Chapter 30

Regulation of Photosynthetic Electron Transport

Peter J. Nixon*
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, U.K.

Conrad W. Mullineaux
Department of Biology, University College London, Darwin Building, Gower St, London WC1E 6BT, U.K.

Summary I. Introduction II. Background Concepts A. Photosynthetic Electron Transport and the Z Scheme B. Coordination of Electron Transport and Metabolism C. Feed-forward Activation of the Calvin Cycle D. The Use of Alternate Electron Sinks III. Feedback Control of the Photosynthetic Electron Transport Chain A. Role of the Lumenal pH in Slowing the Rate of Electron Transfer B. Role of Lumenal pH in Quenching Excitation Energy in PS II C. Regulation of Electron Flow through State Transitions 1. The Triggering Mechanism for State Transitions 2. Alterations in Function of the Light-harvesting Complexes 3. The Genetic Approach to State Transitions 4. Higher Levels of Control IV. Regulation of Photosystem II ActivityThe Bicarbonate Effect A. The Acceptor-side Effect B. The Donor-side Effect V. Cyclic Electron Flow A. Pathways of Cyclic Electron Transfer 1. Ferredoxin Cycles 2. NAD(P)H Cycles a. Identification of the NADH Dehydrogenase (Ndh) Complex b. Substrate Specificity of the Ndh Complex in vitro and in vivo c. Role of the Ndh Complex B. Regulation of Cyclic Electron Flow VI. Chlororespiration and Cyanobacterial Respiration VII. Interaction between Chloroplasts and Mitochondria VIII. Questions for the Future A. Are there other Regulatory Mechanisms still to be Characterized? B. What are the Physiological Roles of the Adaptation Mechanisms? C. Are there Cell- and Tissue-specific Responses in Multicellular Photosynthetic Organisms? References

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*Author for correspondence, email: p.nixon@ic.ac.uk Eva-Mari Aro and Bertil Andersson (eds): Regulation of Photosynthesis, pp. 533555. 2001 Kluwer Academic Publishers. Printed in The Netherlands.

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Summary
Green plants and cyanobacteria possess a complex and diverse set of mechanisms to regulate their photosynthetic electron transport. These include both rapid mechanisms, and long-term responses involving changes in gene expression. Together they are believed to play a number of physiological roles, including the maintenance of a relatively constant flow of electrons in a changing light-environment (homeostasis), the adjustment of the mode of photosynthetic electron transport in response to the changing metabolic needs of the cell (adaptation) and the minimization of the destructive side-effects of light and redox reactions (protection). This Chapter concentrates on the rapid responses (those not involving changes in gene expression). There has been exciting progress in our understanding 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 highlighting the questions that remain to be answered.

I. Introduction
Oxygenic photosynthetic organisms show a number of alternative modes of photosynthetic electron transport (Fig. 1). Linear photosynthetic electron transport involves the Photosystem two (PS II) and Photosystem one (PS I) reaction centers acting in series to transfer electrons from water to ferredoxin. To maintain this mode of electron transport efficiently, the turnover of the reaction centers must be regulated so that the turnover of PS II is roughly the same as that of PS I. In addition, there are alternative modes of electron transport. Various forms of cyclic electron transport around PS I have been proposed. Respiratory electron transfer involving NADH and succinate dehydrogenases (Complexes I and II, respectively), plastoquinone and terminal oxidases also occurs in the thylakoid membrane of cyanobacteria (reviewed by Schmetterer, 1994). There is now increasing evidence for similar activities in chloroplasts (Endo et al., 1997; Burrows et al., 1998). This raises the possibility that the redox state of the plastoquinone pool, which is important for the regulation of photosynthetic 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-harvesting complex; MDA monodehydroascorbate; Ndh NADH dehydrogenase; ORF open reading frame; PET photosynthetic electron transport; PS I Photosystem I; PS II Photosystem II; primary plastoquinone electron acceptor of Photosystem two; secondary plastoquinone electron acceptor of Photosystem two; qE energy-dependent component of nonphotochemical quenching; qI non-photochemical quenching induced by photoinactivation; RC reaction center; WT wild type

photosynthetic and respiratory processes. The coexistence of respiratory and photosynthetic electron transfer chains in the same membrane also means that electrons could be extracted from water and passed back to oxygen via a terminal oxidase. Such pathways are readily observed in cyanobacterial mutants lacking one or other of the reaction centers (Vermaas, 1994) but they may also be important in wild-type cells. A photosynthetic organism must be able to regulate the various possible modes of photosynthetic electron transport. Regulation is necessary for several reasons: First, it is important to maintain photosynthetic electron transport in a varying light environment (homeostasis). Photosynthetic organisms may be subject to huge and rapid changes in the intensity and spectral quality of illumination. They use regulatory mechanisms to buffer the effects of these changes, for example, by activating mechanisms for energy dissipation in strong illumination, or by adjusting the relative turnover of the two reaction centers to compensate for changes in the spectral quality of illumination. Second, regulation is necessary for adjusting the mode of electron transport in response to the metabolic needs of the cell. For example, cyclic electron transport generates a proton gradient (and hence ATP) but no reducing equivalents. Linear electron transport generates both ATP and reducing equivalents. It may therefore be necessary to regulate the extent of linear versus cyclic electron flow according to the requirement for ATP and reducing equivalents. Third, regulation is needed to protect the organism from the destructive side-effects of light and redox

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reactions. Photosynthetic electron transport is a dangerous activity, since there is always a risk of generating reactive by-products such as singlet oxygen and free radicals, which can cause widespread damage in the cell. Mechanisms exist to downregulate photosynthetic electron transport, particularly when the supply of the products exceeds the metabolic requirements of the cell. Regulation often occurs via feedback control mechanisms. Many mechanisms can be effective for more than one of the functions listed above. For example, a mechanism that adjusts the relative turnover of the two photosystems could be employed both for maintaining similar rates of turnover in varying light environment (homeostasis) and for switching between different modes of electron transport (adjustment). Numerous regulatory mechanisms have been proposed in plants and cyanobacteria. Regulation can occur on different timescales. Long-term mechanisms, generally involving changes in gene expression, are complemented by rapid mechanisms occurring on timescales of seconds to minutes. Regulation of gene expression is discussed elsewhere in this volume (see Part II: Gene Expression and Signal Transduction). This Chapter will therefore discuss the rapid mechanisms, concentrating on those mechanisms where there has been significant progress towards understanding the molecular basis of the mechanism.

II. Background Concepts

A. Photosynthetic Electron Transport and the Z Scheme
According to the Z scheme model of photosynthesis, PS I and PS II act in tandem to transfer electrons from water to ferredoxin and then to (Fig. 1). Accordingly, a minimum of eight photons are required (four for each photosystem) to produce one molecule of oxygen from the oxidation of water at PS II, and to generate the four reducing equivalents needed to fix one molecule of in the Benson-Calvin cycle. The Z scheme therefore predicts that the maximum quantum yield for both oxygen evolution and fixation is 0.125. Measured quantum yields in C3 plants are near this maximum value suggesting the operation of the Z scheme in vivo (discussed in Genty and Harbinson, 1996). A recent challenge to the ubiquity of the Z scheme, based on the analysis of PS I deficient mutants of the green alga Chlamydomonas reinhardtii (Greenbaum et al., 1995), has since proved to be unfounded (Redding et al., 1999). Although from a thermodynamic perspective it is feasible that PS II can reduce ferredoxin or recent experiments with well-characterized engineered PS I-deficient mutants of C. reinhardtii appear to exclude these as significant reactions in vivo (Redding et al., 1999). Despite this, the uncoupling of PS II from PS I under certain physiological conditions cannot yet be fully discounted.

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1994). The mechanism involves reduction of disulphide bridges in the target enzyme by thioredoxin which in the chloroplast is reduced using a ferredoxindependent thioredoxin reductase and is thus controlled by photosynthetic electron flow (Buchanan, 1994). Thioredoxin also activates the chloroplast ATP-synthase (Stumpp et al., 1999).

