Regulation of Photosynthetic Electron Transport.pdf

Chapter 30Regulation of Photosynthetic Electron Transport

Peter J. Nixon*Department of Biochemistry, Imperial College of Science,

Technology and Medicine, London SW7 2AY, U.K.

Conrad W. MullineauxDepartment of Biology, University College London,

Darwin Building, Gower St, London WC1E 6BT, U.K.

SummaryIntroductionBackground Concepts

Photosynthetic Electron Transport and the Z SchemeCoordination of Electron Transport and MetabolismFeed-forward Activation of the Calvin CycleThe Use of Alternate Electron Sinks

Feedback Control of the Photosynthetic Electron Transport ChainRole of the Lumenal pH in Slowing the Rate of Electron TransferRole of Lumenal pH in Quenching Excitation Energy in PS IIRegulation of Electron Flow through State Transitions

The Triggering Mechanism for State TransitionsAlterations in Function of the Light-harvesting ComplexesThe Genetic Approach to State TransitionsHigher Levels of Control

Regulation of Photosystem II ActivityThe Bicarbonate EffectA. The Acceptor-side EffectB. The Donor-side Effect

Cyclic Electron FlowA. Pathways of Cyclic Electron Transfer

Ferredoxin CyclesNAD(P)H Cycles

Identification of the NADH Dehydrogenase (Ndh) ComplexSubstrate Specificity of the Ndh Complex in vitro and in vivoRole of the Ndh Complex

B. Regulation of Cyclic Electron FlowChlororespiration and Cyanobacterial RespirationInteraction between Chloroplasts and MitochondriaQuestions for the Future

Are there other Regulatory Mechanisms still to be Characterized?What are the Physiological Roles of the Adaptation Mechanisms?Are there Cell- and Tissue-specific Responses in Multicellular Photosynthetic Organisms?

References

*Author for correspondence, email: [email protected]

I.

Eva-Mari Aro and Bertil Andersson (eds): Regulation of Photosynthesis, pp. 533555. 2001 Kluwer Academic Publishers. Printed in The Netherlands.

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VII.VIII.

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534 Peter J. Nixon and Conrad W. Mullineaux

Summary

Green plants and cyanobacteria possess a complex and diverse set of mechanisms to regulate their photosyntheticelectron transport. These include both rapid mechanisms, and long-term responses involving changes in geneexpression. Together they are believed to play a number of physiological roles, including the maintenance of arelatively constant flow of electrons in a changing light-environment (homeostasis), the adjustment of the modeof photosynthetic electron transport in response to the changing metabolic needs of the cell (adaptation) and theminimization of the destructive side-effects of light and redox reactions (protection). This Chapter concentrateson the rapid responses (those not involving changes in gene expression). There has been exciting progress in ourunderstanding of these mechanisms in recent years through a multi-disciplinary approach combining biophysics,biochemistry and molecular genetics. We describe the major mechanisms that have been characterized to date,concentrating on recent progress towards understanding the mechanism at the molecular level and highlightingthe questions that remain to be answered.

I. Introduction

Oxygenic photosynthetic organisms show a numberof alternative modes of photosynthetic electrontransport (Fig. 1). Linear photosynthetic electrontransport involves the Photosystem two (PS II) andPhotosystem one (PS I) reaction centers acting inseries to transfer electrons from water to ferredoxin.To maintain this mode of electron transport efficiently,the turnover of the reaction centers must be regulatedso that the turnover of PS II is roughly the same asthat of PS I. In addition, there are alternative modesof electron transport. Various forms of cyclic electrontransport around PS I have been proposed. Respiratoryelectron transfer involving NADH and succinatedehydrogenases (Complexes I and II, respectively),plastoquinone and terminal oxidases also occurs inthe thylakoid membrane of cyanobacteria (reviewedby Schmetterer, 1994). There is now increasingevidence for similar activities in chloroplasts (Endoet al., 1997; Burrows et al., 1998). This raises thepossibility that the redox state of the plastoquinonepool, which is important for the regulation ofphotosynthetic electron flow, is dependent on both

Abbreviations: FNR ferredoxin: reductase; FQR ferredoxin:quinone reductase; FTIR Fourier transform infrared;IEC intersystem electron transfer chain; LHC light-harvestingcomplex; MDA monodehydroascorbate; Ndh NADHdehydrogenase; ORF open reading frame; PET photosyntheticelectron transport; PS I Photosystem I; PS II Photosystem II;

primary plastoquinone electron acceptor of Photosystemtwo; secondary plastoquinone electron acceptor ofPhotosystem two; qE energy-dependent component of non-photochemical quenching; qI non-photochemical quenchinginduced by photoinactivation; RC reaction center; WT wildtype

photosynthetic and respiratory processes. The co-existence of respiratory and photosynthetic electrontransfer chains in the same membrane also meansthat electrons could be extracted from water andpassed back to oxygen via a terminal oxidase. Suchpathways are readily observed in cyanobacterialmutants lacking one or other of the reaction centers(Vermaas, 1994) but they may also be important inwild-type cells.

A photosynthetic organism must be able to regulatethe various possible modes of photosynthetic electrontransport. Regulation is necessary for several reasons:First, it is important to maintain photosyntheticelectron transport in a varying light environment(homeostasis). Photosynthetic organisms may besubject to huge and rapid changes in the intensity andspectral quality of illumination. They use regulatorymechanisms to buffer the effects of these changes,for example, by activating mechanisms for energydissipation in strong illumination, or by adjusting therelative turnover of the two reaction centers tocompensate for changes in the spectral quality ofillumination. Second, regulation is necessary foradjusting the mode of electron transport in responseto the metabolic needs of the cell. For example,cyclic electron transport generates a proton gradient(and hence ATP) but no reducing equivalents. Linearelectron transport generates both ATP and reducingequivalents. It may therefore be necessary to regulatethe extent of linear versus cyclic electron flowaccording to the requirement for ATP and reducingequivalents.

Third, regulation is needed to protect the organismfrom the destructive side-effects of light and redox

Chapter 30 Photosynthetic Electron Transport 535

reactions. Photosynthetic electron transport is adangerous activity, since there is always a risk ofgenerating reactive by-products such as singletoxygen and free radicals, which can cause widespreaddamage in the cell. Mechanisms exist to down-regulate photosynthetic electron transport, particu-larly when the supply of the products exceeds themetabolic requirements of the cell.

Regulation often occurs via feedback controlmechanisms. Many mechanisms can be effective formore than one of the functions listed above. Forexample, a mechanism that adjusts the relativeturnover of the two photosystems could be employedboth for maintaining similar rates of turnover invarying light environment (homeostasis) and forswitching between different modes of electrontransport (adjustment).

Numerous regulatory mechanisms have beenproposed in plants and cyanobacteria. Regulationcan occur on different timescales. Long-termmechanisms, generally involving changes in geneexpression, are complemented by rapid mechanismsoccurring on timescales of seconds to minutes.Regulation of gene expression is discussed elsewherein this volume (see Part II: Gene Expression andSignal Transduction). This Chapter will thereforediscuss the rapid mechanisms, concentrating on thosemechanisms where there has been significant progresstowards understanding the molecular basis of themechanism.

II. Background Concepts

A. Photosynthetic Electron Transport and theZ Scheme

According to the Z scheme model of photosynthesis,PS I and PS II act in tandem to transfer electronsfrom water to ferredoxin and then to (Fig. 1).Accordingly, a minimum of eight photons are required(four for each photosystem) to produce one moleculeof oxygen from the oxidation of water at PS II, and togenerate the four reducing equivalents needed to fixone molecule of in the Benson-Calvin cycle.The Z scheme therefore predicts that the maximumquantum yield for both oxygen evolution andfixation is 0.125. Measured quantum yields in C3plants are near this maximum value suggesting theoperation of the Z scheme in vivo (discussed inGenty and Harbinson, 1996). A recent challenge tothe ubiquity of the Z scheme, based on the analysis ofPS I deficient mutants of the green alga Chlamy-domonas reinhardtii (Greenbaum et al., 1995), hassince proved to be unfounded (Redding et al., 1999).Although from a thermodynamic perspective it isfeasible that PS II can reduce ferredoxin orrecent experiments with well-characterized engi-neered PS I-deficient mutants ofC. reinhardtii appearto exclude these as significant reactions in vivo(Redding et al., 1999). Despite this, the uncouplingof PS II from PS I under certain physiologicalconditions cannot yet be fully discounted.


