CHAPTER II

PART A INTRODUCTION TO THE PHOTOSYNTHETIC SYSTEM

PART B : REVIEW OF LITERATURE

PART A INTRODUCTION TO THE PHOTOSYNTHETIC SYSTEM

Photosynthesis is a metabolic process by which all

photoautotrophic organisms i. e. photosynthetic bacteria,

cyanobacteria and higher plants are able to convert light

energy into chemical enegry in the form of carbohydrates.

During oxygenic photosynthesis, in cyanobacteria and higher

plants, light energy is utilized to transport electrons from

water to NADP+ with concomitant evolution of oxygen. The ATP

and NADPH generated during this light driven process are

subsequently used for enzymatic conversion of atmospheric C02

to carbohydrates. Thus, the overall reaction carried out during

photosynthesis may considered to be

The site of the light reactions of photosynthesis 1S the

thylakoid membranes.

2A.l. LIGHT HARVESTING, ENERGY TRANSFER AND PHOTOCHEMISTRY

All photosynthetic pigments i.e. chlorophyll,

phycocyanobilin, carotenes etc. have an extended array of

conjugated bonds. This allows them to interact with and

efficiently absorb electromagnetic radiation in the visible

range. The spectral qualities of these pigments are affected

not only by their chemical composition but also by the protein

milieu around them. Typically, the absorption bands of the

pigments complexed with the proteins are broadened due to

further splitting of energy levels. This leads to a more

5

eff icient harvesting of the light energy. In case of

chlorophyll, absorption of a quantum of light generates a

singlet excited state. Formation of triplet state has a low

probability compared to other systems because photochemistry

effectively competes with the triplet state formation.

An individual chlorophyll molecule absorbs merely 2-3

photons/sec even under direct solar illumination. No living

organism could grow if their reaction centers did not have

light harvesting antenna molecules in excess. As early as

1930s, Emerson and Arnold (1932) demonstrated that hundreds of

chlorophyll molecules cooperate to harvest solar energy during

photosynthesis. However, the ability to undergo charge

separation is endowed on ·certain specialized Chl molecules

present in the reaction centers. Hence, it is important for

the energy absorbed by each· of the chlorophyll molecules to

reach the reaction center. Duysens '(1952) demonstrated that

the absorbed energy migrates between any two chromophores to a

considerable extent via the lowest excited singlet state. Such

intermolecular energy transfers were shown for the following

cases :

1. Between accessory pigments (phycobilins) in red and blue

green algae.

2. From accessory pigments ( ChI b, phycobilins, carotenoids)

to ChI a

3. Between different spectral forms of ChI a

Forster (1948, 1949) has postulated a theory to

explain the phenomenon of intermolecular singlet excited state

transfer. The theory is applicable for energy transfer between

a pair of molecules one of which is excited, their absorption

and fluorescence spectra are smooth and wide allowing

considerable overlap, the interaction energy is very small and

6

the distance between the two molecules is much greater than the

dipole lengths. In case of chlorophylls, optimal conditions

prevail for Forster type of energy transfer and it is believed

to be the primary mechanism by which excitation energy migrates

from antenna to reaction centers (van Grondelle and Amesz,

1986). For two molecules about 20 AO apart, the rate of energy

transfer is _ 5 x 1011 sec-1 . In general, where f r f is the

distance between interacting molecules

The efficiency of utilization of the absorbed

radiation by photochemistry has been suggested to be more than

90% (Bj orkman and Demming f 1987). Two other major routes of

deexcitation that compete with photochemistry are fluorescence

and thermal deactivation. While the quantum yield of

fluorescence from ChI a in ether is about 30% f the maximum

yield in the photosynthetic apparatus is only about 3% when the

reaction centers are closed. The value is only 0.6% when the

photochemistry is fully operative. such a high efficiency of

utilization of the absorbed energy has been achieved in the

reaction centers due to the protein environment in the RC which

accomplishes the following tasks :

1. It. alters the spectral quality of the ChI which has to

undergo charge separation. This pigment absorbs radiation

at a wavelength higher than the antenna ChI and thus acts as

an efficient trap for any exciton that reaches it. The

probability of back transfer of excitation to the antenna is

minimized.

2. The RC polypeptides bind- the cofactors like pheophytin, and

specialized quinones QAf QB etc. in appropriate conformations

such that the excited ChI molecule is soon oxidized and returns

7

to the ground state. The charges thus separated are stabilized

by the vectorial displacement of the charge ~ollowing

sequential reduction and oxidation of the cofactors. This

migration of the charge rather than the charged species itself,

takes place on the order of a few picoseconds. Apart from

stabilizing the charge, it also renders the RC 'open' and ready

to undergo another photoact. The lifetime of an exciton

reaching the RC is about 3 ps (Wasielewski et al., 1989). Such

a high rate for the charge separation prevents backtransfer of

the exciton from the RC to the antenna.

Thus, the protein milieu around the photosynthetic

pigments makes the process of light parvesting and energy

utilization extremely efficient. The two other major routes of

deexcitation besides photochemistry are thermal relaxation and

fluorescence. The quantum yield of photochemistry (0p ) is given

by,

Where, Kp

KD

Kp

Kp

0p =

= = =

Kp + KO + Kp

rate constant for photochemistry

rate constant for thermal relaxation

rate constant for fluorescence

The fact that fluorescence and photochemistry compete

with each other for deexcitation, makes the former a very

useful tool to probe and evaluate the photochemical process

under various conditions.

8

2A.2. PHOTOSYNTHETIC ELECTRON TRANSPORT

In cyanobacteria and chloroplasts of higher plants,

light is absorbed by the pigments present in the antennae of

photosystem I and II. The excitation energy transferred to

the reaction center of the photosystems initiates charge

separation. Secondary electron flow through the photosynthetic

electron transport chain stabilizes the charge separated state.

Electron flow from water, the ultimate electron donor to NADP+

results in the generation of a transthylakoid electrochemical

potential difference for protons. This gradient is eventually

utilized for phosphorylation of ADP. The ATP and NADPH thus

generated are used for C02 fixation. Thus, the

photosynthesis comprises two distinct phases.

process of

The light

reactions carried out in the thylakoids are involved in capture

of light energy and its conversion to ATP and reducing power.

The dark reactions are purely enzymatic in nature and utilize

the energy generated by the light reactions to convert C02 to

carbohydrates. These reactions are carried out in the stroma

of the chloroplast.

The transport of electrons from water to NADP+ is

mediated by a number of proteins, pigment protein complexes and

other organic molecules which function in a highly coordinated

manner. The complex set of reactions has been described in the

Z scheme of electron transport (Fig. 2.1) proposed initially by

Hill and Bendall (1960). The hallmarks of the Z scheme are,

utilization of electromagnetic energy at two sites i.e. PS I

and PS II.

- Transport of electrons in a non cyclic manner from H20 to NADP+

- Generation of ATP and NADPH

9

hv

FIGURE 2. l- An updated version of the Z-scheme of photosynthetic electron transport showing the lifetimes of various electron transfer reactions (after Govindjee and Eaton-Rye, 1986)

In addition to the non-cyclic electron transport, the

photosynthetic apparatus of cyanobacteria is also known to

carry out light mediated cyclic transport of electrons around

PS I (Fig 2.2). Such an electron transport does generate

transthylakoid pH gradient (and subsequently ATP) but is unable

to generate reducing power in the form of NADPH.

The site for the various reactions of photosynthetic

electron transport is the thylakoid membranes present inside a

specialized organelle, the chloroplast, in higher plants. They

are organized into stacked (grana) and unstacked regions.

However, cyanobacteria being prokaryotic do not have any such

organelles and the thylakoidmembranes are dispersed inside the

cell. Further, there is no organization of the thylakoid

membranes into grana I and stromal lamellae. Thylakoid membranes

of cyanobacteria are also the site for respiratory activity;

the two electron transport systems interacting closely as some

of the components are common between them (Scherer et al., 1988; Scherer, 1990». The various supramolecular complexes

involved in the electron transport in cyanobacterial thylakoids

are : photosystem II, photosystem I, cytochrome b6/f complex,

ATPase and the phycobilisomes (light harvesting pigment protein

complexes). In addition to these, two mobile electron carriers

are also present, namely, plastoquinone and plastocyanin.