B. Coordination of Electron Transport and Metabolism

The products of photosynthetic electron transport, ATPland reductant in theformof reduced ferredoxin, are used for a variety of purposes including not only the fixation of into carbohydrate and the process of photorespiration but also key biosynthetic steps such as sulfate and nitrite assimilation. These pathways can be thought of as electron or energy sinks for photosynthetic reductant. In classic light saturationcurves forphotosynthesis, two regions can be defined. At low light intensities, photosynthesis is light-limited. At higher light intensities, the rate of photosynthesis becomes saturated because of limitations in the metabolic demand for reductant. In the absence of metabolic electron sinks, there is a danger that stromal components and the photosynthetic electron transfer chain become over-reduced. This can then lead, by a variety ofmechanisms, to the production of reactive oxygen species throughout the electron transport chain, with the ultimate result that there is an increased photodestruction of protein and pigment especially within PS II (Barber and Andersson, 1992; Heber and Walker, 1992). This potential problem means that metabolic demand for reductant and the photosynthetic electron transfer rate must be closely coordinated. In practice, damage to the photosynthetic electron transport chain is alleviated by (i) feedforward mechanisms to activate stromal metabolism, (ii) feed-back mechanisms to slow photosynthetic electron transport when metabolic demand reduces, (iii) the use of alternate emergency electron sinks that dissipate photosynthetic electron flow relatively harmlessly (Asada, 1999), (iv) the presence of detoxification systems to remove reactive oxygen species (Niyogi, 1999), and (v) rapid replacement of damaged protein, such as the D1 subunit of PS II, through highly coordinated repair processes (Barber and Andersson, 1992).

D. The Use of Alternate Electron Sinks

Several electron sinks within the chloroplast have been viewed as safety valves to prevent overreduction of the photosynthetic electron transport and consequent damage (reviewed by Niyogi, 1999). These sinks act to dissipate the energy of photons that is in excess of that required for useful biosynthetic processes and as such play an important photoprotective role. Molecular oxygen is an important physiological electron acceptor for photosynthetic electron flow. In the Mehler reaction (Mehler, 1951), oxygen is reduced by PS I to produce superoxide This species is hazardous so there is an efficient scavenging system for its removal. It is first converted to hydrogen peroxide and oxygen by superoxide dismutase, then hydrogen peroxide is scavenged through a number of routes in the stroma and on the thylakoid membrane (Asada, 1999). In the thylakoid-associated pathway, hydrogen peroxide is removed by the ascorbate peroxidase catalysed conversion of ascorbate to monodehydroascorbate (Asada, 1999). The regeneration of ascorbate by reduction of monodehydroascorbate (MDA) requires photosynthetic reductant in the form of reduced ferredoxin (Asada, 1999) and so MDA itself acts as an electron sink. Overall the four electrons derived from the oxidation oftwo molecules ofwater to one molecule ofoxygen are accounted for by the reduction of two molecules of oxygen to superoxide and by the reduction of two molecules of MDA to ascorbate. This process has been termed the water-water cycle (Asada, 1999) because electrons derived from the oxidation ofwater ultimately lead to the reduction of oxygen to water without the net production of oxygen. These steps form the basis of pseudo-cyclic electron flow in which linearelectron flow is coupled toATP synthesis without the net production ofreductant. Oxygen also plays a major photoprotective role in C3 plants through its involvement in photorespiration (Osmond, 1981). Excess NADPH can be removed from the stroma

C. Feed-forward Activation of the Calvin Cycle

Upon illumination ofchloroplasts, there is an increase of both pH and the concentration in the stroma. These ionic conditions activate enzymes within the Calvin cycle. Superimposed on this is the specific activation by reduced thioredoxin of several key enzymes, such as fructose-1, 6-bisphosphatase and sedoheptulose-1, 7-bisphosphatase (Buchanan,

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

III. Feedback Control of the Photosynthetic Electron Transport Chain

A. Role of the Lumenal pH in Slowing the Rate of Electron Transfer
Because several of the electron transfer reactions of the photosynthetic electron transfer chain are coupled to deposition of protons in the lumen, a decrease in lumenal pH automatically slows the flow ofelectrons. This has been clearly demonstrated for the oxidation of water at PS II (Bowes and Crofts, 1981) and the oxidation of plastoquinol at the site of the Cyt complex (Kramer and Crofts, 1993). In addition the mid-point redox potential of plastocyanin is dependent on pH so that as the lumenal pH drops, reduction of is less favored (discussed by Kramer and Crofts, 1996). As ATP synthesis at the ATP synthase is coupled to the dissipation of the proton gradient, a high ratio of stromal [ATP]/ which is indicative of a slow down in metabolic activity, will tend to maintain a low lumenal pH.

B. Role of Lumenal pH in Quenching Excitation Energy in PS II

Non-photochemical quenching (otherwise known as non-radiative quenching) represents a collection of mechanisms that regulate the conversion of light energy. In general these mechanisms involve the conversion of excitation energy to heat, thereby reducing the turnover ofthe photosystems and downregulating photosynthetic electron transport. Nonphotochemical quenching has been subdivided into components including energy-dependent quenching (qE) and photoinhibitory quenching (qI). State transitions (Section III.C) are sometimes considered to contribute to non-photochemical quenching. For the qE component, the triggering signal involves changes in the pH of the thylakoid lumen (Krause et al., 1983, Horton et al., 1996). When the pH in the lumen is low, quenching mechanisms are activated, thereby down-regulating electron transport. The reduction in electron flow will decrease the rate of

proton pumping across the thylakoid membrane, and this will in turn allow the lumenal pH to increase. Thus qE should act as a homeostatic mechanism to maintain a relatively constant lumenal pH and rate of electron flux during variable illumination (Fig. 2). It is generally assumed that the major physiological role ofqE is to protect the photosystems (particularly PS II) from photodamage during conditions ofexcess illumination. However, it is also possible that an excessively low pH could damage lumenally-exposed protein complexes. It is now becoming clear that the biochemistry of the lumen is much more complex than was thought (Fulgosi et al., 1998) qE has been observed in numerous green plant species, although the extent ofthe quenching and the rate of induction vary widely according to species and habitat (Demmig-Adams, 1990; Johnson et al., 1993). There is less evidence for such a process in the phycobilin-containing organisms (cyanobacteria and red algae). Quenching mechanisms have been postulated in cyanobacteria (Campbell et al., 1996) and red algae (Delphin et al., 1996) but it is likely that the mechanisms are differentfromthose operating in green plants. qE in green plants is influenced by the levels of xanthophyll carotenoids in the light harvesting antenna (Demmig-Adams, 1990). In particular, there is a correlation between the level of zeaxanthin and the extent and rapidity of non-photochemical quenching (Demmig-Adams, 1990; Phillip et al.,

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1996. This has led to the proposal that the conversion of violaxanthin to zeaxanthin, catalyzed by violaxanthin de-epoxidase, plays a crucial role in qE (Demmig-Adams, 1990). There have been a number of proposals for the mechanism of qE in green plants, including: (i) energy dissipation within the Photosystem II reaction center, either by charge recombination (Krieger et al., 1992) or through the accumulation ofa chlorophyll cation designated (Schweitzer and Brudvig, 1997) bound to the D1 polypeptide, (ii) energy dissipation by direct transfer ofsinglet excitons from chlorophylls to xanthophyll carotenoids (Frank et al., 1994) and (iii) energy dissipation by the formation of quenching aggregates of chlorophylls in the lightharvesting antenna (Horton et al., 1996). The extent to which these different processes contribute to qE in vivo remains controversial. The most complete mechanistic model that has been developed is that of Horton and co-workers (Horton et al., 1996; Gilmore et al., 1998). These authors propose that the conversion of excitation energy to heat occurs in pairs or aggregates of chlorophylls formed as a result of conformational changes in chlorophyll a/b binding proteins of the lightharvesting antenna. The conformational changes are triggered by the protonation of specific amino acids exposed to the lumen, or forming part of a channel that conducts protons away from the water-oxidizing complex of PS II. Specific residues on the minor light-harvesting complex (LHC) components, CP29 and CP26, have been implicated (Walters et al., 1996). It is further proposed that the extent of quenching can be modulated by the xanthophyll pigments associated with the light-harvesting complexes through the action of zeaxanthin as an allosteric activator of the quenching state (Phillip et al., 1996). This contrasts with the view of other authors who propose that zeaxanthin acts as a direct quencher of singlet excitons (Frank et al., 1994). Mutants are now being used to clarify the role of the xanthophyll cycle pigments and to identify proteins involved in qE. Mutants affected in xanthophyll metabolism generally show altered qE, confirming the role ofthe xanthophyll cycle pigments in this mechanism (Niyogi, 1999). However, mutants affected in qE have also been isolated that show normal pigment composition and xanthophyll interconversions (Niyogi, 1999). One such mutant lacks the psbS gene (Li et al., 2000) which codes for a minor chlorophyll-binding subunit associated with

Peter J. Nixon and Conrad W. Mullineaux

the PS II complex (Funk et al., 1995). PsbS could be the major site for the conversion of excitation energy into heat, or it could play a crucial role in sensing a low lumenal pH. For PS I, no such specialized quenching mechanism exists in the antenna system. The difference between PS I and PS II probably lies in the thermodynamic constraints placed on the oxidized primary electron donors. For PS I, the mid-point redox potential ofthe couple is ~0.5 V which means that its presence does not lead to damaging oxidative side reactions. Hence can act as a quencher of excitation (Butler et al., 1979). In contrast, P680 in PS II has a much higher mid-point redox potential (~1.1V) consistent with its role in water oxidation. Unfortunately the use of as a physiological quencher of excess excitation would lead to deleterious oxidative reactions within PS II. To avoid this, PS II has thus developed to be a shallow trap with the exciton more delocalized in the antenna system.