B. Coordination of Electron Transport andMetabolism

The products of photosynthetic electron transport,ATPland reductant in theformof reduced ferredoxin,are used for a variety ofpurposes including not onlythe fixation of into carbohydrate and the processof photorespiration but also key biosynthetic stepssuch as sulfate and nitrite assimilation. Thesepathways can be thought of as electron or energysinks for photosynthetic reductant.

In classic light saturationcurves forphotosynthesis,two regions can be defined. At low light intensities,photosynthesis is light-limited. At higher lightintensities, the rate of photosynthesis becomessaturated because of limitations in the metabolicdemand for reductant. In the absence of metabolicelectron sinks, there is a danger that stromalcomponents and the photosynthetic electron transferchain become over-reduced. This can then lead, by avariety ofmechanisms, to the production of reactiveoxygen species throughout the electron transportchain, with theultimate result that there is an increasedphotodestruction of protein and pigment especiallywithin PS II (Barber and Andersson, 1992; Heberand Walker, 1992). This potential problem meansthat metabolic demand for reductant and thephotosynthetic electron transfer rate must be closelycoordinated. In practice, damage to the photosyntheticelectron transport chain is alleviated by (i) feed-forward mechanisms to activate stromal metabolism,(ii) feed-back mechanisms to slow photosyntheticelectron transport when metabolic demand reduces,(iii) the use of alternate emergency electron sinksthat dissipate photosynthetic electron flow relativelyharmlessly (Asada, 1999), (iv) the presence ofdetoxification systems to remove reactive oxygenspecies (Niyogi, 1999), and (v) rapid replacement ofdamaged protein, such as the D1 subunit of PS II,through highly coordinated repair processes (Barberand Andersson, 1992).

1994). The mechanism involves reduction ofdisulphide bridges in the target enzyme by thioredoxinwhich in the chloroplast is reducedusing a ferredoxin-dependent thioredoxin reductase and is thuscontrolled by photosynthetic electron flow (Bu-chanan, 1994). Thioredoxin also activates thechloroplast ATP-synthase (Stumpp et al., 1999).

D. The Use of Alternate Electron Sinks

Several electron sinks within the chloroplast havebeen viewed as safety valves to prevent over-reduction of the photosynthetic electron transportand consequent damage (reviewed by Niyogi, 1999).These sinks act to dissipate the energy of photonsthat is in excess of that required for useful biosyntheticprocesses and as such play an important photo-protective role.

Molecular oxygen is an important physiologicalelectron acceptor for photosynthetic electron flow. Inthe Mehler reaction (Mehler, 1951), oxygen is reducedby PS I to produce superoxide This species ishazardous so there is an efficient scavenging systemfor its removal. It is first converted to hydrogenperoxide and oxygen by superoxide dismutase, thenhydrogen peroxide is scavenged through a number ofroutes in the stroma and on the thylakoid membrane(Asada, 1999). In the thylakoid-associated pathway,hydrogen peroxide is removed by the ascorbateperoxidase catalysed conversion of ascorbate tomonodehydroascorbate (Asada, 1999). The regen-eration of ascorbate by reduction of monodehy-droascorbate (MDA) requires photosyntheticreductant in the form of reduced ferredoxin (Asada,1999) and so MDA itself acts as an electron sink.Overall the four electrons derived from the oxidationoftwo molecules ofwater to one molecule ofoxygenare accounted for by the reduction of two moleculesof oxygen to superoxide and by the reduction of twomolecules of MDA to ascorbate. This process hasbeen termed the water-water cycle (Asada, 1999)because electrons derived from the oxidation ofwaterultimately lead to the reduction of oxygen to waterwithout the net production of oxygen. These stepsform the basis of pseudo-cyclic electron flow inwhich linearelectron flow is coupled toATP synthesiswithout the net production ofreductant. Oxygen alsoplays a major photoprotective role in C3 plantsthrough its involvement inphotorespiration (Osmond,1981).

Excess NADPH can be removed from the stroma

C. Feed-forward Activation of the Calvin Cycle

Upon illumination ofchloroplasts, there is an increaseof both pH and the concentration in the stroma.These ionic conditions activate enzymes within theCalvin cycle. Superimposed on this is the specificactivation by reduced thioredoxin of several keyenzymes, such as fructose-1, 6-bisphosphatase andsedoheptulose-1, 7-bisphosphatase (Buchanan,

Chapter 30 Photosynthetic Electron Transport

by reducing oxaloacetate to malate in the malatevalve (Scheibe, 1987). This reaction is catalysed bymalate dehydrogenase and is driven by export ofmalate from the chloroplast.

III. Feedback Control of the PhotosyntheticElectron Transport Chain

A. Role of the Lumenal pH in Slowing theRate of Electron Transfer

Because several of the electron transfer reactions ofthephotosynthetic electron transfer chain are coupledto deposition of protons in the lumen, a decrease inlumenal pH automatically slows the flowofelectrons.This has been clearly demonstrated for the oxidationof water at PS II (Bowes and Crofts, 1981) and theoxidation of plastoquinol at the site of the Cytcomplex (Kramer and Crofts, 1993). In addition themid-point redox potential of plastocyanin isdependent on pH so that as the lumenal pH drops,reduction of is less favored (discussed byKramer and Crofts, 1996). As ATP synthesis at theATP synthase is coupled to the dissipation of theproton gradient, a high ratio of stromal [ATP]/

which is indicative of a slow down inmetabolic activity, will tendtomaintain a lowlumenalpH.

B. Role of Lumenal pH in Quenching ExcitationEnergy in PS II

Non-photochemical quenching (otherwise knownas non-radiative quenching) represents a collectionof mechanisms that regulate the conversion of lightenergy. In general these mechanisms involve theconversion of excitation energy to heat, therebyreducing the turnover ofthe photosystems and down-regulating photosynthetic electron transport. Non-photochemical quenching has been subdivided intocomponents including energy-dependent quenching(qE) and photoinhibitory quenching (qI). Statetransitions (Section III.C) are sometimes consideredto contribute to non-photochemical quenching. Forthe qE component, the triggering signal involveschanges in the pH of the thylakoid lumen (Krause etal., 1983, Horton et al., 1996). When the pH in thelumen is low, quenching mechanisms are activated,thereby down-regulating electron transport. Thereduction in electron flow will decrease the rate of

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proton pumping across the thylakoid membrane, andthis will in turn allow the lumenal pH to increase.Thus qE should act as a homeostatic mechanism tomaintain a relatively constant lumenal pH and rate ofelectron flux during variable illumination (Fig. 2). Itis generally assumed that the major physiologicalrole ofqE is to protect the photosystems (particularlyPS II) from photodamage during conditions ofexcessillumination. However, it is also possible that anexcessively low pH could damage lumenally-exposedprotein complexes. It is now becoming clear that thebiochemistry of the lumen is much more complexthan was thought (Fulgosi et al., 1998)

qE has been observed in numerous green plantspecies, although the extent of the quenching and therate of induction vary widely according to speciesand habitat (Demmig-Adams, 1990; Johnson et al.,1993). There is less evidence for such a process in thephycobilin-containing organisms (cyanobacteria andred algae). Quenching mechanisms have beenpostulated in cyanobacteria (Campbell et al., 1996)and red algae (Delphin et al., 1996) but it is likelythat the mechanisms are differentfromthoseoperatingin green plants.

qE in green plants is influenced by the levels ofxanthophyll carotenoids in the light harvestingantenna (Demmig-Adams, 1990). In particular, thereis a correlation between the level of zeaxanthin andthe extent and rapidity of non-photochemicalquenching (Demmig-Adams, 1990; Phillip et al.,


1996. This has led to the proposal that the conversionof violaxanthin to zeaxanthin, catalyzed byviolaxanthin de-epoxidase, plays a crucial role in qE(Demmig-Adams, 1990).

There have been a number of proposals for themechanism of qE in green plants, including: (i)energy dissipation within the Photosystem II reactioncenter, either by charge recombination (Krieger etal., 1992) or through the accumulation ofa chlorophyllcation designated (Schweitzer and Brudvig,1997) bound to the D1 polypeptide, (ii) energydissipation by direct transfer ofsinglet excitons fromchlorophylls to xanthophyll carotenoids (Frank etal., 1994) and (iii) energy dissipation by the formationof quenching aggregates of chlorophylls in the light-harvesting antenna (Horton et al., 1996).