2A.2.1 Photosystem II

This multisubunit pigment protein complex acts as the

water plastoquinol oxidoreductase catalysing the reaction,

10

+ NADPH NADP

I cyt b6/f I f

cytaa~ 4-cytcss3/PC-- ~oo

FIGURE 2.2. Functional interaction of respiratory and photosynthetic electron transport in the cyanobacterium Anabaena variabilis. The lredox components common to the two chains are emboldened. P680, reaction centre of PS II;

P700, reaction centre ~Of PS I; Cyt, cytochrome; PC, plastocyanin; DH, dehy rogenase; fd, ferredoxin; FNR, ferredoxin NADP oxidoredu tase. (Adapted from Scherer, 1990)

Des.pite a considerable evolutionary distance between

cyanobacteria and higher plants, the PS II complex of the two

groups is very similar. More than 22 polypeptides are known to

constitute PS II (see Hansson and Wydryzinsky, 1990; Vermaas

and Ikeuchi, 1991 for reviews). Functional roles have been

assigned to some of them (Table 2.1) while others are presently

thought to be important only from a structural point of view.

The reaction center from purple bacteria has been crystallized

and its detailed structure known (Deisenhoefer and Michel,

1989). This has proven to be a very useful model for PS'II.

The analogy between these two reaction centers ·has been

demonstrated using site directed mutagenesis (Debus et al., 1988a; Debus et al., 1988b; Vermaas et al., 1988b) and

spectroscopic techniques (Schatz and' van Gorkom, 1985;

Rutherford and Zimmerman, 1984). Based on the function, the

organization of PS II (Fig 2.3) may be considered as follows:

1) REACTION CENTER - The minimum unit capable of a stable

photo induced charge separation has been isolated by Nanba and

Satoh (1987) from spinach and by Barber et al., (1987) from

pea. Synechocystis 6803 was the first cyanobacterium from which

the reaction center was isolated (Gounaris et al., 1989). It

comprises a heterodimer of closely related polypeptides 01

and 02 in addition to one.cytochrome b559 haem iron, 8 ChI a

molecules and one pheophytin a molecule. The two polypeptides

01 and 02 provide the protein environment within their core

such that P680 (a specialized ChI dimer) is able to undergo

photooxidation. 01 and 02 also bind other cofactors like

pheophytin, QA, QB etc. in appropriate orientation to effect

stabilization of charge separated state. Many of the known

herbicides like DCMU, atrazine etc. interfere with QB binding

by interacting with the 01 polypeptide (Tischer and Strotmann,

1977; Trebst, 1986).

11

•

TABLE 2.1

NAME

D1

CP47-P5

CP43-P6

D2

Cyt b559

Cyt b559

10 kOa

phosphoprotein

I polypeptide

K polypeptide

L polypeptide

M polypeptide

N polypeptide

OEE1

OEE2

OEE3

10 kOa nuclear protein

Summary of PS II polypeptides

ENCODED BY GENE

psb A

psb S

psb C

psb D

psb E

psb F

psb H

psb I

psb K

psb L

psb M

psb N

psb 0

psb P

psb Q

psb R

SIZE (kDa)

32

47-51

43-47

34

9

4

7.6

4.2

4.3

4.3

3.8

4.7

33

23

16

10

DESCRIPTION

RC core, binds QS' Pheo, special pair Chl, Mn (?)

Chl a binding inner antenna RC core complex

Chl a binding inner antenna RC core complex

RC core, binds QA' Pheo, Chl special pair, Mn (?)

RC Core

RC Core

PS II particle, affects 01/D2 dimer conformation

RC core

Present in PS II core complex, absent in purified O2 evolving preparations

PS II oxygen evolving particles

PS II oxygen evolving core

PS II oxygen evolving core

O2 evolving core, Mn stabilizing OEC component

OEC component

OEC component

Regulatory OEC component

1. psb P, psb Q and psb R products are present only in eukaryotes 2. PS and P6 are apoproteins, CP47 and CP43 are the respective

chlorophyll protein complexes (Adapted from Erickson and Rochaix, 1992)

stroma CP43 02 01

CP47

Pheo

\

lumen

FIGURE 2.3. Schema_tic model of the PS I I 'core' complex from cyanobacteria. The PS II reaction centre environment is created by the 01 (PSII-A) and 02 (PSII-B) proteins which provide the binding environment for the reaction centre chlorophyll P680, pheophytin (Pheo), the plastoqu inones QA and QB and a non heme iron. In addition, 01 and 02 contain redox-active Tyr residues, Z and 0 respectively, and are assumed to provide ligands to Mn involved in water splitting. Also involved in water splitting, but presumably not providing ligands to Mn, is the manganese stabilizing protein (MSP). Two small proteins, Cyt b559 and PSI I-I are closely associated with 01 and 02. CP43 and CP47 represent chlorophyll binding proteins serving as light harvesting antennae ('core antenna') for the PS II reaction centre. A large number of small proteins of obscure function associated with the PS I I complex have been omitted from this figure (Adapted from Vermaas and Ikeuchi, 1991).

2) REGULATORY CAP - The unique feature of PS II as a reaction

center is its ability to evolve 02 by oxidizing H20. In

cyanobacteria, a 33 kDa polypeptide is involved in stabilizing

the manganese atoms necessary for splitting of H20 and is hence

called the manganese stabilizing polypeptide (MSP). In addition

to MSP, higher plants have a 18kDa and 24kDa polypeptides as a

part of the water oxidizing complex located on the lumenal side

of PS II. Since these polypeptides also play a role in

regulating the ionic requirements for 02 evolution, together

they have been termed as the regulatory cap (Hansson and

Wydryzinsky, 1990).

3) PROXIMAL ANTENNA : Two polypeptides, called CP47 and CP4],

constitute the proximal antenna for PS II reaction center. The

exact number of ChI a molecules bound by these polypeptides has

not been clearly established yet. The estimates of ChI a

molecules per CP47 polypeptide range from 6-11 (Tang and Satoh,

1984) to 20-30 (Yamaguchi et al., 1988). For CP43, the number

of Chl a molecules has been reported to be 11 (Akabori et al., 1988) and 26 (Yamaguchi et al., 1988). These ChI a molecules

harvest light energy and transfer it to the reaction center.

They are also supposed to be involved in the energy transfer

from light harvesting complex (phycobilisomes or LHCP II) to

the reaction center. Recent studies suggest the role of CP43

and CP47 in assembly of PS II (Vermaas et al., 1988 a).

Cyanobacterial mutants in which CP43 has been impaired due to

mutation of psb C gene, cannot grow photoautotrophically.

However, the thylakoids from such mutants of Synechocystis 6803 do support light induced reduction of DCIP in presence of

exogenous electron donors like DPe. At this stage, it is not

possible to rule out the involvement of these polypeptides in

the functioning of PS II mediated electron transport.

12

2A.2.2 Plastoquinone

Plastoquinone is a mobile electron carrier shuttling

between PS II and the Cyt b6/f complex (Anderson, 1980; Millner

and Barber, 1984; Whitmarsh, 1986). It is a relatively small

molecule consisting of a quinone ring with two methyl groups

and an isoprenoid chain. The size of the PQ pool has been

estimated to be between 7 (in the cyanobacter ium Anabaena

variabilis) and 40 (in chloroplasts and Anacystis nidulans)

molecules per P700 (Hauska and Hurt, 1982). Each quinone

molecule transfers two electrons from PS II to Cyt b6/f complex

during a reduction-o¥idation reaction. This is accompanied by

uptake of two H+ ions from the stroma (during reduction at.

PS II) and release of the same to the lumen (during oxidation

by Cyt b6/f complex). The flux of electron through the PQ pool

seems to be the rate limiting step in photosynthetic electron

transport. In cyanobacterial thylakoids, PQ pool is also a

component of the respiratory electron transport (see Scherer,

1990) .