C. Regulation of Electron Flow through State Transitions

State 1-state 2 transitions (state transitions for short) are a rapid mechanism that adjusts the function ofthe light-harvesting apparatus in response to signals from the photosynthetic electron transport chain (Allen, 1992). Reduction of electron carriers between PS II and PS I triggers a switch to state 2, in which more excitation energy is transferred to PS I. Oxidation of the electron carriers triggers a switch to state 1, in which more energy is transferred to PS II. Thus the mechanism functions as a homeostatic control to maintain a moderate level of reduction of the intersystem electron carriers (Fig. 3). State transitions are observed both in green plants and cyanobacteria (Allen, 1992). The phenomena observed in Chl-b and phycobilin-containing organisms show many features in common, although it is clear that some features of the mechanism must differ in organisms with fundamentally different lightharvesting complexes.

1. The Triggering Mechanism for State Transitions

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

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

2. Alterations in Function of the Lightharvesting Complexes

State transitions change the association of the lightharvesting complexes with the reaction centers. In green plants, LHCII is predominantly associated with PS II in state 1. In state 2, a proportion of LHCII detaches from PS II and appears to associate with PS I instead (Allen, 1992). In cyanobacteria the phycobilisomes appear to be a mobile light-harvesting antenna (Mullineaux et al., 1997) that can associate with and transfer energy both to PS II and to PS I (Mullineaux, 1994). State transitions change the relative coupling ofphycobilisomes to PS II and PS I (Mullineaux, 1992) although it is likely that other effects are also involved (Olive et al., 1997; EmlynJones et al., 1999). In green plants, the changes in LHCII association are brought about by phosphorylation of the LHCII complexes on threonine residues near the N-terminus (Allen, 1992). This seems to bring about a conformational change in LHCII (Nilsson et al., 1997) that causes its dissociation from PS II and reassociation with PS I. The biochemical mechanism in cyanobacteria is not known. Allen and co-workers correlated changes in the phosphorylation state of two polypeptides with state transitions in the cyanobacterium Synechococcus 6301 (Allen et al., 1985), but it now seems increasingly unlikely that there is a causal relationship (Emlyn-Jones et al., 1999). It is very probable that some form ofcovalent modification is involved, with the phycobilisome core the most probable site of modification. There have been many attempts to isolate the protein kinase responsible for LHCII phosphorylation in green plants. The kinase must be activated as a result of the triggering redox signal, resulting in net phosphorylation of LHCII. The process is reversed by a phosphatase that seems to be constitutively active (Allen, 1992). A number of kinases have been isolated from thylakoid membranes (Coughlan and Hind, 1987; Gal et al., 1992; Snyders and Kohorn, 1999) but we do not know for sure which, if any, of these kinases are involved in state transitions in vivo. Furthermore, we know nothing of the signal transduction pathway that links the triggering redox signal to activation/inactivation of the kinase.

cyanobacteria (Mullineaux and Allen, 1990). In the case of green plants, mutagenesis studies have indicated that the cytochrome complex plays an essential role in state transitions (Wollman and Lemaire, 1988). Flash photolysis studies have shown the involvement of a specific quinol binding site (Vener et al., 1997). It is probable that cyanobacterial state transitions are triggered the same way, although the evidence is less conclusive because no mutants lacking the cytochrome complex are available. Triggering by changes in the redox state of intersystem electron carriers provides a mechanism for homeostatic regulation of electron transport in a varying light-environment (Fig. 3). In cyanobacteria, where the respiratory and photosynthetic electron transport chains intersect (Fig. 1, Scherer et al., 1988), state transitions may be important in regulating the interaction between photosynthetic and respiratory electron transport (Schreiber et al., 1995). State transitions may also be involved in regulating the extent of linear versus cyclic electron transport (Section V.B). Some effects cannot straightforwardly be explained in terms oftriggering by the redox state of intersystem electron carriers. It is therefore likely that other triggers are involved (i.e. a number of different sensing mechanisms trigger the same response). In green plants, it has recently been suggested that a direct light-induced conformational change may play a role in facilitating LHCII phosphorylation (Zer et al., 1999). It is also possible that the ADP/ATP ratio is a triggering signal (Bult

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3. The Genetic Approach to State Transitions

Mutants specifically deficient in state transitions have recently been identified in the cyanobacterium Synechocystis 6803 (Emlyn-Jones et al., 1999) and the green alga C. reinhardtii (Kruse et al., 1999; Fleischmann et al., 1999). In the cyanobacterial case, the mutation results from insertional inactivation of a specific gene that codes for a protein of previously unknown function, showing no homology to any previously-characterized gene product (Emlyn-Jones et al., 1999). The genetic approach shows great promise as a method of identifying the proteins involved in the signal transduction pathway of state transitions. Furthermore, mutants specifically deficient in state transitions are a powerful tool for establishing the physiological role(s) of the mechanism. Cyanobacterial state transition mutants grow slower than the wild-type under very weak yellow illumination, suggesting that the primary role of state transitions in cyanobacteria is to maximize the efficiency of utilization of light absorbed by the phycobilisomes (Emlyn-Jones et al., 1999).

IV. Regulation of Photosystem II Activity The Bicarbonate Effect

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

A. The Acceptor-side Effect

Type II photosynthetic reaction centers (RCs) such as PS II and those found in purple non-sulfur photosynthetic bacteria contain on the acceptor side a non-haem iron located between two quinone molecules termed and (reviewed by Diner et al., 1991 a). is tightly bound to the RC and acts as a one-electron carrier which reduces first to the semiquinone anion, then after a second charge separation reaction within the RC to the quinol (probably in the state The quinol is further protonated and released from the RC in the neutral form, Because depletion of bicarbonate primarily slows electron transfer between and after formation of it is likely that bicarbonate participates in the protonation reactions involved in formation of (Blubaugh and Govindjee, 1988). Despite the similarity in redox factors on the acceptor in type II RCs, only PS II shows this bicarbonate effect. Fourier transform infrared (FTIR) experiments (Hinerwadel and Berthomieu, 1995) indicate that bicarbonate acts as a bidentate ligand to the nonhaem iron in PS II. In bacterial RCs a glutamate residue, at position 232 in the M subunit of Rhodopseudomonas viridis, fulfils this role. Bicarbonate binding is reversible so that a wide range of anions including formate, glycolate, glyoxylate, and oxalate compete with bicarbonate for binding to the non-haem iron and slow electron transfer between and (Petrouleas et al., 1994). The presence of bicarbonate close to the site is thus consistent with a role in the protonation of either directly or indirectly. Mutation of a number of potentially positively charged amino-acid residues

4. Higher Levels of Control

In green plants the level of LHCII phosphorylation is maintained by the balance between the activities of a protein kinase that is activated by a redox signal, and a constitutively-active phosphatase (Allen, 1992). Such a mechanism has a clear metabolic cost. The maintenance of any level of LHCII phosphorylation will require continual hydrolysis of ATP. Higher levels of kinase and/or phosphatase activity would result in more rapid state transitions and therefore a swifter response to rapidly changing illumination conditions. However, this would come at the expense of more rapid ATP hydrolysis. It is therefore possible that there are mechanisms to up-regulate or downregulate the enzymes involved, depending on growth conditions. It could be imagined that the genes coding for the enzymes would be strongly expressed under low or variable illumination, but repressed under strong, constant illumination. The subject needs further investigation. In green plants, the phosphorylation of LHCII is inactivated in high light (Rintamki et al., 1997). In the cyanobacterium Synechocystis 6803, state transitions are only observed in low-light grown cells (C. W. Mullineaux, unpublished).