The extent to which these different processescontribute to qE in vivo remains controversial. Themost complete mechanistic model that has beendeveloped is that of Horton and co-workers (Hortonet al., 1996; Gilmore et al., 1998). These authorspropose that the conversion of excitation energy toheat occurs in pairs or aggregates of chlorophyllsformed as a result of conformational changes inchlorophyll a/b binding proteins of the light-harvesting antenna. The conformational changes aretriggered by the protonation of specific amino acidsexposed to the lumen, or forming part of a channelthat conducts protons away from the water-oxidizingcomplex of PS II. Specific residues on the minorlight-harvesting complex (LHC) components, CP29and CP26, have been implicated (Walters et al.,1996). It is further proposed that the extent ofquenching can be modulated by the xanthophyllpigments associated with the light-harvestingcomplexes through the action of zeaxanthin as anallosteric activator of the quenching state (Phillip etal., 1996). This contrasts with the view of otherauthors who propose that zeaxanthin acts as a directquencher of singlet excitons (Frank et al., 1994).

Mutants are now being used to clarify the role ofthe xanthophyll cycle pigments and to identifyproteins involved in qE. Mutants affected inxanthophyll metabolism generally show altered qE,confirming the role ofthe xanthophyll cycle pigmentsin this mechanism (Niyogi, 1999). However, mutantsaffected in qE have also been isolated that shownormal pigment composition and xanthophyllinterconversions (Niyogi, 1999). One such mutantlacks the psbS gene (Li et al., 2000) which codes fora minor chlorophyll-binding subunit associated with

the PS II complex (Funk et al., 1995). PsbS could bethe major site for the conversion ofexcitation energyinto heat, or it could play a crucial role in sensing alow lumenal pH.

ForPS I, no such specialized quenching mechanismexists in the antenna system. The difference betweenPS I and PS II probably lies in the thermodynamicconstraints placed on the oxidized primary electrondonors. For PS I, the mid-point redox potential ofthe

couple is ~0.5 V which means that itspresence does not lead to damaging oxidative sidereactions. Hence can act as a quencher ofexcitation (Butler et al., 1979). In contrast, P680 inPS II has a much higher mid-point redox potential(~1.1V) consistent with its role in water oxidation.Unfortunately the use of as a physiologicalquencher of excess excitation would lead todeleterious oxidative reactions within PS II. To avoidthis, PS II has thus developed to be a shallow trapwith the exciton more delocalized in the antennasystem.

C. Regulation of Electron Flow through StateTransitions

State 1-state 2 transitions (state transitions for short)are a rapid mechanism that adjusts the function ofthelight-harvesting apparatus in response to signals fromthe photosynthetic electron transport chain (Allen,1992). Reduction of electron carriers between PS IIand PS I triggers a switch to state 2, in which moreexcitation energy is transferred to PS I. Oxidation ofthe electron carriers triggers a switch to state 1, inwhich more energy is transferred to PS II. Thus themechanism functions as a homeostatic control tomaintain a moderate level of reduction of theintersystem electron carriers (Fig. 3).

State transitions are observed both in green plantsand cyanobacteria (Allen, 1992). The phenomenaobserved in Chl-b and phycobilin-containingorganisms show many features in common, althoughit is clear that some features of the mechanism mustdiffer in organisms with fundamentally different light-harvesting complexes.

1. The Triggering Mechanism for StateTransitions

State transitions are triggered by changes in theredox state of electron carriers between PS II andPS I, both in green plants (Allen, 1992) and in


cyanobacteria (Mullineaux and Allen, 1990). In thecase of green plants, mutagenesis studies haveindicated that the cytochrome complex plays anessential role in state transitions (Wollman andLemaire, 1988). Flash photolysis studies have shownthe involvement of a specific quinol binding site(Vener et al., 1997). It is probable that cyanobacterialstate transitions are triggered the same way, althoughthe evidence is less conclusive because no mutantslacking the cytochrome complex are available.

Triggering by changes in the redox state ofintersystem electron carriers provides a mechanismfor homeostatic regulation of electron transport in avarying light-environment (Fig. 3). In cyanobacteria,where the respiratory and photosynthetic electrontransport chains intersect (Fig. 1, Scherer et al.,1988), state transitions may be important in regulatingthe interaction between photosynthetic and respir-atory electron transport (Schreiber et al., 1995).State transitions may also be involved in regulatingthe extent of linear versus cyclic electron transport(Section V.B). Some effects cannot straightforwardlybe explained in terms oftriggering by the redox stateof intersystem electron carriers. It is therefore likelythat other triggers are involved (i.e. a number ofdifferent sensing mechanisms trigger the sameresponse). In green plants, it has recently beensuggested that a direct light-induced conformationalchange may play a role in facilitating LHCIIphosphorylation (Zer et al., 1999). It is also possiblethat the ADP/ATP ratio is a triggering signal (Bult

et al., 1990), but as yet we have no information on themolecular mechanisms that may be involved.

2. Alterations in Function of the Light-harvesting Complexes

State transitions change the association of the light-harvesting complexes with the reaction centers. Ingreen plants, LHCII is predominantly associatedwith PS II in state 1. In state 2, a proportion of LHCIIdetaches from PS II and appears to associate withPS I instead (Allen, 1992). In cyanobacteria thephycobilisomes appear to be amobile light-harvestingantenna (Mullineaux et al., 1997) that can associatewith and transfer energy both to PS II and to PS I(Mullineaux, 1994). State transitions change therelative coupling ofphycobilisomes to PS II and PS I(Mullineaux, 1992) although it is likely that othereffects are also involved (Olive et al., 1997; Emlyn-Jones et al., 1999).

In green plants, the changes in LHCII associationare brought about by phosphorylation of the LHCIIcomplexes on threonine residues near the N-terminus(Allen, 1992). This seems to bring about aconformational change in LHCII (Nilsson et al.,1997) that causes its dissociation from PS II andreassociation with PS I. The biochemical mechanismin cyanobacteria is not known. Allen and co-workerscorrelated changes in the phosphorylation state oftwo polypeptides with state transitions in thecyanobacterium Synechococcus 6301 (Allen et al.,1985), but it now seems increasingly unlikely thatthere is a causal relationship (Emlyn-Jones et al.,1999). It is very probable that some form ofcovalentmodification is involved, with the phycobilisomecore the most probable site of modification.

There have been many attempts to isolate theprotein kinase responsible for LHCII phosphorylationin green plants. The kinase must be activated as aresult of the triggering redox signal, resulting in netphosphorylation of LHCII. The process is reversedby a phosphatase that seems to be constitutivelyactive (Allen, 1992). A number ofkinases have beenisolated from thylakoid membranes (Coughlan andHind, 1987; Gal et al., 1992; Snyders and Kohorn,1999) but we do not know for sure which, if any, ofthese kinases are involved in state transitions in vivo.Furthermore, we know nothing of the signaltransduction pathway that links the triggering redoxsignal to activation/inactivation of the kinase.

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3. The Genetic Approach to State Transitions

Mutants specifically deficient in state transitionshave recently been identified in the cyanobacteriumSynechocystis 6803 (Emlyn-Jones et al., 1999) andthe green alga C. reinhardtii (Kruse et al., 1999;Fleischmann et al., 1999). In the cyanobacterial case,the mutation results from insertional inactivation ofa specific gene that codes for a protein of previouslyunknown function, showing no homology to anypreviously-characterized gene product (Emlyn-Joneset al., 1999). The genetic approach shows greatpromise as a method of identifying the proteinsinvolved in the signal transduction pathway of statetransitions. Furthermore, mutants specificallydeficient in state transitions are a powerful tool forestablishing the physiological role(s) of themechanism. Cyanobacterial state transition mutantsgrow slower than the wild-type under very weakyellow illumination, suggesting that the primary roleof state transitions in cyanobacteria is to maximizethe efficiency of utilization of light absorbed by thephycobilisomes (Emlyn-Jones et al., 1999).