2A.2.3 cytochrome b6/f

This complex functions as a membrane embedded

plastoquinone plastocyanin oxidoreductase catalysing the

reaction,

PQH2 + 2PCox ---> PQ + 2PCred + 2H+

The complex is homologous to the Cyt b-c complex of

mitochondria involved in respiratory electron transport chain.

with Cyt b-c complex, it has significant but not complete

homology. There are four polypeptides i.e. Cyt f (33 kDa) , Cyt

b6 (23 kDa) , Rieske protein (20 kDa) and subunit IV (17 kDa).

13

The stoichiometry of heam b : heam C : [2 Fe - 2 S) center is

2: 1: 1. The two he am groups are coordinated within a single

polypeptide.

Cyt b6/ f complex has also been considered as a

junction point for the two electron transport pathways,

photosynthetic and respiratory, in cyanobacteria where the

thylakoids are also a site for respira~ion. The unique aspect

of the electron transport network of Cyt b6/f complex is the

possibility of feed back loops involving electron donation from

ferredoxin during cyclic electron flow around PS I (Cramer et

al., 1987) and oxidation of cytoplasmic electron donors such as

sulphide during anoxygenic photosynthesis in cyanobacteria

(Shahak et al., 1987).

2A.2.4 Electron Donors to PS I Plastocyanin and Cyt c553

Plastocyanin, a copper containing peripheral membrane

protein (10.5 kDa) is localized' on the lumenal surface of the

thylakoids (Katoh, 1977; Haehnel, 1980) and mediates the

electron transport between Cyt b6/f complex and PS I. However,

in many cyanobacteria, the ro~e of plastocyanin is taken over

by Cyt c553 (10-12 kDa). Some cyanobacteria also possess the

ability to synthesize both plastocyanin and Cyt c553 and

regulate their amounts depending on the availability of iron

and copper (Bricker et al., 1986).

2A.2.5 Photosystem I

PS I catalyses the photochemical reduction of

ferredoxin using reduced plastocyanin as the source of

electrons.

14

FdOX + PCred + hv -> Fdred + PCOX

studies on PS I complexes isolated from organisms belonging to

diverse groups revealed that the composition as well as

biochemical, biophysical and immunological properties of PS I

have been highly conserved during evolution (Bruce and Malkin,

1988a; 1988b; Lundell et al., 1985; Nechustai et al., 1983 and

1985) .

The PS I core complex contains photoxidizable ChI

P700, about 100 molecules of ChI a as antenna, 12 to 16

molecules of ~ carotene, 2 molecules of phylloquinone and three

4Fe-4S centers (see Bryant, -1992; Golbeck, 1992 for reviews).

More than a dozen polypeptides are involved in maintaining the

structure and function of PS I (Fig. 2.4). Two of these with a

molecular weight of _ 70 kDa each on the SDS-PAGE comprise a

heterodimer which binds the entire chlorophyll present in the

PS I core complex along with all the cofactors i.e. P700, Ao '

A1' 4Fe-4S clusters etc. (see Golbeck, 1992 for a review). The

functions known or postulated for other polypeptides are given

in Table 2.2. In case of higher plants, additional chlorophyll

binding proteins are present organized as LHCP I.

In PS I, charge separation

photooxidation of the pigment P700 which

starts

is a

wi th the

ChI a dimer

(Norris et al., 1971; Golbeck and Bryant, 1991). The electron

moves sequentially through Ao , shown to be a ChI a molecul~

(Shuvalov et al., 1986), and A1, a vitamin K molecule, to the

first 4Fe-4S cluster Fx. From Fx the electron is transferred to

two other iron-sulphur clusters, shown to be 4Fe-4S by Scheller

et al., (1989) and Petrouleas et al., (1989), coordinated on

subunit C on the stromal side of PS I. Finally ferredoxin (Fd)

takes over the electron for transport to NADP+ reductase .

. 15

STI{O;\IA

I PsaA XJ.2-kl>a

--------"----------LUMEN

PsaE

I'saB 82.S-kDa

I'saL

FIGURE 2.4. The architecture for the PS I reaction centre (adapted from Golbeck, 1992). The symbols are described in

Table 2.2.

TABLE 2.2 Components of PS I Core Complex

GENE MASS COFACTORS NUMBER FUNCTION/PROPERTIES

psa A 83.0 ChI a -100 Antenna chlorophyll and photochemical charge separation

psa B 82.4 Quinone 2 Charge stabilization [4Fe-4S) 1 Charge stabilization

f3 carotene 12-16 Antenna and photoprotection

psa I 4.0 ? Transmembrane helix

psa J 5.1 ? Transmembrane helix

psa K 8.4 None Intrinsic membrane protein

psa L 18.0 ? Intrinsic membrane protein

. psa C 8.9 [4Fe-4S) 2 Terminal electron acceptor

psa D 17.9 None Ferredoxin docking protein

psa E 9.7 None ?

psa F 17.3 None P1astocyanin docking protein

psa G 10.8 None ?

psa H 10.2 None LHC I linker protein ?

psa M 3.5 ? Intrinsic membrane protein

psa N 4.8 ? Intrinsic membrane protein

1. Numbers given are per reaction center heterodimer 2. Mass is in kDa, 3. psa G and psa H 4. psa M and psa N 5. In eukaryotes, the. holocomplex

deduced from the gene sequence gene products are present only in eukaryotes gene products are present only in cyanobacteria core complex together with LHC I constitutes

( Adapted from Golbeck, 1992 )

Recently, three dimensional structure of PS I,

isolated from synechococcus spp., has been elucidated (Krauss

et al., 1993) by X ray crystallography. The crystals were in

the form of PS I trimers. Electron densities corresponding to

various cofactors of the electron transport chain of PS I were

shown to be arranged on the central axis defined by the

heterodimer. The electron densities were sequentially arranged,

from lumenal to the stomal side, for P700, Ao, A1, 4Fe-4S

centers etc. as expected for the elctron migration from lumenal

side to the stromal side of PS I complex.

2A.2.6 ATP Synthase

The transport of electrons from PS II to PS I is

accompanied by a vectorial transport of H+ from stroma to the

lumen of the thylakoids. In addition, the protons are also

released into the lumen due to oxidation of H20 by PS II. The

electrochemical gradient thus generated is utilized by the ATP

synthase of the thylakoids. Typically, the ATP synthase

comprises two morphologically and functionally distinct parts.

The hydrophi 11 ic part F1' emerges from the membrane and

harbors the catalytic sites responsible for ATP synthesis

and/or hydrolysis. It consists of 5 subunits ~ , fl , Y , 6 £. The other part, Fo, is embedded in the lipid bilayer and

acts as a proton channel. In chloroplasts, the Fo comprises 4 .

subunits I, II, III, IV.

Primary sequence analysis shows broad structural and

sequence similarity between chloroplast ATP synthase and those

from bacteria and mitochondria. Reconstitution studies

involving subunit components from diverse organisms, higher

16

plants, cyanobacteria and bacteria, lend credence to

evolutionary relatedness (Bar-Zvi et al., 1985; Hick et al., 1986; Richter et al., 1986; Gatenby et al., 1988).

2A.2.7 Phycobilisomes

These are supramolecular pigment protein complexes

found in cyanobacteria and red algae (see Glazer, 1982; 1984;

1985; 1989; Gantt, 1988; Bryant 1991 for reviews.). Due to the

presence of bilins as chromophores, these antenna systems are

able to harvest the energy from light regimes where ChI a does

not absorb.

The bilin chromophores (phycocyanobilin,

phycoerythrobilin and others) have an open chain tetrapyrrole

structure and are linked covalently (by a thioether bond) to

the cystein residues of the apoprotein. Spectral

characteristics of the various biliproteins (,rable 2.3) are

widely different owing to the differences in,

1. The chemical structure of the chromophore

2. Its specific association with the apoprotein

3. Interactions with other biliproteins and linker polypeptides

As a result, the phycobi lisomes are capable of

absorbing light energy from nearly 550nm to 650nm. The other

important feature is the directed transfer of this absorbed

energy to the RC. Phycobil isomes seem to be extremely

efficient in this respect because of a very ordered structure.