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considered to perform two types of photophosphorylation: cyclic and non-cyclic. In cyclic electron flow, ATP synthesis is coupled to light-induced electron flow in a closed system around PS I without the net production ofNADPH whereas in non-cyclic mode, or linear electron flow, both ATP and NADPH are synthesized. Cyclic photophosphorylation is therefore an ATP generating process that serves to increase the ratio of ATP/NADPH produced in photosynthetic electron flow (reviewed in detail by Fork and Herbert, 1993; Bendall and Manasse, 1995). In principle the metabolic needs of the cell can be met through appropriate modulation of the ratio of linear to cyclic electron flow. Although the waterwater cycle also increases the ATP/NADPH ratio, cyclic electron flow is a more energy efficient way of generating ATP. In general, cyclic electron flow involves the reduction 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 nonphotochemical reduction are sometimes used to describe the various non-PS II electron-transfer pathways able to feed electrons into the IEC. In addition the term cyclic is often used loosely to describe all PS II-independent pathways for the reduction of PS I, including those leading to reduction of the IEC through dehydrogenases (Complex I and II) (Fig. 1). According to the Z scheme, the linear transport of an electron from water to is thought to translocate one proton at PS II and two protons at Cyt ifa Q cycle operates constitutively (Rich, 1988). In total three protons are translocated per electron transported. If the synthesis of one molecule ofATP requires the influx offour protons at the ATP synthase (van Walraven et al., 1996), then the ratio of ATP/ NADPH produced by linear electron flow is about 1.5. This value will depend on the amount ofpassive proton leakage across the thylakoid membrane at high pH, which would lower the effective number of protons pumped by the electron transport chain (Berry and Rumberg, 1999), and the precise number of protons required to synthesize ATP (3, 4 or indeed a non-integral value). For C3 plants in ambient air, the ratio ofNADPH/ ATP needed to fix and perform photorespiration is about 1.56 (discussed by Foyer and Harbinson, 1997). Hence ATP production through cyclic electron flow is not required for C3 plants during steady-state photosynthesis and indeed there is little evidence for

within the D2 polypeptide (Diner et al., 1991b; Cao et al., 1991), modeled to be in the vicinity ofthe nonhaem iron, reduce the affinity of PS II for bicarbonate. Based on the isolation of D1 mutants that are resistant to formate inhibition (Xiong et al., 1997, 1998), it has also been suggested that there is an additional bicarbonate bound within the niche which is responsible for protonation of (van Rensen et al., 1999). Whether the bicarbonate effects observed for PS II in vitro also occur in vivo is a matter of debate. values for bicarbonate are estimated to be (Snel and van Rensen, 1984). The concentration of bicarbonate in the stroma is highly dependent on pH and temperature but has been estimated to be about at pH 6.3 and at pH 8.0 (van Rensen et al., 1999). Thus under optimal rates of photosynthesis, when the stromal pH reaches pH 8, the levels of bicarbonate do not appear to be controlling PS II activity. Only when levels of carbon dioxide, hence bicarbonate, decrease, such as upon the closure of stomata through for example drought or high temperature, would PS II activity be downregulated. This downregulation of PS II would also act to reduce the level of oxygen within the cell which otherwise would lead to enhanced photorespiration in C3 plants. Binding ofbicarbonate to PS II has also been linked to enhanced resistance to photoinhibition (Sundby, 1990; Klimov et al., 1997).

B. The Donor-side Effect

A variety of indirect evidence has also hinted to the possibility that bicarbonate may have a role on the donor side of PS II (reviewed by Stemler, 1999; van Rensen et al., 1999; Klimov and Baranov, 2001). Recently Klimov and co-workers have interpreted FTIR difference spectra obtained for the donor side of PS II in terms of actual ligation of bicarbonate to the Mn cluster (Yruela et al., 1998). In the absence of bicarbonate assembly of the Mn cluster is also less efficient (Allakhverdiev et al., 1997). Further work is required to clarify these donor side effects particularly as it is plausible that binding of bicarbonate on the acceptor side of PS II causes structural effects that extend across the membrane to the donor side.

V. Cyclic Electron Flow

Historically, thylakoid membranes have been

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its existence under these conditions (Herbert et al., 1990), It has been suggested that the main role for cyclic electron flow in C3 plants is to regulate electron flow through acidification of the lumen under conditions where electron sinks for photosynthetic electron transport are depleted (Heber and Walker, 1992;Katona et al., 1992; Heber et al., 1995). For C4 photosynthesis, there is a requirement of 2.5-3 ATP/ NADPH, much greater than that can be generated in linear mode. The extra ATP is considered to be generated by cyclic electron flow around PS I. For algae and cyanobacteria, which need to adapt to large fluctuations in environmental conditions through energy dependent processes, cyclic electron flow appears to be an important component of photosynthetic electron flow (reviewed by Bendall and Manasse, 1995). The precise rates of cyclic electron transfer in vivo are, however, difficult to assess because of the technical problems deconvoluting linear and cyclic electron flow, although a variety of techniques have been 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).

Peter J. Nixon and Conrad W. Mullineaux

stromal reductant ofthe membrane bound intersystem chain of electron carriers in chloroplasts (Arnon, 1991). This assignment stemmed from experiments in vitro in which ferredoxin was added back to thylakoid membranes in the absence ofother stromal components (Tagawa et al., 1963). Whether this or some other cycle actually operated in vivo could not be assessed. For many years the mechanism by which reduced ferredoxin reduced the intersystem chain was thought to involve the direct reduction of cytochrome This hypothesis was based on the mistaken assumption that the binding site of antimycin A, a known inhibitor of ferredoxin-mediated cyclic electron flow, was the Cyt complex (discussed in Bendall and Manasse, 1995). The actual binding site for antimycin is still uncertain although there is some preliminary evidence to indicate an association with PS I (Davies and Bendall, 1987) and also that there may be an additional weaker binding site in the Ndh complex (Endo et al., 1998). The inhibition of ferredoxin-mediated cyclic electron flow by inhibitors of the site of the Cyt complex, together with other data (Bendall and Manasse, 1995), has led to models in which cyclic electron flow involved reduction ofthe plastoquinone pool followed by its oxidation by Cyt Reduction ofthe plastoquinone pool by reduced ferredoxin was therefore hypothesized to be catalyzed by a ferredoxin-plastoquinone reductase (FQR) (Moss and Bendall, 1984). The molecular identity of FQR still remains unknown. Recent evidence indicates that FQR may include a cytochrome, termed Cyt b559(Fd) (Miyake et al., 1995). Whether this cytochrome is truly part ofFQR or is reduced through redox equilibration with the plastoquinol pool is uncertain. Given that there may be an antimycin-binding site close to PS I, attention has focused on whether PS I contains FQR activity (Bendall and Manasse, 1995). Mutation ofthe psaE gene, which encodes a stromally exposed subunit of PS I (Klukas et al., 1999), in Synechococcus (Yu et al., 1993) impairs cyclic electron flow when assayed by the dark reduction kinetics of However photoacoustic spectroscopy has failed to detect changes in cyclic electron flow in this mutant (Charlebois and Mauzerall, 1999). Because PsaE is thought to be involved in the binding of ferredoxin (Barth et al., 1998) and ferredoxin: oxidoreductase (FNR) to PS I (van Thor et al., 1999a), the role of PsaE in cyclic electron flow may be to stabilize the binding of these

A. Pathways of Cyclic Electron Transfer

As yet the precise pathways ofcyclic electron transfer remain unclear although the consensus favors multiple routes (Hosler and Yocum, 1985; Ravenel et al., 1994). Currently two cycles are thought to dominate. One mediated by the Ndh complex involves the pyridine nucleotide pool and the other consists of ferredoxin-mediated reduction of the IEC. From studies on cyanobacteria (Yu et al., 1993) and algae (Ravenel et al., 1994) in vivo, the rates of cyclic electron flow have been estimated to be less than 10% of the maximum linear rates. Of this, recent estimates using isolated barley thylakoids suggest that the rates ofNdh-mediated cyclic electron flow is about 3% ofthe linear rates and the rate offerredoxindependent cyclic electron flow is about 5% (Teicher and Scheller, 1998). Under stress conditions such as photoinhibition and nutrient deprivation, the contribution of cyclic electron flow may, however, become more important (discussed by Fork and Herbert, 1993).

1. Ferredoxin Cycles
Historically reduced ferredoxin was considered the

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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 subcomplex of the Ndh complex, which displays an NADPH:ferricyanide oxidoreductase activity, has been isolated from Synechocystis PCC 6803 (Matsuo et al., 1998). Of the nine subunits present, only NdhH could be assigned.

components close enough to a quinone binding site within the PS I core complex to allow plastoquinone reduction.