4. Higher Levels of Control

In green plants the level of LHCII phosphorylation ismaintained by the balance between the activities of aprotein kinase that is activated by a redox signal, anda constitutively-active phosphatase (Allen, 1992).Such a mechanism has a clear metabolic cost. Themaintenance of any level of LHCII phosphorylationwill require continual hydrolysis of ATP. Higherlevels of kinase and/or phosphatase activity wouldresult in more rapid state transitions and therefore aswifter response to rapidly changing illuminationconditions. However, this would come at the expenseof more rapid ATP hydrolysis. It is therefore possiblethat there are mechanisms to up-regulate or down-regulate the enzymes involved, depending on growthconditions. It could be imagined that the genes codingfor the enzymes would be strongly expressed underlow or variable illumination, but repressed understrong, constant illumination. The subject needsfurther investigation. In green plants, the phos-phorylation of LHCII is inactivated in high light(Rintamki et al., 1997). In the cyanobacteriumSynechocystis 6803, state transitions are onlyobserved in low-light grown cells (C. W. Mullineaux,unpublished).

Peter J. Nixon and Conrad W. Mullineaux

IV. Regulation of Photosystem II ActivityThe Bicarbonate Effect

It has long been known that carbon dioxide itself, inthe form of bicarbonate, is able to modulate theactivity ofthe photosynthetic electron transport chain(recently reviewed by van Rensen et al., 1999). Morespecifically depletion of bicarbonate leads to thedownregulation of PS II activity which can be restoredby subsequent addition of carbon dioxide/bicarbonate.Current models indicate two distinct effects ofbicarbonate within PS II: an acceptor side effect atthe iron quinone complex and a donor side effect atthe manganese cluster.

A. The Acceptor-side Effect

Type II photosynthetic reaction centers (RCs) suchas PS II and those found in purple non-sulfurphotosynthetic bacteria contain on the acceptor sidea non-haem iron located between two quinonemolecules termed and (reviewed by Diner etal., 1991 a). is tightly bound to the RC and acts asa one-electron carrier which reduces firstto the semiquinone anion, then after a secondcharge separation reaction within the RC to thequinol (probably in the state The quinol isfurther protonated and released from the RC in theneutral form, Because depletion of bicarbonateprimarily slows electron transfer between andafter formation of it is likely that bicarbonateparticipates in the protonation reactions involved information of (Blubaugh and Govindjee, 1988).Despite the similarity in redox factors on the acceptorin type II RCs, only PS II shows this bicarbonateeffect.

Fourier transform infrared (FTIR) experiments(Hinerwadel and Berthomieu, 1995) indicate thatbicarbonate acts as a bidentate ligand to the non-haem iron in PS II. In bacterial RCs a glutamateresidue, at position 232 in the M subunit ofRhodopseudomonas viridis, fulfils this role.Bicarbonate binding is reversible so that a widerange of anions including formate, glycolate,glyoxylate, and oxalate compete with bicarbonatefor binding to the non-haem iron and slow electrontransfer between and (Petrouleas et al., 1994).The presence of bicarbonate close to the site isthus consistent with a role in the protonation ofeither directly or indirectly. Mutation of a number ofpotentially positively charged amino-acid residues


within the D2 polypeptide (Diner et al., 1991b; Caoet al., 1991), modeled to be in the vicinity ofthe non-haem iron, reduce the affinity of PS II for bicarbonate.Based on the isolation of D1 mutants that are resistantto formate inhibition (Xiong et al., 1997, 1998), ithas also been suggested that there is an additionalbicarbonate bound within the niche which isresponsible for protonation of (van Rensen et al.,1999).

Whether the bicarbonate effects observed for PS IIin vitro also occur in vivo is a matter of debate.values for bicarbonate are estimated to be(Snel and van Rensen, 1984). The concentration ofbicarbonate in the stroma is highly dependent on pHand temperature but has been estimated to be about

at pH 6.3 and at pH 8.0 (van Rensenet al., 1999). Thus under optimal rates of photo-synthesis, when the stromal pH reaches pH 8, thelevels ofbicarbonate do not appear to be controllingPS II activity. Only when levels of carbon dioxide,hence bicarbonate, decrease, such as upon the closureof stomata through for example drought or hightemperature, would PS II activity be downregulated.This downregulation of PS II would also act toreduce the level of oxygen within the cell whichotherwise would lead to enhanced photorespirationin C3 plants. Binding ofbicarbonate to PS II has alsobeen linked to enhanced resistance to photoinhibition(Sundby, 1990; Klimov et al., 1997).

V. Cyclic Electron Flow

considered to perform two types of photophos-phorylation: cyclic and non-cyclic. In cyclic electronflow, ATP synthesis is coupled to light-inducedelectron flow in a closed system around PS I withoutthe net production ofNADPH whereas in non-cyclicmode, or linear electronflow, both ATP and NADPHare synthesized. Cyclic photophosphorylation istherefore an ATP generating process that serves toincrease the ratio of ATP/NADPH produced inphotosynthetic electron flow (reviewed in detail byFork and Herbert, 1993; Bendall and Manasse, 1995).In principle the metabolic needs of the cell can bemet through appropriate modulation of the ratio oflinear to cyclic electron flow. Although the water-water cycle also increases the ATP/NADPH ratio,cyclic electron flow is a more energy efficient way ofgeneratingATP.

In general, cyclic electron flow involves thereduction of the intersystem electron transfer chain(IEC) linking PS II and PS I (Biggins, 1974) (Fig. 1).The terms PS II-independent electron flow or non-photochemical reduction are sometimes used todescribe the various non-PS II electron-transferpathways able to feed electrons into the IEC. Inaddition the term cyclic is often used loosely todescribe all PS II-independent pathways for thereduction of PS I, including those leading to reductionof the IEC through dehydrogenases (Complex I and II)(Fig. 1).

According to the Z scheme, the linear transport ofan electron from water to is thought totranslocate one proton at PS II and two protons at Cyt

ifa Q cycle operates constitutively (Rich, 1988).In total three protons are translocated per electrontransported. If the synthesis of one molecule ofATPrequires the influx offourprotons at the ATP synthase(van Walraven et al., 1996), then the ratio of ATP/NADPH produced by linear electron flow is about1.5. This value will depend on the amount ofpassiveproton leakage across the thylakoid membrane athigh pH, which would lower the effective number ofprotons pumped by the electron transport chain (Berryand Rumberg, 1999), and the precise number ofprotons required to synthesize ATP (3, 4 or indeed anon-integral value).

For C3 plants in ambient air, the ratio ofNADPH/ATP needed to fix and perform photorespirationis about 1.56 (discussed by Foyer and Harbinson,1997). HenceATP production through cyclic electronflow is not required for C3 plants during steady-statephotosynthesis and indeed there is little evidence forHistorically, thylakoid membranes have been

B. The Donor-side Effect

A variety of indirect evidence has also hinted to thepossibility that bicarbonate may have a role on thedonor side of PS II (reviewed by Stemler, 1999; vanRensen et al., 1999; Klimov and Baranov, 2001).Recently Klimov and co-workers have interpretedFTIR difference spectra obtained for the donor sideof PS II in terms of actual ligation of bicarbonate tothe Mn cluster (Yruela et al., 1998). In the absence ofbicarbonate assembly of the Mn cluster is also lessefficient (Allakhverdiev et al., 1997). Further work isrequired to clarify these donor side effects particularlyas it is plausible that binding of bicarbonate on theacceptor side of PS II causes structural effects thatextend across the membrane to the donor side.


its existence under these conditions (Herbert et al.,1990), It has been suggested that the main role forcyclic electron flow in C3 plants is to regulate electronflow through acidification of the lumen underconditions where electron sinks for photosyntheticelectron transport are depleted (Heber and Walker,1992;Katona et al., 1992; Heber et al., 1995). For C4photosynthesis, there is a requirement of 2.5-3 ATP/NADPH, much greater than that can be generated inlinear mode. The extra ATP is considered to begenerated by cyclic electron flow around PS I. Foralgae and cyanobacteria, which need to adapt tolarge fluctuations in environmental conditions throughenergy dependent processes, cyclic electron flowappears to be an important component of photo-synthetic electron flow (reviewed by Bendall andManasse, 1995).