The biliproteins absorbing higher energy radiation are

spatially most distal to the RC while those which absorb a

lower energy radiation are most proximal. Also, the

biliproteins are arranged in arrays such that the emission

17

TABLE 2.3

Properties of Major Cyanobacterial Phycobillprotelnl"

I'ItHlfl'S'

Subunit Bilin Con- Moll'culM t\b~"rpti"" l·l'I"-... '

[A'signa- Genc tent pcr M,ISS M,l\illlUIlI M,l\iIIHIIII ('mll'in tion" Locus Subunit' Aggregation Stilt,,,, " 10 I D,I' (11111)' (1IIil)'

Cur\' l'OmpOnl'nts (0'\1'1:1'\1'), 100 b:;(l hhll Allophycol'yanin a AI' IIl'fA 1 PCB

fjAI' 1111('8 1 PCB AllllphYl'll(:yanin U 0' "I'·U 11,...-0 1 PCB (0 AI""{3AI') , IN h7ll' hlH h7S

fj AI' 1111('8 1 PCB Allophycol'yanill fj'X a AI' IIl'cA 1 PCB (a AI'{3'X)" (/I = 1-2) 35-70 blh t>-lO

fj Ix IIII('F 1 PCB Con' linker phycobili- LCM all('[ 1 PCB 72-120 hfl5 f>!-\ll

pnltl'in P,t'riphl'ral rud l-omponl'nts

Phycocyanin a''l' fllCA 1 PCB (a 1'l'fJ l'l)" (/I = 1-6) 30.5-220 b20 h-ltl fj l'l' cIIC8 2 PCB

Phycol'rythrocyanin o''t:C II('CA 1 PXB (a 1'F.(·fj I'F("), 120 ~, 5'X) (sh)' h25 fJ''fT 11('('8 2 PCB

rhYl'llt.'rythrin a''E cpt'A 2 PEB (a I'FpI'f.)" (/I = 3.6) 120, 240 565 57S fjl'f. cpt'B 3 PEB

" [l.llol lak,'n fr"m Coh,'n-IIdLire olnd Bryanl (1982). Glazer (1985). and Bryanl (1987), • Sut>unil d,'sign"lions follow Ih" n'commendalions of Glazer (19115), The abbrevialions Ar. Ar-B. !'C. rEc. and I'E.lr\, USlod (or Ih,' phy,'ot>iliprol"in" ,,110'

phVl'Ul·Yolnin. olliophyl'Ucyanin B. phycocyanin. phycoerylhrocyanin. and phycoerylhrin. K'Spt'CIiVl'ly. and a"', fJ"', l'Ie. (or Ih,'" olnd fJ subunils oi Ih,'''' Prl l '

l<'ins. Link"r polypt'plidl's "r\' "bbrl'viallod L, wilh a superscripl denoting Ihe size of Ihe polypeptide and a sut>scripl Ihdl d,'noll's Ihe 10l',,'ion o( Ih,' p"lvpq'lid," I( pt'riph,'roll rod subslru,'lufl'; RC. rud-wfl' junction; C. con' substruclure; CM. core-membrane junction, Thl' d,'Signdli,1O iJ" is u~od 10 indll".,'" .In AP·ll·Ii!..,' p"IYP"'plid,' wilh oln appafl'nl Il\ilSS of abuul lllkDa.

Abbfl'vialions (or phywbilins: !'CB. phycocyanobilin; rEB. phycoerythrubilin; rXB, phycobiliviolin-type chromophor\'. J Aggfl'gation slal,'s giv .. 'n art' Ihose for purifil'CI complexes at mO<krate ionic stn:~th, protein concenl ration , and n,'ar-n,'ulral pll 1>.'I~ (u, mul,',·ul ... mol", abSl>rphon molximum, ~nd fluon'!ICl'n('\' nwdmum .ore the"..· for tI", aAAfl'Kdlion ,, .. ," ,huw"

, Sh, ,huuld .. ·,. "'.'

(Adapted from Bryant, 1991)

spectrum of one kind has a large overlap with the absorption

spectrum of the other biliportein thus facilitating energy

transfer towards Re.

The basic subunit of the apoprotein is a heterodimer

of polypeptides ex and J3 The (j.f3 unit has a tendency to

form trimers and hexamers (~fo)6. Many hexamers are linked by

linker polypeptides to form rods which are arranged around a

core comprised of allophycocyanin hexamers (Fig 2.5). The core

is most proximal to the reaction center and also has an

emission spectrum that overlaps well with the ChI a absorption • spectrum. Thus the core, -along with a linker polypeptide, is

responsible for the final step of energy transfer from PBS to

the PS II.

One of the important features of cyanobacterial

phycobilisiomes is that their size and composition may be

altered depending on the growth conditions, especially the

spectral quality of the irradiating light (Hattori and Fujita,

1959; Bennett and Bogorad, 1973; and Tandeau de Marsac, 1991).

Nutritional status of the growth medium also affects the size

and composition of the PBS to a great extent (see Grossman et

al., 1993 for a review).

2A.2.& Tbylakoid Lipids

The predominant lipids of thylakoids are the uncharged

galactolipids, monogalactosyldiacylglycerol (MGDG) and

digalactosyldiacylglycerol (DGDG). Together they constitute

about 75% of the total thylakoid lipid (Webb and Green, 1991).

The remaining 25% is comprised of anionic lipids

sulfoquinovocyldiacylglycerol (SQDG) and phosphatidylglycerol

(PG) (Allen and Gord, 1971). No sterol are present and the

18

~ ~

D··· " IS . . .. • (IlP)PC.L ·L . . • . • 6 R It

APB AP AP I (Ill al~) )-LC

AP AP IU 99 2 (a2 ~2 ~ LCM)

AP 3 (al3),

4 (al3)~L~

FIGURE 2.5. Schematic representation of the hemidiscoidal phycobilisome of Synechococcus PCC 7002. AP, allophycocyanin; APB, allophycocyanin B; PC, phycocyanin;

AP IJ KP ,., 12 Ol. , r etc. for the "" and,- subunits of these proteins. Linker polypeptides are abbreviated L, v,'ith the superscipt denoting the size in kilodaltons and a subscript that specifies the location of the polypeptide R, peripheral rod substructure; RC, rod-core junction; C, core substructure; CM, core-membrane junction. In species containing phycoerythrin or phycoerythrocyanin, some of the phycofyanin in the peripheral rods is replaced by these proteins or else the rods are extended. (Adapted from Bryant, 1991)

presence of phophatidylcholine is debatable. The remainder

includes a wide variety of lipophillic pigments including

ChI a! xanthophylls, fl carotene and the quinones.

The fatty acids most commonly esterified to membrane

lipids in higher plant thylakoids are palmitic acid (16: 0) ,

stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2)

and linolenic acid (18: 3). Many cyanobacteria are devoid of

polyunsaturated fatty acids (see Webb and Green, 1991 for a

review). The thylakoid lipids of Anacystis nidulans are

comprised exclusively of saturated and monounsaturated fatty

acids (Kenyon and stanier, 1970; Kenyon, 1972) .

. 19

PART B REVIEW OF LITERATURE

2B.1. DYNAMIC NATURE OF THE THYLAKOIDS

The Z scheme very elegantly explains the coordinated

functioning of various proteins and pigment protein complexes

in the thylakoids. However, certain features of the thylakoid

structure and functions were assumed to be implicit in the Z

scheme.

1. It was assumed that the spatial organization of the

complexes in vivo is similar to that proposed by the Z scheme

i. e. they are sequentially arranged and homogeneously

distributed in the thylakoids. This assumption was supported

by the measurements of the electrochromic shift at 518nm

(Schiephake et al., 1968). However, Anderson and Boardman

(1966) and Arntzen et ale (1972) demonstrated that the grana

fraction was rich in PS II electron transport activity while

the PS I activity resided primarily in the stroma exposed

lamellae. such a heterogeneous distribution was found for Cyt

b6/f complex and ATPase also.