2. NAD(P)H Cycles
a. Identification of the NADH Dehydrogenase (Ndh) Complex
Following the sequencing of the tobacco (Shinozaki et al., 1986) and liverwort (Ohyama et al., 1986) chloroplast genomes, several open reading frames (ORFs) were identified with significant sequence similarities to mitochondrial and eubacterial genes encoding subunits of the respiratory NADH:ubiquinone oxidoreductase (also known as Complex I or type I NADH dehydrogenase) (Fearnley and Walker, 1992). These ORFs were consequently designated ndh genes (NADH dehydrogenase). ndh genes have now been identified in cyanobacteria and in the chloroplast genomes of some but not all green algae (Turmel et al., 1999). The absence of ndh genes from various plastomes may reflect transfer to the nucleus or deletion from the organism. In the case of black pine, ofthe 11 ndh genes usually found in land plants, four have been lost from the plastome and seven remain as pseudogenes (Wakasugi et al., 1994). Despite little evidence at the time to suggest a role for the 11 ndh genes in encoding a chloroplast analogue ofComplex I (Fearnley and Walker, 1992), recent biochemical (Sazanov et al., 1998) and genetic studies (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998) have provided strong evidence for this assignment (also reviewed by Nixon, 2000). The low abundance of the Ndh complex in the chloroplast, estimated to be at 1.5% the levels of PS II in tobacco (Burrows et al., 1998), helps to explain why it had escaped detection for so long. For chloroplasts, theNdh complex is located in the stromal lamellae (Nixon et al., 1989; Sazanov et al., 1998) whereas in cyanobacteria there is still uncertainty about its location although it would appear to be in the thylakoid (Norling et al., 1998) and possibly cytoplasmic (Berger et al., 1991) membranes of Synechocystis 6803. Analysis of the cyanobacterial Ndh complex is also complicated by the presence of multigene families which may reflect structural and functional heterogeneity (Ohkawa et al., 1998; Klughammer et al., 1999). Biochemical analysis ofthe Ndh complex remains incomplete. A 550 kDa Ndh complex with an associated NADH:ferricyanide oxidoreductase

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

The enzyme activity of the Ndh complex has been assayed mainly through the use of pyridine nucleotides as electron donors and artificial electron acceptors such as water-soluble quinones and ferricyanide, which probably accept electrons from the iron-sulfur clusters within the complex rather than from the natural quinone binding site (Sazanov et al., 1998). Biochemical characterization ofthe Ndh complex has proved difficult because of its low abundance in chloroplast thylakoid membranes, its lability, the lack of a convincinginhibitor, possible mitochondrial contamination in chloroplast samples and the presence of multiple NAD(P)H dehydrogenase activities, including a high level of NADPH:ferricyanide oxidoreductase activity due to ferrodoxin NADP reductase (FNR). This latter difficulty may explain why FNR has been suggested to be a component of the Ndh complex (Guedeney et al., 1996). Consequently it is extremely difficult to assign enzyme activities within the thylakoid membrane to the Ndh complex without additional purification or verification. The instability ofthe Ndh complex also raises the possibility that the activity of the Ndh complex and its possible role in cyclic photophosphorylation may have been overlooked in early experiments. Attempts to study the Ndh complex in tobacco thylakoid membranes, for which there are engineered ndh knock-out mutants available to help assign the activities of the complex, have proved difficult because of the apparent instability of the complex (Endo et al., 1998). Such behavior may explain why reduction of the plastoquinone pool in isolated thylakoid membranes using NAD(P)H is so low (Rich et al., 1998). In membranes the reduction of the plastoquinone pool by NADPH is known to be stimulated by ferredoxin (Mills et al., 1979; Rich et al., 1998). Rather than an Ndh-catalyzed reaction,

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the possible pathway may involve the membranebound FNR-mediated reduction of ferredoxin by NADPH followed by reduction ofthe plastoquinone pool using FQR. The nature of the stromal reductant oxidized by the isolated detergent-solubilized plastid Ndh complex, of different degrees of purification, is still under debate with there being evidence for a specificity for NADH (Sazanov et al., 1998; Elortza et al., 1999), NADPH (Guedeney et al., 1996) or both NADH and NADPH (Quiles and Cuello, 1998; Funk et al., 1999). For an isolated sub-complex of the Synechocystis 6803 Ndh complex, a specificity for NADPH has been reported (Matsuo et al., 1998; whilst for the complex in thylakoid membranes, NADPH, NADH and reduced ferredoxin have all been implicated (Mi et al., 1995). It is possible that in vivo the Ndh complex may be rather promiscuous interacting with a number of different reductants depending on plant species and physiological state. The resulting holocomplex(es) containing the plastid ndh gene products plus the as yet uncharacterized electron input module(s) (Friedrich et al., 1995) may be rather unstable. The identity of the subunits that constitute the subcomplex involved in NAD(P)H oxidation is particularly intriguing. As yet no obvious homologues of the NADH-binding subunits found in other Complex I species have been identified (Friedrich et al., 1995). Although there has been controversy over whether plants can tolerate the inactivation of plastid ndh genes (Maliga and Nixon, 1998; Koop et al., 1998) the consensus now appears that under normal growth conditions loss ofthe Ndh complex does not result in an obvious growth defect (Burrows et al., 1998; Shikanai et al., 1998). Plastid ndh mutants do however appear to be more sensitive to the effects ofhigh light damage, for which the reason is not yet clear (Endo et al., 1999). For cyanobacteria, the photoautotrophic growth of several, but not all, ndh mutants shows a requirement for enhanced levels (Ohkawa et al., 1998; Klughammer et al., 1999). This observation is consistent with a role for Ndh subunits in generating the ATP needed for the active transport of dissolved inorganic carbon into the cell.

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NAD(P)H:plastoquinone oxidoreductase has led to speculation that it may have a dual function in photosynthetic systems (Burrows et al., 1998). In the light one role for the Ndh complex may be to catalyze cyclic electron flow around PS I (either directly or through appropriate redox poising of the IEC), whereas in the dark the Ndh complex may function in respiration. Figure 4 summarizes the possible electron pathways involving an NADH-specific Ndh complex of the chloroplast. By analogy to the situation in purple bacteria, it is also possible that the Ndh complex may catalyze the reverse reaction in which the plastoquinol-mediated reduction of to NAD(P)H is coupled to the proton motive force. Experimental data to support a role of the Ndh complex in cyclic electron flow first came from studies on cyanobacterial ndh mutants (Mi et al, 1992; Yu et al., 1993) and more lately from analysis of chloroplast ndh mutants for which: (i) the postillumination reduction of the plastoquinone pool is absent or strongly attenuated (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998), (ii) the rate of re-reduction of is slower in the dark after far-red induced oxidation of P700 (Burrows et al., 1998) and (iii) accumulates more rapidly under far-red illumination, after a period of white light illumination to reduce the pool of stromal reductant (Shikanai et al., 1998). Interestingly under conditions of water stress, when the levels of electron acceptors for linear flow are limited, the ndh mutants show a reduced ability to quench chlorophyll fluorescence non-photochemically during the early stages of the induction of photosynthesis in dark-adapted plants (Burrows et al., 1998). Such behavior is consistent with a role for the Ndh complex under conditions when the Calvin cycle enzymes are not fully activated and there is a build up of reductant in the stroma. Cyclic electron flow under these conditions would contribute to the pH gradient across the thylakoid enhancing qE, thus downregulating PS II, as well as removing stromal reductant which could lead to the generation of reactive oxygen species. Further indirect evidence to support a role for the Ndh complex in cyclic electron flow has come from the finding that Ndh proteins are located in the stromal lamellae close to PS I (Nixon et al., 1989) and show elevated levels in the bundle sheath cells of C4 plants which lack PS II and carry out high levels of 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 precise substrate specificity of the Ndh complex, a role as an