The precise rates of cyclic electron transfer in vivoare, however, difficult to assess because of thetechnical problems deconvoluting linear and cyclicelectron flow, although a variety of techniques havebeen applied including photoacoustic spectroscopy(Herbert et al., 1990) and the rate of re-reduction of

using optical spectroscopy (Fork and Herbert,1993; Bendall and Manasse, 1995).

stromal reductant ofthe membrane bound intersystemchain of electron carriers in chloroplasts (Arnon,1991). This assignment stemmed from experimentsin vitro in which ferredoxin was added back tothylakoid membranes in the absence ofother stromalcomponents (Tagawa et al., 1963). Whether this orsome other cycle actually operated in vivo could notbe assessed.

For many years the mechanism by which reducedferredoxin reduced the intersystem chain was thoughtto involve the direct reduction of cytochromeThis hypothesis was based on the mistakenassumption that the binding site of antimycin A, aknown inhibitor of ferredoxin-mediated cyclicelectron flow, was the Cyt complex (discussed inBendall and Manasse, 1995). The actual binding sitefor antimycin is still uncertain although there is somepreliminary evidence to indicate an association withPS I (Davies and Bendall, 1987) and also that theremay be an additional weaker binding site in the Ndhcomplex (Endo et al., 1998).

The inhibition of ferredoxin-mediated cyclicelectron flow by inhibitors of the site of the Cyt

complex, together with other data (Bendall andManasse, 1995), has led to models in which cyclicelectron flow involved reduction ofthe plastoquinonepool followed by its oxidation by Cyt Reductionofthe plastoquinone pool by reduced ferredoxin wastherefore hypothesized to be catalyzed by aferredoxin-plastoquinone reductase (FQR) (Moss andBendall, 1984). The molecular identity of FQR stillremains unknown. Recent evidence indicates thatFQR may include a cytochrome, termed Cyt b-559(Fd) (Miyake et al., 1995). Whether thiscytochrome is truly part ofFQR or is reduced throughredox equilibration with the plastoquinol pool isuncertain.

Given that there may be an antimycin-binding siteclose to PS I, attention has focused on whether PS Icontains FQR activity (Bendall and Manasse, 1995).Mutation ofthepsaE gene, which encodes a stromallyexposed subunit of PS I (Klukas et al., 1999), inSynechococcus (Yu et al., 1993) impairs cyclicelectron flow when assayed by the dark reductionkinetics of However photoacoustic spectros-copy has failed to detect changes in cyclic electronflow in this mutant (Charlebois andMauzerall, 1999).Because PsaE is thought to be involved in the bindingof ferredoxin (Barth et al., 1998) and ferre-doxin: oxidoreductase (FNR) to PS I (vanThor et al., 1999a), the role ofPsaE in cyclic electronflow may be to stabilize the binding of these

A. Pathways of Cyclic Electron Transfer

As yet the precise pathways ofcyclic electron transferremain unclearalthough the consensus favorsmultipleroutes (Hosler and Yocum, 1985; Ravenel et al.,1994). Currently two cycles are thought to dominate.One mediated by the Ndh complex involves thepyridine nucleotide pool and the other consists offerredoxin-mediated reduction of the IEC. Fromstudies on cyanobacteria (Yu et al., 1993) and algae(Ravenel et al., 1994) in vivo, the rates of cyclicelectron flow have been estimated to be less than10% of the maximum linear rates. Of this, recentestimates using isolated barley thylakoids suggestthat the rates ofNdh-mediated cyclic electron flow isabout 3% ofthe linear rates and the rate offerredoxin-dependent cyclic electron flow is about 5% (Teicherand Scheller, 1998). Under stress conditions such asphotoinhibition and nutrient deprivation, thecontribution of cyclic electron flow may, however,become more important (discussed by Fork andHerbert, 1993).

1. Ferredoxin Cycles

Historically reduced ferredoxin was considered the


Following the sequencing of the tobacco (Shinozakiet al., 1986) and liverwort (Ohyama et al., 1986)chloroplast genomes, several open reading frames(ORFs) were identified with significant sequencesimilarities to mitochondrial and eubacterial genesencoding subunits of the respiratory NADH:ubi-quinone oxidoreductase (also known as Complex Ior type I NADH dehydrogenase) (Fearnley andWalker, 1992). These ORFs were consequentlydesignated ndh genes (NADH dehydrogenase). ndhgenes have now been identified in cyanobacteria andin the chloroplast genomes of some but not all greenalgae (Turmel et al., 1999). The absence of ndh genesfrom various plastomes may reflect transfer to thenucleus or deletion from the organism. In the case ofblack pine, ofthe 11 ndh genes usually found in landplants, four have been lost from the plastome andseven remain as pseudogenes (Wakasugi et al., 1994).

Despite little evidence at the time to suggest a rolefor the 11 ndh genes in encoding a chloroplastanalogue ofComplex I (Fearnley and Walker, 1992),recent biochemical (Sazanov et al., 1998) and geneticstudies (Burrows et al., 1998; Kofer et al., 1998;Shikanai et al., 1998) have provided strong evidencefor this assignment (also reviewed by Nixon, 2000).The low abundance of the Ndh complex in thechloroplast, estimated to be at 1.5% the levels of PSII in tobacco (Burrows et al., 1998), helps to explainwhy it had escaped detection for so long. Forchloroplasts, theNdh complex is located in the stromallamellae (Nixon et al., 1989; Sazanov et al., 1998)whereas in cyanobacteria there is still uncertaintyabout its location although it would appear to be inthe thylakoid (Norling et al., 1998) and possiblycytoplasmic (Berger et al., 1991) membranes ofSynechocystis 6803. Analysis of the cyanobacterialNdh complex is also complicated by the presence ofmultigene families which may reflect structural andfunctional heterogeneity (Ohkawa et al., 1998;Klughammer et al., 1999).

Biochemical analysis ofthe Ndh complex remainsincomplete. A 550 kDa Ndh complex with anassociated NADH:ferricyanide oxidoreductase

The enzyme activity of the Ndh complex has beenassayed mainly through the use of pyridinenucleotides as electron donors and artificial electronacceptors such as water-soluble quinones andferricyanide, which probably accept electrons fromthe iron-sulfur clusters within the complex ratherthan from the natural quinone binding site (Sazanovet al., 1998).

Biochemical characterization ofthe Ndh complexhas proved difficult because of its low abundance inchloroplast thylakoid membranes, its lability, thelack of a convincinginhibitor, possiblemitochondrialcontamination in chloroplast samples and thepresence of multiple NAD(P)H dehydrogenaseactivities, including a high level of NADPH:ferri-cyanide oxidoreductase activity due to ferrodoxinNADP reductase (FNR). This latter difficulty mayexplain why FNR has been suggested to be acomponent of the Ndh complex (Guedeney et al.,1996). Consequently it is extremely difficult to assignenzyme activities within the thylakoid membrane tothe Ndh complex without additional purification orverification. The instability ofthe Ndh complex alsoraises the possibility that the activity of the Ndhcomplex and its possible role in cyclic photophos-phorylation may have been overlooked in earlyexperiments.

Attempts to study the Ndh complex in tobaccothylakoid membranes, for which there are engineeredndh knock-out mutants available to help assign theactivities of the complex, have proved difficultbecause of the apparent instability of the complex(Endo et al., 1998). Such behavior may explain whyreduction of the plastoquinone pool in isolatedthylakoid membranes using NAD(P)H is so low(Rich et al., 1998). In membranes the reduction ofthe plastoquinone pool by NADPH is known to bestimulated by ferredoxin (Mills et al., 1979; Rich etal., 1998). Rather than an Ndh-catalyzed reaction,

components close enough to a quinone binding sitewithin the PS I core complex to allow plastoquinonereduction.

2. NAD(P)H Cyclesa. Identification of the NADH Dehydrogenase(Ndh) Complex

activity has been isolated from pea thylakoids(Sazanov et al., 1998). Of the estimated 16 subunits,five have been so far assigned to plastid Ndh proteins.For cyanobacteria, a 376 kDa hydrophilic sub-complex of the Ndh complex, which displays anNADPH:ferricyanide oxidoreductase activity, hasbeen isolated from Synechocystis PCC 6803 (Matsuoet al., 1998). Of the nine subunits present, onlyNdhH could be assigned.

b. Substrate Specificity of the Ndh Complex invitro and in vivo


the possible pathway may involve the membrane-bound FNR-mediated reduction of ferredoxin byNADPH followed by reduction ofthe plastoquinonepool using FQR.