2. The second assumption was that the various complexes are

present in a stoichiometry of 1 1 in the thylakoids.

However, careful analysis by Fujita (1976), Kawamura et al., (1979), Melis and Brown (1980) and Melis and Thielen (1980)

showed that PS II

from unity.

PS I stoichiometry deviates significantly

Photosynthetic organisms are subjected to many stress

conditions that lead to an imbalance in the electron transport

20

rate through the two photosysterns. Such stress factors could be

environmental and genetic including

and quantity of light, nutritional

size of antenna due to mutations

alterations in the quality

stress, alterations in the

etc. The structure and

function of. the thylakoid membrane gets altered under stress

conditions (Melis et al., 1985; Melis, 1991). The alterations

are both short term and long term depending on the nature and

duration of the stress.

2B.1.1 Short Term Changes

The phenomenon by which photosynthetic organisms

achieve a redistribution of excitation energy between the two

photosystems under light conditions that overexcite either PS I

or PS II, has been termed state transi ti ton. Such a

redistribution occurs on the order of a few minutes and is

purely reorganizational in nature. (see Fork and Satoh, 1986

for a review).

state transitions were first observed independently by

Murata (1969a) and Bonaventura and Myers (1969) in red and

green algae respectively. In a study involving slow changes in

the fluorescence kinetics, they observed that preferential

excitation of PS I or PS II resulted in large changes in the

distribution of excitation energy between the two photosystems.

Changes in the oxygen evolution from PS II were also reported

by Bonaventura and Myers (1969) and indicated adjustment of the

electron transport reactions. Under conditions that overexcite

PS I, activity of PS II was found to be enhanced. Such a state

was termed State 1. Under State 2 condi tions, i. e.

overexcitation of PS II, energy gets distributed in favour of

PS I.

21

An alteration in the distribution of excitation energy

between the two photosystems can be effected in the following

two ways :

1. Change in the transfer of excitation energy from PS II to

PS I (spillover). During spillover, the transfer of energy

proceeds in the thermodynamically favoured direction i.e. from

PS II to PS I and not vice versa. Such a transfer necessarily

requires a close proximity between the antennae of the two

photosystems and also would be affected by the redox state of

the PS II reaction centers.

2. Change in the' fraction of light energy absorbed by a

particular photosystem (absorption cross section). Such a

change is effected by movement of the light harvesting antenna

leading to an alteration in the energetic coupling between the

antenna and photosystems. Thus the amount of energy delivered

to a photosystem may be increased or decreased.

Even though it has not been investigated in

cyanobacter ia, the randomization of the photosystems in

unstacked membranes in the absence of cations leads to an

increase in the spillover of energy from PS II to PS I in case

of higher plant thylakoids. A similar mechanism involving

variations in the concentration of cations was believed to

regulate the energy redistribution during state transitions in vivo. Such a belief was based on the following considerations

1. cations like Mg++ affect the stacking and destacking of the

thylakoids and also the proportion of excitation energy

delivered to PS II and PS I (Murata, 1969b).

22

2. Barber (1982) proposed a model for the mechanism of long

range migration of LHC based upon a consideration of membrane

surface charge densities and the effects of screening of

changes by various cations.

3. Kyle et al.(1983) demonstrated by freeze facture techniques

that stacking and destacking of thylakoids involves lateral

migration of LHC and its specific association and dissociation

with PS II in the thylakoids.

However, in case of leaves, the state changes were shown to

operate via a mechanism involving changes in absorption cross

section of the photosystems rather than the alterations in

spillover (Canaani and Malkin, 1984; Malkin et al., 1986)

STATE TRANSITIONS IN HIGHER PLANTS

In case of higher plants, a mechanism of alterations

in the absorption cross sections of the photosystems mediated

by phosporylation of LHC II seems to be operative (Bennett,

1977; Bennett et al., 1980). Under conditions that lead to

overreduction of the PQ pool (high PS II activity) LHC kinase

is activated. The phosphorylated form of LHC II gets

dissociated from PS II and migrates into the unappressed zones.

In its new environment, it probably transfers the 'excitation

energy to PS I. conversely, if the prevailing light condition

is such that PS I is more active than PS II, oxidation of PQ

pool leads to activation of phosphatase which is able to

dephosphorylate the LHC. In its dephosphorylated form, the LHC

migrates to the appressed zones and gets energetically coupled

to PS II units thus increasing the absorption cross section of

PS II (Allen et al., 1981; Allen, 1992ai 1992b).

23

STATE TRANSITIONS IN CYANOBACTERIA

The mechanism responsible for redistribution of

excitation energy between the photosystems in cyanobacteria and

red algae is presently controversial (Biggins and Bruce, 1989;

Allen, 1992a). These organisms differ from the higher plants

in the organization of the photosysthetic apparatus in two

prominent ways,

1. There is a lack of lateral heterogeneity. Various

supramolecular complexes are randomly distributed in the

thylakoids.

2. The light harvesting antenna of PS II i.e. phycobilisome

is totally extrinsic in nature.

Compared to the higher plants the state transitions in

cyanobacteria have a faster kinetics. Also the effect of state

transitions on the redistribution of excitation energy is much

more marked (Fork and Satoh, 1986). However, all the three

aspects related to the mechanism of state transition i.e.

primary signal, mediation of the signal and the manner by which

energy is redistributed are controversial. Some of the ideas

proposed ln context· of the mechanism for short term

redistribution of excitation energy and experimental evidence

in support of these is given as follows :

1. Cyclic electron flow around PS I regulates the light state transi tion : uncouplers like CCCP, but not inhibitors

of phosphorylation, reduced the extent of state 1 transition.

Inhibitors of cyclic electron flow e.g. Antimycin A decreased

the rate but not the extent of State 1 transition; acceptors of

electrons from PS I e.g. MV prevented state 1 transition (Satoh

and Fork, 1983b).

24

2. Redox state of PQ pool regulates state transitions Amongst a variety of chemical and light treatments used,

state 2 was induced by all those which led to reduction of the

PQ pool. Treatments leading to oxidation of the PQ pool

favoured a transition to state 1 as monitored by the 77K

fluorescence emission spectra (Mullineaux and Allen, 1990).

3. Phosphorylation of 18.5 kDa and 15 kDa proteins plays an important role in state transitions state 2 transition in

synechococcus 6301 correlated with phosphorylation of 18.5 kDa

(soluble or weakly membrane bound) protein and 15 kDa (membrane

bound) protein. The two proteins are components of PBS and

PS II core. (Allen et al., 1985). Sodium fluoride, a potent

inhibi tor of most phosphatases, prevented a transition from

state 2 to State 1 implicating the role of reversible

phosphorylation during state transitions (Canaani, 1986).

4. state transitions are independent of phosphorylation of the phycobilisome components : The fluorescence change at 77K

in Synechococcus 6301 thylakoids upon illumination was not

dependent upon phosphorylation. Also, the protein kinase of

the thylakoid membranes did not require light for

activation (Biggins and Bruce, 1987). Phycobilisome less mutant

of Synechococcus 7002 has the ability of undergo state

transition. (Bruce et al., 1989).

5. Phycobilisome can ransfer the harvested light energy to PS II (in state 1) or PS I (in state 2) During state 2, the

population of PS II units energetically uncoupled with PBS,

increases as shown by room temperature fluorescence induction.

(Mullineaux and Holzwarth, 1990). During state 2, there is a

60% decrease in the amplitude of PS II fluorescence emission

accompanied by an increase in the apparent amplitude of the

emission from PBS as measured by time resolved fluorescence

25

spectroscopy. The life times of fluorescence decay from PS II

core and PBS were unaffected upon transition to state 2 from

State 1 (Mullineaux' et al., 1990). A laser induced optoacoustic

study indicated that the energy diverted away from PS II during

State 2 is not quenched as heat. The PBS possibly transfer

this energy to PS I (Mullineaux et al., 1991). For the cells

adapted to light state 2, the PBS made a major contribution to

the light harvesting capacity of PS I measured by flash unduced

absorbance changes at 700 nm. (Mullineaux, 1992).