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

B. Regulation of Cyclic Electron Flow

Although many of the components involved in cyclic electron flow have yet to be clarified, it is clear that for optimum rates of cyclic electron transport, there must be appropriate redox poising of the system so that it is neither too oxidizing nor too reducing. This is probably achieved in vivo by concurrent cyclic and linear electron flow (Arnon and Chain, 1975). A dynamic picture of cyclic electron flow would then be of cycle(s) in which electrons that leak out are replaced through both photochemical (PS II) and respiratory processes (e.g. oxidation of NADH by the Ndh complex). The mechanism whereby cyclic flow is switched on at the expense of linear electron flow is unknown. Under normal steady state linear electron flow, when the Calvin cycle is fully activated, the cycling of electrons from NAD(P)H or reduced ferredoxin to

the IEC is unlikely to compete with PS II-dependent reduction ofthe plastoquinone pool. In any event, the ferredoxin and NADP pools are relatively oxidized under these conditions because of forward electron transfer. Cyclic electron flow would however be promoted if the levels of NADPH and reduced ferredoxin increased because of a reduction in the rate of fixation. Such physiological situations include (i) the early stages in the induction of photosynthesis in dark-adapted plants (Takahama et al., 1981), (ii) a reduction in the levels ofthe electron acceptors, and through for example the water-stress induced closure of stomata (Katona et al., 1992), and (iii) the inhibition of the Calvin cycle enzymes through for instance heat stress (Weis, 1981; Havaux, 1996). An increase in the activity of PS I compared with PS II may also act to enhance cyclic electron flow by poising the plastoquinone pool in a more oxidized state. This may occur physiologically when (i) PS II is 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 of PS II complex such as in the bundle sheath cells of C4 plants. How is the ratio of cyclic to linear electron flow controlled by the metabolic demands of the cell? In

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principle, changes in the level of ATP/ADP may regulate cyclic electron flow through allosteric modulation of FQR or the Ndh complex. However, neither ATP norADP modulate FQR activity (Cleland and Bendall, 1992). Effects of ATP on Ndh activity have not yet been tested although preliminary work has indicated that the thylakoid NAD(P)H dehydrogenase activity, assumed to be due to the Ndh complex, is activated by light and incubation in lowionic-strength buffer (Teicher and Scheller, 1998). There is good evidence, particularly in green algae, that the metabolic control ofcyclic over linear electron flow appears to be exerted through state transitions (Section III.C). For C. reinhardtii state transitions are more dramatic than in higher plants (Delosme et al., 1996). State 2 is linked with cyclic electron flow whereas State 1 promotes linear electron flow (Finazzi et al., 1999). In state 2 there is little PS II-dependent oxygen evolution (Finazzi et al., 1999) which reflects not only a reallocation of excitation energy to PS I but also the migration of Cyt away from PS II to PS I (Vallon et al., 1991) so that linear electron flow is further diminished in favor of cyclic electron flow. Thus when ATP demand is high, state 2 would be favored. Such a case occurs when nitrogen-limited cells of the green alga Selenastrum minutum are transferred into an replete medium (Turpin and Bruce, 1990). assimilation requires a high ATP/ NADPH ratio with the extra ATP provided by cyclic electron flow. Chlorophyll fluorescence measurements confirm that without changing light quality the cells go into state 2 to enhance PS I activity. Concomitantly dark respiration is stimulated and fixation is decreased probably because of the need to transfer carbon skeletons from the Calvin cycle (in the chloroplast) to the TCA cycle (in the mitochondrion) in order to synthesize amino acids (Elrifi and Turpin, 1986). Consequently the availability of electron acceptors in the chloroplast declines to produce redox conditions that would further favor cyclic electron transport and cyclic photophosphorylation. Transition to state 2 also occurs in response to hyperosmotic stress in Chlamydomonas (Endo et al., 1995). The role of state transitions in regulating cyclic electron flow can now be tested more directly using recently isolated state transition mutants (Kruse et al., 1999). How cellular ATP levels are sensed within the chloroplast and converted into a state transition is uncertain. In principle metabolite translocators within the inner envelope membrane of chloroplasts allow

Peter J. Nixon and Conrad W. Mullineaux

changes in metabolite levels outside the chloroplast to be detected within the stroma. This signal can then be transduced into an appropriate response within the thylakoid membrane to regulate photosynthetic electron flow.Forinstanceinhibitionofmitochondrial respiration in C. reinhardtii in the dark, to prevent ATP synthesis, leads to a more reduced thylakoid plastoquinone pool (Gans and Rebeille, 1990; Bennoun, 1994) and a transition to state 2 (Gans and Rebeille, 1990) probably via a redox-controlled LHCII kinase. Cyclic electron flow would then be favored upon re-illumination. The mechanism of plastoquinone reduction has been suggested (Gans and Rebeille, 1990; Bult et al., 1990) to be (i) a reduction in cellular ATP levels which is sensed in the chloroplast by a drop in ATP, (ii) an increase in the rates of starch degradation and chloroplast glycolysis to synthesize chloroplastic ATP, (iii) an increase in chloroplast NAD(P)H pools through glycolysis and the oxidative pentose phosphate pathway, and (iv) reduction of the plastoquinone pool using an NAD(P)H:PQ oxidoreductase. Whether it is the ATP level (Bult et al., 1990) or the redox state of the plastoquinone pool which exerts the major control on state transitions in C. reinhardtii in vivo is a matter of debate (Finazzi et al., 1999). It is possible that the need for enhanced cyclic electron flow results in the synthesis of new electron transfer components or the enhanced accumulation of pre-existing proteins. Under salt stress conditions, Synechocystis 6803 produces a flavodoxin which can substitute for ferredoxin and which promotes cyclic over linear electron flow (Hagemann et al., 1999), Under photooxidative stress conditions, when PS II activity declines, expression of the NdhA subunit of the Ndh complex increases (Martin et al., 1996). Given that PS II and PS I in chloroplasts are spatially segregated (Andersson and Anderson, 1980), it is possible that cyclic electron flow is restricted to the stromal lamellae and linear flow to the grana. Upon state transitions, a further redistribution of electron transfer complexes within the membrane may 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 a direct role in cyclic electron flow but as yet there is no definitive evidence (Bendall and Manasse, 1995). In cyanobacteria, there are two populations of FNR, one directly associated with the thylakoid membrane and one attached to the phycobilisomes (van Thor et

Chapter 30 Photosynthetic Electron Transport

al, 1999b). It is possible that the two populations of FNR have different electron transport roles. As a cyanobacterial psaE/ndhF double mutant shows little cyclic electron flow (Yu et al., 1993), it would appear that cyclic electron flow in this particular case is dominated by the Ndh and PsaE pathways. But what regulates the type of cycle? It can be hypothesized that the relative reduction levels of NAD(P)H and ferredoxin is an important determinant in the type of cycle, whether ferredoxinor Ndh-mediated, used around PS I. Because the reduction of by reduced ferredoxin (via FNR) is favored, in the event that there is an accumulation of reductant on the acceptor side of PS I, it would be anticipated that the ratio of would increase before that of reduced ferredoxin/oxidized ferredoxin. Such a simplistic analysis would suggest that the Ndh-mediated cycle is more likely to occur in vivo than ferredoxin cycles. Analysis of tobacco ndh mutants support this view (Burrows, 1998). Under moderate illumination the post-illumination reduction of the plastoquinone pool is absent in ndh mutants compared with WT (Burrows et al., 1998; Kofer et al., 1998; Shikanai et al., 1998), suggesting a block in the cycling of electrons into the pool. Under more extreme illumination conditions some post-illumination reduction of the pool now occurs, perhaps mediated by reduced ferredoxin that could not accumulate under the lower intensity light (Burrows, 1998). Where the Ndh complex is specific for NADH rather than NADPH, then cyclic electron flow could involve a transhydrogenase reaction to produce NADH from NADPH (possibly via FNR) or the use of NAD- and NADP-dependent dehydrogenases in a substrate cycle (Sazanov et al., 1998). Recently chloroplasts have been shown to contain an NADlinked malate dehydrogenase (Berkemeyer et al., 1998) in addition to the light-activated NADPdependent malate dehydrogenase.

