.

Bjorkman, O. and J. Berry. "High EfficiencyPhotosynthesis." Scientific American, October1973.

Galbraith, D.I. et al. Biology. Principles, Patterns,and Processes. Toronto: John Wiley & Sons,1989. Forthcoming.

Govindjee, R. "The Primary Events ofPhotosynthesis." Scientific American, Decem-ber 1974.

Hinkle, P.C. and R.E. McCarty. "How Cells MakeATP." Scientific American, March 1978.

Kimball, J.W. Biology. Reading, Mass.: Addison-Wesley, 1983.

Levine, R.P. "The Mechanism ofPhotosynthesis." Scientific American, Decem-ber 1969.

McCarty, R.E. "H+-ATPases in Oxidative andPhotosynthetic Phosphorylation." BioScience,January 1985.

Miller, K.R. "The Photosynthetic Membrane."Scientific American, October 1979.

Prince, R.C. "Redox-Driven Proton Gradients."BioScience, January 1985.

Salisbury, F.B. and C.W. Ross Plant Physiology,Belmont, Calif.: Wadsworth, 1985.

Whittingham, C.P. "Photosynthesis," CarolinaBiology Reader. Burlington, N.C.: CarolinaBiological Supply Co., 1977.

Youvan, D.C. and B.L. Marrs. "Molecular Mecha-nisms of Photosynthesis." Scientific American,June 1987.

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