6. The extent of energy transfer from PS II to PS I (spillover) is higher in state 2 than state 1 This is

controlled by the proximity of PS II and PS I. An alteration

in the distribution of excitation energy absorbed by Chl a

antenna of PS II~as observed during fluorescence induction at

room temperature (Mullineaux and Allen, 1988). Transition to

state 1 accompanied by a large increase in the apparent

fluorescent lifetime of Chl a associated with PS II, measured

by time resolved fluorescence speetroscopy, was reported by

Bruce et al.(1985). Laser induced optoacoustic calorimetry

of cells adapted to state 1 or State 2 indicated that the

fraction of absorbed energy released as heat was similar for

cells in State 1 and State 2. This was interpreted to suggest

that efficiency of excitation energy storage was not affected

by the state transition i. e. PBS are not energetically

decoupled from the photosystems (Bruce and Salehian, 1992).

Thus, none of the currently proposed models for the state

transitions in cyanobacteria can fully explain the data and all

the three aspects of state transitions in cyanobacteria i. e.

signal for the transition, mediation of the signal and the

final reorganization of the photosynthetic apparatus that

effects a redistribution of excitation energy, are presently

controversial.

26

2B.1. 2 Long Term Changes

A variety cif environmental, developmental and genetic

factors may cause a prolonged stress to the photosynthetic

apparatus 0 Under such a situation, the composition of the

thylakoids gets altered. This process involves alterations in

the rate of biosynthesis, assembly and degradation of various

supramolecular complexes of the thylakoids. Antenna sizes of

the two photosystems and relative stoichiometry of the

complexes is subject to be altered during this process of

acclimation (Melis, 1991). Further, these changes can be

reversed if the stress is alleviated.

Specific responses of cyanobacteria, algae and higher

plants towards diverse stress factors have been examined during

the past few years. The factors that lead to an imbalance in

the electron transport rate through the two photosystems have

been shown to regulate the photosynthetic apparatus.

1. LIGHT INTENSITY

The intensity of light during growth of the plants

seems to regulate three different aspects of the photosynthetic

apparatus :

The antenna sizes of both PS I and PS II are larger for

low light grown plants. PS II antenna size is affected to a

greater extent. This has been shown independently by

Lichtenthaler et ale (1982) Hodges and Barber (1983) and

Leong and Anderson (1983, 1984a, 1984b) for higher plants. In

case of cyanobacteria, the size of phycobilisomes (antenna for

PS II) increases under low light irradience (Glazer, 1984).

27

The PS II : PS I stoichiometry decreases under low light

intensity in cyanobacteria (Kawamura et al., 1979; Murakami and

Fujita, 1991), chlamydomonas spp (Neale and Melis, 1986) and

chloroplasts from several species (Melis and Harvey, 1981;

Wild, et al., 1986; Leong and Anderson, 1986). However, this is

not a universal feature. In diatoms and brown algae, the

PS II PS I ratio is higher when grown under low light

condition (Falkowski et al., 1981; smith and Melis, 1988) n

The content of the Cyt b6/f complex is higher relative to

the reaction centers when the photosynthetic organisms are

grown at a higher intensity of light (Melis, 1991). Hence, its

content may be considered to reflect the electron transport

rate through the two photo systems (Wilhelm and Wild, 1984).

2. LIGHT QUALITY

The action spectrum of the two reaction

quite different. This is primarily because of the

ChI b in the antenna of PS II in higher plants.

centers is

presence of

In case of

cyanobacteria and red algae, the phycobilisomes have absorption

properties very distinct from chlorophyll and the energy

harvested by them is preferentially transferred to PS II (Ley

and Butler, 1980). The light harvesting antenna of PS I in all

photosynthetic organisms has a predominance of ChI a. Such

major differences in the action spectrum of the two

photosystems imply that the quality of irradiating light has

the potential to exert a very strong effect on the relative

activity of the two photosystems.

Pea plants grown under PS II light had small grana

stacks and extensive stromal lamellae. These structural

changes were accompanied by adjustments in the phot~system

28

stoichiometry. PS II PS I was found to be low under

condition of PS II excitation (Melis, 1991). Many studies

suggest that the shade plants and low light grown plants have

larger PS II antenna (Lichtenthaler et al., 1981; Chu and

Anderson, 1984)

Cyanobacteria grown in light that overexcites ,PS II

(yellow light) have a lower PS II PS I ratio compared to

those grown in light which overexcites PS I (far red light).

Th i s was observed independently by Myers et al. (1980) ,

Manodori and Melis (1986), and Fujita and Murakami (1987). The

antenna sizes for PS II and PS I seem to remain unaltered under

these conditions.

The composition of the phycobilisomes in terms of

relative amounts of phycocyanin and phycoerythrin may be

altered depending on the ambient light conditions. The

phenomenon is termed complementary chromatic adaptation and has

been reviewed recently (Tandeau de Marsac, 1991; Grossman et

al., 1993).

3. MUTATIONS AFFECTING THE LIGHT HARVESTING ANTENNA

Virescent (ChI b deficient) mutants are known for many

plants like tobacco, soybean, sugarbeet etc. These mutants

lack functional LHC due to the deficiency of ChI b. Since LHC

comprises 80% of the PS II antenna and only about 40% of the PS

I antenna, the light harvesting capacity of PS II is affected

more severely than PS I. Such mutants show an altered

structure and function of the thylakoids. The most prominent

changes are an increase in ChI alb and PS II : PS I ratio while

Chl/P700 and Chl/QA decrease (Melis, 1991).

29

Similarly, AN112, a mutant of A. nidulans, was

isolated by nitrosoguanidine mutagenesis. Due to the lack of a

linker polypeptide, this mutant has smaller phycobilisomes than

the wild type. contrary to expectations, the RC II RC I

ratio in this mutant remains unaltered. However a reduction in

the PS II : PBS stoichiometry was evident. Thus the reduction

in the size of the phycobilisome is compensated for by the

increase in the number of PBS units and the overall ability of

light utilization by PS II remains unaltered (Manodori et al., 1984) .

4. PARTIAL INHIBITION OF PS II BY HERBICIDES

Partial inhinbition of PS II by various herbicides

results in an impaired PS II electron transport capacity. In

higher plants, long term treatment with various herbicides like

bentazon, metabenzthiazuron (MBT) , DCMU and atrazine etc. was

shown to induce shade type reorganization of the photosynthetic

apparatus characterized by an increase in the stacking and

grana formation, decline in ChI alb ratio, Im.,er amounts of

carotene etc. (Fedke et al., 1977; Kleudgen, 1978;

Lichtenthaler, 1979; Lichtenthaler et al., 1980; Mattoo et al., 1984b). Subsequently, it was shown that treatment of plants

with SAN 9785, a pyridazinone derivative, leads to an overall

reduction in ChI alb ratio, and increased synthesis of PS II

units and alterations in the structure-function relationship of

thylakoids (Bose et al., 1984; 1992). Also, the thylakoids were

stacked to a larger extent with a lower proportion of stromal

lamellae.

In case of cyanobacteria, various herbicides like

atrazine, terbutryn, sUbstituted ureas, triazines etc. were

used to partially block the PS II activity in Synechococcus

30

6301. All the herbicides interacting with the D1 polypeptide

of PS II were reported to cause shade type adaptation

characterized by an increased PC ChI ratio (Koenig 1987a,

1987b, 1990a). Similar effect of DCMU was also observed for

Anacystis nidulans by Hatfield et ale (1989)

5. MUTATIONS CAUSING RESISTANCE TO PS II INHIBITORS

All the known PS II inhibitors seem to interact with

the D1 protein of PS II (Tisher and Strotmann, 1977) sharing

common binding determinants with each other and with the QB

site (Vermaas et al., 1984; Trebst, 1986). Mutations in the

QB binding domain of the D1 protein are known to confer

resistance towards herbicide action in plants and

cyanobacteria. As a consequence, the electron transport form

QA to QB also gets affected in these mutants. Pfister and

Arntzen (1979) showed that the rate of electron transport

through PS II is reduced by a factor of ten in resistant

biotypes compared to the susceptible ones. However, by

analysing the decay time of the fluorescence induced due to

single turnover flashes, Jansen and Pfister (1989) have shown

that the reoxidation of QA - takes about thrice the time in

plants resistant to triazine compared to the susceptible ones.

comparision of photosynthetic apparatus of herbicide

resistant and susceptible varieties has shown that the

chloroplasts from herbicide resistant plants have a higher

degree of thylakoid stacking and a paucity of stromal lamellae.