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of the plastoquinone pool in green algae in darkness. It was proposed that the plastoquinone pool could be reduced through the action of an NAD(P)H dehydrogenase and on the basis of inhibitor studies that it could be oxidized by oxygen at an oxidase. At the same time an electrochemical gradient could be generated across the thylakoid. While there is now ample evidence for non-photochemical reduction of the plastoquinone pool in a range of plants (Groom et al., 1993) the original conclusions concerning the presence of thylakoid oxidases are less convincing (Bennoun, 1998). It is now clear that the inhibitors of the putative chloroplast oxidase were in fact inhibiting the mitochondrial oxidase which then caused the indirect reduction of the chloroplast plastoquinone pool (Bennoun, 1994). Because of mitochondrial/ chloroplast interactions (Section VII), mitochondrial oxidases are in principle able to drive the oxidation of the plastoquinone pool (Bennoun, 1998) through reverse electron flow from plastoquinol to at a proton pumping NAD(P)H dehydrogenase, with the reaction driven by the proton electrochemical gradient across the membrane. Despite the fact that the original data (and possibly later results from higher plants) can no longer be interpreted unambiguously, there are now biochemical and genetic data to support the concept of chlororespiration. First, the Ndh complex found in higher plant chloroplasts, but not yet in C. reinhardtii, appears to fulfill the role of the NAD(P)H dehydrogenase (see above). Second, a protein, designated Immutans, with strong sequence similarities to the alternative oxidases of plant mitochondria has been detected in chloroplasts of Arabidopsis thaliana (Wu et al., 1999; Carol et al., 1999). It is plausible that these two components together with plastoquinone may form a chloroplast respiratory chain (Fig. 4). Additional or alternative plastoquinone reductase and oxidase activities may be present depending on the species and physiological state. For instance there appears to be multiple thylakoid NADH dehydrogenase activities in tobacco thylakoids (Cornac et al., 1998). A plastoquinol peroxidase has also been suggested to be a component of chlororespiration (Casano et al., 2000). The rate of chlororespiratory electron transfer in the dark is, however, quite small with rates in sunflower estimated to be at only about 0.3% of the light-saturated photosynthetic electron flow (Feild et al., 1998). The role of chlororespiration is uncertain although speculation has focused on metabolism in

VI. Chlororespiration and Cyanobacterial Respiration

In 1982, Pierre Bennoun coined the term chlororespiration to describe respiration within the chloroplast as distinct from mitorespiration which occurs in the mitochondrion (Bennoun, 1982). A chloroplast respiratory chain was invoked to explain a number of observations concerning the redox state

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the dark such as the maintenance of a proton motive force (for ATP synthesis and activation of the ATP synthase) and the regulation of pyridine nucleotide levels for efficient starch mobilization. Given the low rates of electron transfer in chlororespiration, it is unlikely that it makes a significant contribution to ATP synthesis in the light in mature chloroplasts. In immature or non-photosynthetic plastids, the contribution may become important. However the Ndh component of the chain may have a role in cyclic electron flow in the light as described above, possibly directly or by poising the IEC in an appropriate state for cyclic electron transfer via ferredoxin-mediated pathways. The immutans mutant lacking the putative chloroplast alternative oxidase shows a variegated phenotype with leaves consisting of white and green sectors. The white sectors develop because insufficient carotenoid is made to protect from photooxidative stress (Carol et al., 1999; Wu et al., 1999). Lack of Immutans may inhibit phytoene desaturation because the plastoquinone pool is too reduced. For cyanobacteria there are a number of potential thylakoid plastoquinone reductases and oxidases (early work reviewed by Schmetterer, 1994). In Synechocystis 6803, there are three open reading frames encoding potential type 2 NADH dehydrogenases, designated ndbA-C (Howitt et al., 1999). In contrast to the type 1 dehydrogenases, type 2 dehydrogenases consist of single subunits, lack ironsulfur centers and do not pump protons across the membrane. Because the activity of the Synechocystis ndb gene products appears to be low under laboratory growth conditions it has been suggested that they may act as redox sensors and serve a regulatory function (Howitt et al., 1999). The activity of the Ndb proteins under stress conditions, such as irondepletion when the Ndh complex may be less active, awaits examination as does confirmation that the Ndb subunits are located in the thylakoid rather than cytoplasmic membrane. The genome of Synechocystis 6803 also contains three 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 of engineered mutants are that CtaI is the major oxidase in thylakoid membranes, Cyd is mainly located in the cytoplasmic membrane and that CtaII plays only

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a minor role in cellular respiration under the conditions tested (Howitt and Vermaas, 1998). Interestingly there is no obvious cyanobacterial homologue of Immutans.

VII. Interaction between Chloroplasts and Mitochondria

Chloroplasts, like mitochondria, possess a number of protein transporters within the inner envelope membrane which enable the selective passage of metabolites into and out of the organelle (Flgge, 1998). Although there is a chloroplast ADP/ATP antiport 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 which can be oxidized by and malate dehydrogenases in a number of cell compartments to yield NADH and NADPH respectively. The chloroplast is thus in metabolic communication with the rest of the cell and potentially (on a slower time scale) with the rest of the plant. The potential importance of mitochondrialchloroplast interactions in photosynthesis has been highlighted by the isolation of a photoautotrophic suppressor of the chloroplast mutant FUD50 of C. reinhardtii which lacks the chloroplast ATP synthase (Lemaire et al., 1988). The suppressor strain still lacks the chloroplast ATP synthase and has therefore had to develop an alternative route to accumulate the necessary ATP in the chloroplast for the Calvin cycle. A possible pathway involves the export from the chloroplast to the cytosol of reducing equivalents in the form of dihydroxyacetone phosphate which is first converted to glyceraldehyde 3-phosphate and then dehydrogenated to produce cytosolic NADPH. NADPH can be then oxidized at the external surface of the inner mitochondrial membrane and ATP synthesized through oxidative phosphorylation. ATP is then exported from the mitochondrial matrix to the chloroplast stroma using ADP/ATP translocators in both organelles. This pathway is energetically more costly than ATP synthesis by the chloroplast ATP synthase and does not seem to operate in the WT. The role of the mitochondrial electron transport chain in photosynthetic electron flow has also been implicated from studies of PS I and Cyt deficient

Chapter 30 Photosynthetic Electron Transport

strains of C. reinhardtii. Using mass spectrometry measurements to differentiate between oxygen uptake and oxygen release, a permanent electron flow could be established between water oxidation at PS II and oxygen reduction at mitochondrial terminal oxidases (Peltier and Thibault, 1988). In the absence of PS I, the reductant NAD(P)H was hypothesized to be produced by an NAD(P)H dehydrogenase driven in reverse by the proton motive force (Peltier and Thibault, 1988). As yet the identity of this complex is unknown although it would appear not to be the Ndh complex (Cornac et al., 1998). Transport of the reductant to the mitochondrion for oxidation would occur through metabolite shuttles as described above. Evidence that the mitochondrial ATP synthesis may be important for high rates of photosynthesis in WT has come from studies on barley leaf protoplasts (Krmer et al., 1988). Selective inhibition of mitochondrial respiration using oligomycin resulted in an approximate 40% inhibition in oxygen evolution. The role of the mitochondrion in photosynthetic electron flow may be important at two levels. First, from energetic considerations it has been suggested that ATP production through the mitochondrial oxidation of reductant produced by photosynthetic electron transport would be more efficient than that through cyclic electron flow in the chloroplast (Krmer et al., 1988). Thus when the ATP/NADPH ratio needs to be increased, reductant may be oxidized within the mitochondrion. Indeed the mitochondrial oxidation of photosynthetic reductant is considered the basis of the phenomenon oflight-enhanced dark respiration(Xue et al., 1996). Second, mitochondrial activity acts as an additional electron sink for photosynthetic electron transport and so may have a role in protecting the photosynthetic electron transport chain from damage (through the malate valve).

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A. Are there other Regulatory Mechanisms still to be Characterized?

Even unicellular photosynthetic organisms are exceptionally complex systems, capable of responding to their environment in numerous and subtle ways. This is nicely illustrated by the complete sequencing of the genome of the cyanobacterium Synechocystis 6803 (Kaneko et al., 1996). In addition to a very large number of genes of completely unknown function, there are more than 80 open reading-frames coding for components of putative two-component signal transduction systems (Mizuno et al., 1996). We still have no idea of the function of the majority of these genes. It seems almost certain that any photosynthetic organism will respond to any environmental change with a whole suite of responses. We may have very little hope of dissecting out all these responses using conventional physiological and biophysical techniques. Specific mutants may offer the best way forward. Mutants can be used to address the question in two ways: First, the inactivation of specific genes may lead to the characterization of new regulatory mechanisms. For example, the inactivation of a putative DNA-binding response regulator in Synechocystis produces a phenotype suggesting that the response regulator is involved in a long-term mechanism for regulating the interaction of phycobilisomes with reaction centers, a mechanism that had not previously been suspected (Ashby and Mullineaux, 1999). Second, if a specific regulatory mechanism is inactivated by a mutation, what other responses can be seen in the mutant? The inactivation of one or more regulatory pathways can be a good way to reveal other control mechanisms that were masked in the wild-type. For example, a mutational study shows that state transitions in cyanobacteria in fact consist of at least two independent responses (Emlyn-Jones et al., 1999).

VIII. Questions for the Future

It is likely that the power of the genetic approaches now being applied in cyanobacteria, green algae and higher plants will lead to the identification of the genes and gene products required for all the known photosynthetic regulatory mechanisms within the next few years. This in turn should lead to the determination of the basic biochemistry of the mechanisms. Some other questions are then likely to come to the fore.