The nature of the stromal reductant oxidized bythe isolated detergent-solubilized plastid Ndhcomplex, of different degrees of purification, is stillunder debate with there being evidence for aspecificity for NADH (Sazanov et al., 1998; Elortzaet al., 1999), NADPH (Guedeney et al., 1996) orboth NADH and NADPH (Quiles and Cuello, 1998;Funk et al., 1999). For an isolated sub-complex ofthe Synechocystis 6803 Ndh complex, a specificityfor NADPH has been reported (Matsuo et al., 1998;whilst for the complex in thylakoid membranes,NADPH, NADH and reduced ferredoxin have allbeen implicated (Mi et al., 1995).

It is possible that in vivo the Ndh complex may berather promiscuous interacting with a number ofdifferent reductants depending on plant species andphysiological state. The resulting holocomplex(es)containing the plastid ndh gene products plus the asyet uncharacterized electron input module(s)(Friedrich et al., 1995) may be rather unstable. Theidentity of the subunits that constitute the sub-complex involved in NAD(P)H oxidation isparticularly intriguing. As yetno obvious homologuesof the NADH-binding subunits found in otherComplex I species have been identified (Friedrich etal., 1995).

Although there has been controversy over whetherplants can tolerate the inactivation of plastid ndhgenes (Maliga and Nixon, 1998; Koop et al., 1998)the consensus now appears that under normal growthconditions loss ofthe Ndh complex does not result inan obvious growth defect (Burrows et al., 1998;Shikanai et al., 1998). Plastidndh mutants do howeverappear to be more sensitive to the effects ofhigh lightdamage, for which the reason is not yet clear (Endoet al., 1999). For cyanobacteria, the photoautotrophicgrowth of several, but not all, ndh mutants shows arequirement for enhanced levels (Ohkawa et al.,1998; Klughammer et al., 1999). This observation isconsistent with a role for Ndh subunits in generatingthe ATP needed for the active transport of dissolvedinorganic carbon into the cell.

NAD(P)H:plastoquinone oxidoreductase has led tospeculation that it may have a dual function inphotosynthetic systems (Burrows et al., 1998). In thelight one role for the Ndh complex may be to catalyzecyclic electron flow around PS I (either directly orthrough appropriate redox poising of the IEC),whereas in the dark the Ndh complex may functionin respiration. Figure 4 summarizes the possibleelectron pathways involving an NADH-specific Ndhcomplex of the chloroplast. By analogy to thesituationin purple bacteria, it is also possible that the Ndhcomplex may catalyze the reverse reaction in whichthe plastoquinol-mediated reduction of toNAD(P)H is coupled to the proton motive force.

Experimental data to support a role of the Ndhcomplex in cyclic electron flow first came fromstudies on cyanobacterial ndh mutants (Mi et al,1992; Yu et al., 1993) and more lately from analysisof chloroplast ndh mutants for which: (i) the post-illumination reduction of the plastoquinone pool isabsent or strongly attenuated (Burrows et al., 1998;Kofer et al., 1998; Shikanai et al., 1998), (ii) the rateof re-reduction of is slower in the dark afterfar-red induced oxidation of P700 (Burrows et al.,1998) and (iii) accumulates more rapidly underfar-red illumination, after a period of white lightillumination to reduce the pool of stromal reductant(Shikanai et al., 1998).

Interestingly under conditions of water stress, whenthe levels of electron acceptors for linear flow arelimited, the ndh mutants show a reduced ability toquench chlorophyll fluorescence non-photochem-ically during the early stages of the induction ofphotosynthesis in dark-adapted plants (Burrows etal., 1998). Such behavior is consistent with a role forthe Ndh complex under conditions when the Calvincycle enzymes are not fully activated and there is abuild up of reductant in the stroma. Cyclic electronflow under these conditions would contribute to thepH gradient across the thylakoid enhancing qE, thusdownregulating PS II, as well as removing stromalreductant which could lead to the generation ofreactive oxygen species.

Further indirect evidence to support a role for theNdh complex in cyclic electron flow has come fromthe finding that Ndh proteins are located in thestromal lamellae close to PS I (Nixon et al., 1989)and show elevated levels in the bundle sheath cells ofC4 plants which lack PS II and carry out high levelsof cyclic photophosphorylation (Kubicki et al., 1996).In the latter case, it is possible that a significant

c. Role of the Ndh Complex

Despite the lack ofa consensus concerning the precisesubstrate specificity of the Ndh complex, a role as an


electron transfer pathway involves the oxidation bythe Ndh complex of NAD(P)H derived from malatedehydrogenation rather than the oxidation ofNAD(P)H produced by PS I activity. A role for theNdh complex outside photosynthesis is also suggestedfrom its detection in non-photosynthetic tissue inhigher plants (Berger et al., 1993).

B. Regulation of Cyclic Electron Flow

Although many of the components involved in cyclicelectron flow have yet to be clarified, it is clear thatfor optimum rates of cyclic electron transport, theremust be appropriate redox poising of the system sothat it is neither too oxidizing nor too reducing. Thisisprobably achieved in vivo by concurrent cyclic andlinear electron flow (Arnon and Chain, 1975). Adynamic picture of cyclic electron flow would thenbe of cycle(s) in which electrons that leak out arereplaced through both photochemical (PS II) andrespiratory processes (e.g. oxidation of NADH bythe Ndh complex).

The mechanism whereby cyclic flow is switchedon at the expense of linear electron flow is unknown.Under normal steady state linear electronflow, whenthe Calvin cycle is fully activated, the cycling ofelectrons from NAD(P)H or reduced ferredoxin to

the IEC is unlikely to compete with PS II-dependentreduction ofthe plastoquinone pool. In any event, theferredoxin and NADP pools are relatively oxidizedunder these conditions because of forward electrontransfer. Cyclic electron flow would however bepromoted if the levels of NADPH and reducedferredoxin increased because of a reduction in therate of fixation. Such physiological situationsinclude (i) the early stages in the induction ofphotosynthesis in dark-adapted plants (Takahama etal., 1981), (ii) a reduction in the levels ofthe electronacceptors, and through for example thewater-stress induced closure of stomata (Katona etal., 1992), and (iii) the inhibition of the Calvin cycleenzymes through for instance heat stress (Weis, 1981;Havaux, 1996).

An increase in the activity of PS I compared withPS II may also act to enhance cyclic electron flow bypoising the plastoquinone pool in a more oxidizedstate. This may occur physiologically when (i) PS IIis downregulated through means ofa state-transition,(ii) PS II has been inactivated through light damage(Canaani et al., 1989), and (iii) there are low levels ofPS II complex such as in the bundle sheath cells ofC4 plants.

How is the ratio of cyclic to linear electron flowcontrolled by the metabolic demands of the cell? In


principle, changes in the level of ATP/ADP mayregulate cyclic electron flow through allostericmodulation of FQR or the Ndh complex. However,neither ATP norADP modulate FQR activity (Clelandand Bendall, 1992). Effects ofATP on Ndh activityhave not yet been tested although preliminary workhas indicated that the thylakoid NAD(P)H dehy-drogenase activity, assumed to be due to the Ndhcomplex, is activated by light and incubation in low-ionic-strength buffer (Teicher and Scheller, 1998).

There is good evidence, particularly in green algae,that the metabolic control ofcyclic over linear electronflow appears to be exerted through state transitions(Section III.C). For C. reinhardtii state transitionsare more dramatic than in higher plants (Delosme etal., 1996). State 2 is linked with cyclic electron flowwhereas State 1 promotes linear electron flow (Finazziet al., 1999). In state 2 there is little PS II-dependentoxygen evolution (Finazzi et al., 1999) which reflectsnot only a reallocation of excitation energy to PS Ibut also the migration of Cyt away from PS II toPS I (Vallon et al., 1991) so that linear electron flowis further diminished in favor ofcyclic electron flow.

Thus when ATP demand is high, state 2 would befavored. Such a case occurs when nitrogen-limitedcells of the green alga Selenastrum minutum aretransferred into an replete medium (Turpin andBruce, 1990). assimilation requires a high ATP/NADPH ratio with the extra ATP provided by cyclicelectron flow. Chlorophyll fluorescence measure-ments confirm that without changing light qualitythe cells go into state 2 to enhance PS I activity.Concomitantly dark respiration is stimulated and

fixation is decreased probably because of theneed to transfer carbon skeletons from the Calvincycle (in the chloroplast) to the TCA cycle (in themitochondrion) in order to synthesize amino acids(Elrifi and Turpin, 1986). Consequently theavailability of electron acceptors in the chloroplastdeclines to produce redox conditions that wouldfurther favor cyclic electron transport and cyclicphotophosphorylation. Transition to state 2 alsooccurs in response to hyperosmotic stress inChlamydomonas (Endo et al., 1995). The role ofstate transitions in regulating cyclic electron flowcan now be tested more directly using recently isolatedstate transition mutants (Kruse et al., 1999).