Also, the resistant varieties had a larger amount of LHCP and

hence a lower ChI alb ratio. This was shown for the atrazine

resistant varieties of Chenopodium album, Amaranthus retroflexus, Solanum nigrum and Brassica campestris etc.

(Vaughn and Duke, 1984; Lemoine et al., 1986).

31

Anacystis nidul ans mutants resistant to DCMU were

shown to have shade type phenotype characterized by a high

PC : ChI ratio even in the presence of strong light and absence

of any inhibitor (Koenig, 1987 a). However no other aspects

related to excitation energy distribution in herbicide

resistant mutants have been reported yet.

2B.2. PYRIDAZINONE DERIVATIVES AS TOOLS TO INVESTIGATE THE PHOTOSYNTHETIC APPARATUS

pyridazinone is a six membered heterocyclic compound.

More than forty derivatives of this compound have been tested

for their interaction with the plant systems (st. John et al., 1979) . Given below is the molecular structure of the

pyridazinone derivatives. R1, R2 and R3 represent the three

positions where different substitutions have been made.

Though some of the pyridazinone derivatives like

pyrazon (5-amino-4-chloro-2-phenyl pyridazin-3-one) have been

used commercially for weed control, most are used as

experimental photosynthetic herbicides. Depending upon the

nature of sUbstituent groups, the pyridazinone derivatives

32

induce alterations in the pigment content, lipid content or

photosynthetic parameters of the thylakoid membranes. Effects

of this group of herbicides known so far are :

1. Inhibition of photosynthetic electron transport

2. Alterations in the lipid matrix of thylakoids

3. Interference with pigment biosynthesis

4. Alterations in the structure function relationship of the

thylakoid membranes.

2B.2.1 Inhibition of Photosynthetic Electron Transport

Many of the sUbstituted pyridazinones have been shown

to inhibit Hill reaction in isolated thylakoid membranes,

isolated leaf mesophyll cell and intact algal cells (Hilton et

al., 1969; Tischer and Strotmann, 1977; Herczeg et al., 1979;

Karapetyan et al., 1981; Mannan and Bose, 1985).

The·· eff iciency to inhibit the electron transport

varies depending on the sUbsitution in the pyridazinone. The

ISO value for the inhibition of photosynthetic electron

transport in isolated chloroplasts for SAN 6706 was reported to

be 5pM while that for SAN 9789 was 90 pM.

Experiments by Hilton et ale (1969) indicated the ISO

of SAN 9785 as 14 pM. However, later Mannan and Bose (1985)

reported the ISO for this derivative to be 20 pM. For SAN

9785, both the binding constant and the inhibition constant are

100-150 folds higher than DeMU (Tischer and Strotmann, 1977).

Hence, the pyridazinone derivatives have been characterised as

weak inhibitors of the photosynthetic electron transport.

33

EVen though much

derivatives seem to bind

weaker as inhibitors,

to the same protein

pyridazinone

as OCMU and

atrazine as was suggested by Tischer and Strotmann (1977) on

the basis of competition experiments using radioactively

labelled and unlabelled inhibitors. Experiments conducted by

Pf ister and Arntzen (1979) and Droppa et al. (1981) further

indicated that pyridazinone derivatives act on the reducing

side of PS II in a manner analogous to OCMU. Inhibi tion of

electron transport by SAN 9785 could not be reversed by

donation of electrons on addition of OPC (Mannan and Bose,

1985) supporting the belief that pyridazinones act on the

reducing side of PS II.

Nature of binding and inhibition by PS II herbicides

Trebst and Draber (1979) have identified two features

common to all inhibitors of the PS II dependent electron

tran3port.

X \\

1. Presence of the group >N-C- where X represents an oxygen

or nitrogen atom but not sulphur

2. A hydrophobic residue in close vicinity to the above

mentioned group.

Pyridazinone derivatives fulfill both these structural

requirements to be able to inhibit PS II dependent electron

transport.

Photoaffinity studies with an azido-analogue of

atrazine indicated that the receptor site contained a protein

(01) of molecular weight 32 kOa (Gardner, 1981). A model has

been proposed by Gardner (1989) that encompasses biochemical,

biophysical and structure activi ty relationships of the

34

inhibitors. In essence, the PS II inhibitors have been

considered to be non-reducible analogues of PQ or its

semiquinone anion. It implies that,

1. since the PQ molecule is asymmetric due to the presence of

an isoprenoid chain, the strongly hydrophobic regions of the

inhibitor should correspond to this isoprenoid tail of PQ.

2. Due to the presence of two carbonyl atoms in the PQ

molecule, depending on the structure, the essential element of

the inhibitor (sp2 carbon) can interact with the receptor in

two different ways. Thus the binding sites for the inhibitors

may be overlapping but not necessarily identical.

The exact nature of binding of the PS II herbicides

to the. PQ site has hitherto rema ined unkonwn. However,

recently a triazine resistant mutantT4 from Rhodopseudomonas viridis has been found to be sensitive to bothureas and

phenolics (photosynthetic activity of wild type Rps. viridis is

not sensi ti ve to ureas and phenolic herbicides). structural

details of DCMU binding to T4 as determined by X-ray

crystallographic analysis have been elucidated (Sinning, 1992)

and shed some light on the nature of interaction between PS II

reaction centre and the PS II inhibitors. Fig. 2.6 shows a

hypothetical model of interaction of atrazine with the 01

protein.

Various site specific mutants· of D1 polypeptide in

cyanobacterium synechocystis 6803 and Synechococcus 7942 have

been analysed (Ohad et al., 1990; Ohad and Hirschberg, 1992).

This analysis has helped to extend the structural analogy

between secondary quinone binding site in 01 polypeptide and in

the L subunit of the photosynthetic reaction centre of purple

bacter ia. Some distinctions have also been made between the

binding sites of QB and the herbicides.

35

FIGURE 2.6 Schematic diagram herbicide binding pocket of 01

of the plastoquinone and protein. Dashed lines

represent hydrogen bonds; dotted lines represent hydrophobic interactions. A. Plastoquinone binds to the 01 protein, accepts two electrons and two protons and is released as plastohydroquinone. B. Atrazine binds to the 01 protein and prevents the binding of pl~stoquinone. (Adapted from Fuerst and Norman, 1991).

2B.2.2 Alterations in the Lipid Matrix

Substituted pyridazinones induce a specific decrease

ln the content of linolenic acid (18:3) with a concomitant

increase in the linoleic acid (18:2) (st. John, 1976; st. John

et al., 1976; Willemot, 1977; Frosch et al., 1979; Ashworth et

al., 1981). Out of forty four sUbstituted pyridazinones tested,

SAN 9785 was found to be most efficient in inducing a specific

decrease in the linolenic acid (18:3) content (st. John et al., 1979). This specific decrease in the linolenic acid (18:3) was

due to the inhibition of desaturation of linoleic acid (18:2)

to form linolenic· acid (18:3) as shown by Murphy et ale (1980) and Willemot et ale (1982) using 14c acetate and leaf discs.

2B.2.3 Alterations in the content of Photosynthetic Pigments

Depending on the nature of substitution, the

pyridazinone derivatives induce a bleaching of photosynthetic

pigments to a varying degree in algal cells and higher plants

(Kleudgen, 1979; Kummel and Grimme, 1975; Mannan and Bose,

1985) . certain features in the structure of substituted

pyridazinones were identified (Urbach et al., 1976) which are

important for conferring the ability to cause efficient

bleaching. Based on these criteria, SAN 9785 was expected to

be rather inefficient in causing bleaching of the

photosynthetic pigments. This prediction was found to be ture

when tested experimentally (Urbach et al., 1976; st. John,

1976) . Even at a concentration of 100 pm, SAN 9785 did not

cause significant bleaching. In contrast, SAN 9789 induced 99%

inhibition of carotenoid biosynthesis (Lehoczki et al., 1982). Under these conditions the plants can grow provided the light

intensi ty is extremely low. This is necessary to avoid

photodegradation of chlorophyll.

36

The mechanism of bleaching of photosynthetic pigments

by pyridazinone compounds is still not clear (Karapetyan, 1993)

However, three hypothesis have been proposed to explain the

phenomenon.

1. Direct inhibition of carotenoid biosynthesis : Sandmann et

ale (1980) showed that SAN 9789 inhibits phytoene synthetase.

Ben-Aziz and Koren (1974) observed that SAN 6706 interfered

with the cyclization reactions in the carotenoid biosynthesis

whereas Bartels and McCullough (1982) and Bartels and Watson

(1978) reported that this compound inhibits dehydrogenation

reactions of carotenoid biosynthesis in wheat seedlings. Since

carotenoids are known to have a protective role against

chlorophyll bleaching (Frosch et al., 1979), chlorophyll

molecules get photodegraded under the influence of

pyridazinones.

2. According

chlorophyll

to the second hypothesis, the carotenoid and

biosynthesis are highly coordinated. Thus

inhibjtion of carotenoid biosynthesis by pyridazinones leads to

a reduction in the biosynthesis of chlorophyll as well. Such a

regulation was demonstrated in Euglena gracilis (Vaisberg and

Schiff, 1976). However, Pardo and Schiff (1980) observed that

inhibition of carotenoid biosynthesis by SAN 9789 in etiolated

bean and maize leaves had little effect on the formation of

prolamellar bodies containing the usual phototransformable

protochlorphyllide species. They inferred that in higher

plants the biosynthesis of carotenoids and chlorophylls is not

necessarily coordinated.

3. The third hypothesis considers a more generalized effect

of pyridazinone derivatives. It is proposed that pyridazinone

derivatives decrease the content of 70S ribosomes. Under such a

37

situation, the enzymes involved ln the biosynthesis of

carotenoids and chlorophylls are not formed leading eventually

to a decrease in the content of pigments. Direct evidence in

favour of this hypothesis is lacking.

2B.2.4 Alterations in the structure and Function of Thylakoids

Growth of plants in presence of various derivatives of

pyridazinones induces several alterations in the structure and

function of the thylakoids. There is a lot of variation in the

changes induced 'by these herbicides depending upon the

nature of substitution, plant species and the growth

conditions.

SAN 9785 induced an increased grana stacking (Khan, et

al., 1979; Leech et al., 1985; Bose et al., 1992). On the

contrary, Laskay and Lehoczki (1986) observed a disorganization

of thylakoid membrane structure in barley leaves greened in

presence of SAN 6706 and SAN 9789.

Differential effect of pyridazionone derivatives was

also evident when their effect on PS II units was considered.

While Scenedesmus cells grown in presence of norflurazon (SAN

9789) exhibited an inactivation of PS II units (Karapetyan,

1993), wheat seedlings grown in presence of SAN 9785 exhibited

an increased synthesis of PS II units leading to an enhancement

of PS II : PS I ratio (Bose et al., 1984).

Some of the prominent effects of SAN 9785 on the

thylakoid structure and function reported by Mannan and Bose

(1985), Leech et al. (1985) and Bose et al. (1984; 1992) are,

38

1. a decrease in the ChI alb ratio

2. an increase in the PS II : PS I ratio

3. an increase in the Cyt f.

4. an inhibition of cation induced changes in the excitation

energy distribution.

5. inabili ty of SAN 9785 treated thylakoids to undergo

destacking in absence of cations.

Bose et al. (1992) correlated these structural and functional

changes and concluded that observed inhibition of cation

induced spillover changes occurs due to loss of the ability of

SAN 9785 treated thylakoids to undergo unstacking in low salt

medium.

2B.3. ABOUT THIS INVESTIGATION

Long term effects of various PS II inhibitors have

been investigated on the photosyntheticapparetus of higher

plants (Fedke et al., 1977; Kleudgen, 1978; Lichtenthaler,

1979; Lichtenthaler et al., 1980; Mattoo et al., 1984b). These

studies have shown that the structure, function and

organization of' the thylakoids alters under conditions of in si tu partial inhibition of PS II by these inhibitors. The

alterations are particularly evident in ChI alb ratio and the

degree of stacking of the thylakoids. How these alterations are

effected at the molecular level is not presently known.

Cyanobacteria, being prokaryotic and much simpler in

genetic and cellular organization compared to higher plants,

39

offer a special advantage in determining how the photosynthetic

apparatus responds to an in situ partial inhibition of PS II.

Koenig (1987a; 1987b; 1990a) has reported an enhancement in the

PC : ChI a ratio in the cells of Anacystis nidulans when grown

in the presence of DCMU type inhibitors. Under such a

situation, the turnover of D1 polypeptide was found to be

lowered and proposed to be the signal for adaptive changes in

the thylakoids. However, so far, no detailed investigation has

been carried out to determine other alterations in the

structure and function of the thylakoids. In view of this, we

were encouraged to focus our attention on the excitation energy

redistribution between the photosystems of cyanobacteria caused

due to a prolonged partial inhibition of PS II.

SAN 9785 is a PS II inhibitor acting on the reducing

side of PS II in a manner analogous to DCMU (Tischer and

Strotmann, 1977). However, it is a much weaker PS II inhibitor

than DCMU. The in vivo action of SAN 9785 on the

photosynthetic apparatus of higher plants has been investigated

earlier (Bose et al., 1984; Mannan and Bose, 1985; Leech et

al., 1985; Karapetyan, 1993; Bose et al., 1992) and has been

shown to cause several alterations in the structure and

function of the thylakoids. SAN 9785, therefore, appears to be

a useful tool to study the structural and functional changes

produced in response to in situ partial inhibition of PS II.

The ability of SAN 9785 to inhibit desaturation of

linoleic acid (18:2) to form linolenic acid (18:3) was reported

by st. John (1976). The consequent decrease in the content of

unsaturated fatty acids was proposed to be the factor that

leads to the observed alterations in the photosynthetic

apparatus (Graf et al. I 1984; 1987). Linolenic acid (18: 3) is

known to be absent or present in very low amounts in the

thylakoids of cyanobacterial· strains belonging to the genus

40

Synechococcus (Kenyon and stanier, 1970; Kenyon, 1972). Thus,

SAN 9785 is not likely to alter the lipid matrix of

Synechococcus 7942 by specifically inhibiting the linoleic acid

desaturase. Hence, this organism was considered to be

particularly suited to ascertain whether the SAN 9785 induced

alterations in the photosynthetic apparatus arise due to its

ability to inhibit PS II or are an indirect effect of the

altered lipid matrix.

In the present investigation, long term changes in the

structure and function of Synechococcus 7942 photosynthetic

apparatus were characterized as a response towards prolonged

partial inhibition of PS II. The Synechococcus 7942 cells were

grown in the presence of sublethal concentration of SAN 9785 to

effect an in situ partial inhibition.

As described above, in the paucity of linolenic acid

(18: 3) in the thylakoids, the changes were expected to be

caused primarily due to the PS II inhibitory activity of SAN

9785 on Synechococcus 7942 cells. However, to clearly estcblish

that PS II inhibition is the sole factor that leads to the SAN

9785 induced adaptive changes, effect of SAN 9785 was also

investigated on the photosynthetic apparatus of a mutant of

synechococcus 7942 resistant to PS II inhibition, designated as

R2TaqI (Golden and Haselkorn, 1985). The mutation changed ser

264 to ala in the Dl polypeptide of PS II thus rendering R2TaqI

to be resistant to PS II inhibition by several DCMU type

inhibitors acting on the reducing side of PS II.

41