How cellular ATP levels are sensed within thechloroplast and converted into a state transition isuncertain. Inprinciple metabolite translocators withinthe inner envelope membrane of chloroplasts allow

changes in metabolite levels outside the chloroplastto be detected within the stroma. This signal can thenbe transduced into an appropriate response withinthe thylakoid membrane to regulate photosyntheticelectron flow.Forinstanceinhibitionofmitochondrialrespiration in C. reinhardtii in the dark, to preventATP synthesis, leads to a more reduced thylakoidplastoquinone pool (Gans and Rebeille, 1990;Bennoun, 1994) and a transition to state 2 (Gans andRebeille, 1990) probably via a redox-controlledLHCII kinase. Cyclic electron flow would then befavored upon re-illumination. The mechanism ofplastoquinone reduction has been suggested (Gansand Rebeille, 1990; Bult et al., 1990) to be (i) areduction in cellular ATP levels which is sensed inthe chloroplast by a drop in ATP, (ii) an increase inthe rates of starch degradation and chloroplastglycolysis to synthesize chloroplastic ATP, (iii) anincrease in chloroplast NAD(P)H pools throughglycolysis and the oxidative pentose phosphatepathway, and (iv) reduction of the plastoquinonepool using an NAD(P)H:PQ oxidoreductase. Whetherit is the ATP level (Bult et al., 1990) or the redoxstate of the plastoquinone pool which exerts themajor control on state transitions in C. reinhardtii invivo is a matter of debate (Finazzi et al., 1999).

It is possible that the need for enhanced cyclicelectron flow results in the synthesis ofnew electrontransfer components or the enhanced accumulationof pre-existing proteins. Under salt stress conditions,Synechocystis 6803 produces a flavodoxin which cansubstitute for ferredoxin and which promotes cyclicover linear electron flow (Hagemann et al., 1999),Under photooxidative stress conditions, when PS IIactivity declines, expression of the NdhA subunit ofthe Ndh complex increases (Martin et al., 1996).

Given that PS II and PS I in chloroplasts arespatially segregated (Andersson and Anderson, 1980),it is possible that cyclic electron flow is restricted tothe stromal lamellae and linear flow to the grana.Upon state transitions, a further redistribution ofelectron transfer complexes within the membranemay occur, such as that observed for Cyt in C.reinhardtii and maize thylakoids (Vallon et al., 1991),to promote cyclic over linear electron flow.

FNR has also been widely speculated as having adirect role in cyclic electron flow but as yet there isno definitive evidence (Bendall and Manasse, 1995).In cyanobacteria, there are two populations of FNR,one directly associated with the thylakoid membraneand one attached to the phycobilisomes (van Thor et


al, 1999b). It is possible that the two populations ofFNR have different electron transport roles.

As a cyanobacterial psaE/ndhF double mutantshows little cyclic electron flow (Yu et al., 1993), itwould appear that cyclic electron flow in thisparticular case is dominated by the Ndh and PsaEpathways. But what regulates the type of cycle? Itcan be hypothesized that the relative reduction levelsof NAD(P)H and ferredoxin is an importantdeterminant in the type of cycle, whether ferredoxin-or Ndh-mediated, used around PS I. Because thereduction of by reduced ferredoxin (via FNR)is favored, in the event that there is an accumulationof reductant on the acceptor side of PS I, it would beanticipated that the ratio of wouldincrease before that of reduced ferredoxin/oxidizedferredoxin. Such a simplistic analysis would suggestthat the Ndh-mediated cycle is more likely to occurin vivo than ferredoxin cycles. Analysis of tobaccondh mutants support this view (Burrows, 1998).Under moderate illumination the post-illuminationreduction of the plastoquinone pool is absent in ndhmutants compared with WT (Burrows et al., 1998;Kofer et al., 1998; Shikanai et al., 1998), suggestinga block in the cycling of electrons into the pool.Under more extreme illumination conditions somepost-illumination reduction of the pool now occurs,perhaps mediated by reduced ferredoxin that couldnot accumulate under the lower intensity light(Burrows, 1998).

Where the Ndh complex is specific for NADHrather than NADPH, then cyclic electron flow couldinvolve a transhydrogenase reaction to produceNADH from NADPH (possibly via FNR) or the useof NAD- and NADP-dependent dehydrogenases in asubstrate cycle (Sazanov et al., 1998). Recentlychloroplasts have been shown to contain an NAD-linked malate dehydrogenase (Berkemeyer et al.,1998) in addition to the light-activated NADP-dependent malate dehydrogenase.

In 1982, Pierre Bennoun coined the term chloro-respiration to describe respiration within thechloroplast as distinct from mitorespiration whichoccurs in the mitochondrion (Bennoun, 1982). Achloroplast respiratory chain was invoked to explaina number of observations concerning the redox state

of the plastoquinone pool in green algae in darkness.It was proposed that the plastoquinone pool could bereduced through the action of an NAD(P)Hdehydrogenase and on the basis of inhibitor studiesthat it could be oxidized by oxygen at an oxidase. Atthe same time an electrochemical gradient could begenerated across the thylakoid. While there is nowample evidence for non-photochemical reduction ofthe plastoquinone pool in a range of plants (Groom etal., 1993) the original conclusions concerning thepresence of thylakoid oxidases are less convincing(Bennoun, 1998). It is now clear that the inhibitors ofthe putative chloroplast oxidase were in fact inhibitingthe mitochondrial oxidase which then caused theindirect reduction of the chloroplast plastoquinonepool (Bennoun, 1994). Because of mitochondrial/chloroplast interactions (Section VII), mitochondrialoxidases are in principle able to drive the oxidationof the plastoquinone pool (Bennoun, 1998) throughreverse electron flow from plastoquinol toat a proton pumping NAD(P)H dehydrogenase, withthe reaction driven by the proton electrochemicalgradient across the membrane.

Despite the fact that the original data (and possiblylater results from higher plants) can no longer beinterpreted unambiguously, there are now biochem-ical and genetic data to support the concept ofchlororespiration. First, the Ndh complex found inhigher plant chloroplasts, but not yet in C. reinhardtii,appears to fulfill the role of the NAD(P)H dehydro-genase (see above). Second, a protein, designatedImmutans, with strong sequence similarities to thealternative oxidases of plant mitochondria has beendetected in chloroplasts of Arabidopsis thaliana (Wuet al., 1999; Carol et al., 1999). It is plausible thatthese two components together with plastoquinonemay form a chloroplast respiratory chain (Fig. 4).Additional or alternative plastoquinone reductaseand oxidase activities may be present depending onthe species and physiological state. For instancethere appears to be multiple thylakoid NADHdehydrogenase activities in tobacco thylakoids(Cornac et al., 1998). A plastoquinol peroxidase hasalso been suggested to be a component of chloro-respiration (Casano et al., 2000).

The rate of chlororespiratory electron transfer inthe dark is, however, quite small with rates insunflower estimated to be at only about 0.3% of thelight-saturated photosynthetic electron flow (Feild etal., 1998). The role of chlororespiration is uncertainalthough speculation has focused on metabolism in

VI. Chlororespiration and CyanobacterialRespiration


the dark such as the maintenance of a proton motiveforce (for ATP synthesis and activation of the ATPsynthase) and the regulation of pyridine nucleotidelevels for efficient starch mobilization. Given thelow rates of electron transfer in chlororespiration, itis unlikely that it makes a significant contribution toATP synthesis in the light in mature chloroplasts. Inimmature or non-photosynthetic plastids, thecontribution may become important. However theNdh component of the chain may have a role incyclic electron flow in the light as described above,possibly directly or by poising the IEC in anappropriate state for cyclic electron transfer viaferredoxin-mediated pathways.

The immutans mutant lacking the putativechloroplast alternative oxidase shows a variegatedphenotype with leaves consisting of white and greensectors. The white sectors develop because insufficientcarotenoid is made to protect from photooxidativestress (Carol et al., 1999; Wu et al., 1999). Lack ofImmutans may inhibit phytoene desaturation becausethe plastoquinone pool is too reduced.

For cyanobacteria there are a number of potentialthylakoid plastoquinone reductases and oxidases(early work reviewed by Schmetterer, 1994). InSynechocystis 6803, there are three open readingframes encoding potential type 2 NADH dehydro-genases, designated ndbA-C (Howitt et al., 1999). Incontrast to the type 1 dehydrogenases, type 2dehydrogenases consist of single subunits, lack iron-sulfur centers and do not pump protons across themembrane. Because the activity of the Synechocystisndb gene products appears to be low under laboratorygrowth conditions it has been suggested that theymay act as redox sensors and serve a regulatoryfunction (Howitt et al., 1999). The activity of theNdb proteins under stress conditions, such as iron-depletion when the Ndh complex may be less active,awaits examination as does confirmation that theNdb subunits are located in the thylakoid rather thancytoplasmic membrane.

The genome of Synechocystis 6803 also containsthree sets of genes for terminal respiratory oxidases:a cytochrome cytochrome c oxidase (CtaI),a possible cytochrome bo-type quinol oxidase (CtaII)and a putative cytochrome bd quinol oxidase (Cyd).Recent conclusions based on the analysis ofengineered mutants are that CtaI is the major oxidasein thylakoid membranes, Cyd is mainly located inthe cytoplasmic membrane and that CtaII plays only

a minor role in cellular respiration under theconditions tested (Howitt and Vermaas, 1998).Interestingly there is no obvious cyanobacterialhomologue of Immutans.

Chloroplasts, like mitochondria, possess a numberof protein transporters within the inner envelopemembrane which enable the selective passage ofmetabolites into and out of the organelle (Flgge,1998). Although there is a chloroplast ADP/ATPantiport system, there is no translocatorofNAD(P)H.Instead reducing equivalents are transported indirectly(Heineke et al., 1991). For instance the oxaloacetate/malate antiporter allows the transport of malate whichcan be oxidized by andmalate dehydrogenases in a number of cellcompartments to yield NADH and NADPH respec-tively. The chloroplast is thus in metaboliccommunication with the rest of the cell and potentially(on a slower time scale) with the rest of the plant.

The potential importance of mitochondrial-chloroplast interactions in photosynthesis has beenhighlighted by the isolation of a photoautotrophicsuppressor of the chloroplast mutant FUD50 ofC. reinhardtii which lacks the chloroplast ATPsynthase (Lemaire et al., 1988). The suppressor strainstill lacks the chloroplast ATP synthase and hastherefore had to develop an alternative route toaccumulate the necessary ATP in the chloroplast forthe Calvin cycle. A possible pathway involves theexport from the chloroplast to the cytosol of reducingequivalents in the form of dihydroxyacetonephosphate which is first converted to glyceraldehyde3-phosphate and then dehydrogenated to producecytosolic NADPH. NADPH can be then oxidized atthe external surface of the inner mitochondrialmembrane and ATP synthesized through oxidativephosphorylation. ATP is then exported from themitochondrial matrix to the chloroplast stroma usingADP/ATP translocators in both organelles. Thispathway is energetically more costly than ATPsynthesis by the chloroplast ATP synthase and doesnot seem to operate in the WT.

The role of the mitochondrial electron transportchain in photosynthetic electron flow has also beenimplicated from studies of PS I and Cyt deficient

VII. Interaction between Chloroplasts andMitochondria


strains of C. reinhardtii. Using mass spectrometrymeasurements to differentiate between oxygen uptakeand oxygen release, a permanent electron flow couldbe established between water oxidation at PS II andoxygen reduction at mitochondrial terminal oxidases(Peltier and Thibault, 1988). In the absence of PS I,the reductant NAD(P)H was hypothesized to beproduced by an NAD(P)H dehydrogenase driven inreverse by the proton motive force (Peltier andThibault, 1988). As yet the identity of this complex isunknown although it would appear not to be the Ndhcomplex (Cornac et al., 1998). Transport of thereductant to the mitochondrion for oxidation wouldoccur through metabolite shuttles as described above.

Evidence that the mitochondrial ATP synthesismay be important for high rates of photosynthesis inWT has come from studies on barley leaf protoplasts(Krmer et al., 1988). Selective inhibition ofmitochondrial respiration using oligomycin resultedin an approximate 40% inhibition in oxygen evolution.

The role of the mitochondrion in photosyntheticelectron flow may be important at two levels. First,from energetic considerations it has been suggestedthat ATP production through the mitochondrialoxidation of reductant produced by photosyntheticelectron transport would be more efficient than thatthrough cyclic electron flow in the chloroplast(Krmer et al., 1988). Thus when the ATP/NADPHratio needs to be increased, reductant may be oxidizedwithin the mitochondrion. Indeed the mitochondrialoxidation of photosynthetic reductant is consideredthe basis of the phenomenon oflight-enhanced darkrespiration(Xue et al., 1996). Second, mitochondrialactivity acts as an additional electron sink forphotosynthetic electron transport and so may have arole in protecting the photosynthetic electron transportchain from damage (through the malate valve).

It is likely that the power of the genetic approachesnow being applied in cyanobacteria, green algae andhigher plants will lead to the identification of thegenes and gene products required for all the knownphotosynthetic regulatory mechanisms within thenext few years. This in turn should lead to thedetermination of the basic biochemistry of themechanisms. Some other questions are then likely tocome to the fore.

Even unicellular photosynthetic organisms areexceptionally complex systems, capable of res-ponding to their environment in numerous and subtleways. This is nicely illustrated by the completesequencing of the genome of the cyanobacteriumSynechocystis 6803 (Kaneko et al., 1996). In additionto a very large number of genes of completelyunknown function, there are more than 80 openreading-frames coding for components of putativetwo-component signal transduction systems (Mizunoet al., 1996). We still have no idea of the function ofthe majority of these genes. It seems almost certainthat any photosynthetic organism will respond to anyenvironmental change with a whole suite of responses.We may have very little hope of dissecting out allthese responses using conventional physiologicaland biophysical techniques. Specific mutants mayoffer the best way forward. Mutants can be used toaddress the question in two ways: First, theinactivation of specific genes may lead to thecharacterization of new regulatory mechanisms. Forexample, the inactivation of a putative DNA-bindingresponse regulator in Synechocystis produces aphenotype suggesting that the response regulator isinvolved in a long-term mechanism for regulatingthe interaction of phycobilisomes with reactioncenters, a mechanism that had not previously beensuspected (Ashby and Mullineaux, 1999). Second, ifa specific regulatory mechanism is inactivated by amutation, what other responses can be seen in themutant? The inactivation of one or more regulatorypathways can be a good way to reveal other controlmechanisms that were masked in the wild-type. Forexample, a mutational study shows that statetransitions in cyanobacteria in fact consist of at leasttwo independent responses (Emlyn-Jones et al.,1999).

A. Are there other Regulatory Mechanismsstill to be Characterized?

VIII. Questions for the Future

B. What are the Physiological Roles of theAdaptation Mechanisms?

Our current thinking on these questions is basedlargely on plausible guesswork, combined withcomparative studies of the occurrence of theregulatory mechanisms in organisms in differentenvironments. The isolation of mutants with highlyspecific phenotypes will allow a much more direct


approach. If we take a mutant specifically deficientin a particular regulatory mechanism, and place it ina particular environment, how will it fare incomparison to the wild type? This approach has beenused, for example, to show that state transitions incyanobacteria are important for regulating theefficiency of utilization of phycobilisome-absorbedlight (Emlyn-Jones et al, 1999).

C. Are there Cell- and Tissue-specific Responsesin Multicellular Photosynthetic Organisms?

Mechanisms for regulating photosynthetic electrontransport have mainly been characterized inunicellular organisms, or in isolated chloroplasts. Itseems almost certain that, in intact plants, there willbe different responses in different cells and differenttissues. Fluorescence video imaging can be used toshow this at the macroscopic level (Scholes andRolfe, 1996). This could be extended to microscopicscales by confocal fluorescence microscopy andmicrovolume spectroscopy. Tissue-specific geneexpression studies should also provide a powerfulapproach to this question.

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