B. What are the Physiological Roles of the Adaptation Mechanisms?

Our current thinking on these questions is based largely on plausible guesswork, combined with comparative studies of the occurrence of the regulatory mechanisms in organisms in different environments. The isolation of mutants with highly specific phenotypes will allow a much more direct

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approach. If we take a mutant specifically deficient in a particular regulatory mechanism, and place it in a particular environment, how will it fare in comparison to the wild type? This approach has been used, for example, to show that state transitions in cyanobacteria are important for regulating the efficiency of utilization of phycobilisome-absorbed light (Emlyn-Jones et al, 1999).

Peter J. Nixon and Conrad W. Mullineaux

Light can be bad for photosynthesis. Trends Biochem Sci 17: 6166 Barth P, Lagoutte B and Stif P (1998) Ferredoxin reduction by Photosystem I from Synechocystis sp. PCC 6803: Toward an understanding of the respective roles of subunits PsaD and PsaE in ferredoxin binding. Biochemistry 37: 1623316241 Bendall DS and Manasse RS (1995) Cyclic photophosphorylation and electron transport. Biochim Biophys Acta 1229: 2338 Bennoun P (1982) Evidence for a respiratory chain in the chloroplast. Proc Natl Acad Sci USA 79: 43524356 Bennoun P (1994) Chlororespiration revisited: Mitochondrialplastid interactions in Chlamydomonas. Biochim Biophys Acta 1186: 5966 Bennoun P (1998) Chlororespiration, sixteen years later. In: Rochaix J-D, Goldschmidt-Clermont M and Merchant S (eds) The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp 675683. Kluwer Academic Publishers, Dordrecht Berger S, Ellersiek U and Steinmller K (1991) Cyanobacteria contain a mitochondrial complex I-homologous NADH dehydrogenase. FEBS Lett 286: 129132 Berger S, Ellersiek U, Westhoff P and Steinmller P (1993) Studies on the expression of NDH-H, a subunit of the NAD(P)Hplastoquinone oxidoreductase of higher-plant chloroplasts. Planta 190: 2531 Berkemeyer M, Scheibe R and Ocheretina O (1998) A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L. J Biol Chem 273: 2792727933 Berry S and Rumberg B (1999) Proton to electron stoichiometry in electron transport of spinach thylakoids. Biochim Biophys Acta 1410: 248261 Biggins J (1974) The role of plastoquinone in the in vivo photosynthetic cyclic electron transport pathway in algae. FEBS Lett 38: 311314 Blubaugh DJ and Govindjee (1988) The molecular mechanism of the bicarbonate effect at the plastoquinone reductase site of photosynthesis. Photosynth Res 19: 85128 Bowes JM and Crofts AR (1981) The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence. Biochim Biophys Acta 637: 464472 Buchanan BB (1994) The ferredoxin-thioredoxin system: update on its role in the regulation of oxygenic photosynthesis. Adv Mol Cell Biol 10: 337354 Bult L, Cans P, Rebill F and Wollman F-A (1990) ATP control on state transitions in vivo in Chlamydomonas reinhardtii. Biochim Biophys Acta 1020: 7280 Burrows PA (1998) Functional characterization of the plastid ndh gene products. PhD Thesis, University of London Burrows PA, Sazanov LA, Svab Z, Maliga P and Nixon PJ (1998) Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J 17: 868876 Butler WL, Tredwell CJ, Malkin R and Barber J (1979) The relationship between the lifetime and yield of the 735 nm fluorescence of chloroplasts at low temperatures. Biochim Biophys Acta 545: 309315 Campbell D, Bruce D, Carpenter C, Gustafsson P and quist G (1996) Two forms of the Photosystem II D1 protein alter

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

Mechanisms for regulating photosynthetic electron transport have mainly been characterized in unicellular organisms, or in isolated chloroplasts. It seems almost certain that, in intact plants, there will be different responses in different cells and different tissues. Fluorescence video imaging can be used to show this at the macroscopic level (Scholes and Rolfe, 1996). This could be extended to microscopic scales by confocal fluorescence microscopy and microvolume spectroscopy. Tissue-specific gene expression studies should also provide a powerful approach to this question.

References
Allakhverdiev SI, Yruela I, Picorel R and Klimov VV (1997) Bicarbonate is an essential constituent of the water-oxidizing complex of Photosystem II. Proc Natl Acad Sci USA 94: 50505054 Allen JF (1992) Protein phosphorylation in regulation of photosynthesis. Biochim Biophys Acta 1098: 275335 Allen JF , Sanders CE and Holmes NG (1985) Correlation of membrane protein phosphorylation with excitation energy distribution in the cyanobacterium Synechococcus 6301. FEBS Lett l93: 271275 Andersson B and Anderson JM (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593: 427440 Arnon DI (1991) Photosynthetic electron transport: Emergence of a concept, 1949-59. Photosynth Res 29: 117131 Arnon DI and Chain RK (1975) Regulation of ferredoxin-catalysed photosynthetic phosphorylations. Proc Natl Acad Sci USA 72: 49614965 Asada K (1999) The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50: 601639 Ashby MK and Mullineaux CW (1999) Cyanobacterial ycf27 gene products regulate energy transfer from phycobilisomes to Photosystems I and II. FEMS Microbiology Lett 181: 253260 Barber J and Andersson B (1992) Too much of a good thing:

Chapter 30

Photosynthetic Electron Transport

551
Elrifi IR and Turpin DH (1986) Nitrate and ammonium induced photosynthetic suppression in N-limited Selenastrum minutum. Plant Physiol 81: 273279 Emlyn-Jones D, Ashby MK and Mullineaux CW (1999) A gene required for the regulation of photosynthetic light-harvesting in the cyanobacterium Synechocystis 6803. Mol Microbiol 33: 10501058 Endo T, Schreiber U and Asada K (1995) Suppression of quantum yield of Photosystem II by hyperosmotic stress in Chlamydomonas reinhardtii. Plant Cell Physiol 36: 12531258 Endo T, Mi H, Shikanai T and Asada K (1997) Donation of electrons to plastoquinone by NAD(P)H dehydrogenase and ferredoxin-quinone reductase in spinach chloroplasts. Plant Cell Physiol 38: 12721277 Endo T, Shikanai T, Sato F and Asada K (1998) NAD(P)H dehydrogenase-dependent, antimycin A-sensitive electron donation to plastoquinone in tobacco chloroplasts. Plant Cell Physiol 39: 12261231 Endo T, Shikanai T, Takabayashi A, Asada K and Sato F (1999) The role of chloroplastic NAD(P)H dehydrogenase in photoprotection. FEBS Lett 457: 58 Fearnley IM and Walker JE (1992) Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim Biophys Acta 1140: 105134 Feild TS, Nedbal L and Ort DR (1998) Non-photochemical reduction of the plastoquinone pool in sunflower leaves originates from chlororespiration. Plant Physiol 116: 12091218 Finazzi G, Furia A, Barbagallo RP and Forti G (1999) State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii. Biochim Biophys Acta 1413: 117129 Fleischmann MM, Ravanel S, Delosme R, Olive J, Zito F, Wollman F-A and Rochaix J-D (1999) Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition. J Biol Chem 274: 3098730994 Flgge U-I (1998) Metabolite transporters in plastids. Curr Opin Plant Biol 1: 201206 Fork DC and Herbert SK (1993) Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria. Photosynth Res 36: 149169 Foyer CH and Harbinson J (1997) The photosynthetic electron transport system: Efficiency and control. In: Foyer CH and Quick WP (eds) A molecular approach to primary metabolism in higher plants, pp 3-39. Taylor and Francis Ltd, London Frank HA, Cua A, Chynwat V, Young A, Gosztola D and Wasielewski MR (1994) Photophysics of the carotenoids associated with the xanthophyll cycle in photosynthesis. Photosynth Res 41: 389395 Friedrich T, Steinmller K and Weiss H (1995) The protonpumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts. FEBS Lett 367: 107111 Fulgosi H, Vener AV, Altschmied L, Herrmann RG and Andersson B (1998) A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen. EMBO J 17: 15771587 Funk C, Schrder WP, Napiwotzki A, Tjus SE, Renger G and Andersson B (1995) The PS II-S protein of higher plants: A new type of pigment-binding protein. Biochemistry 34: 1113311141

energy dissipation and state transitions in the cyanobacterium Synechococcus sp. PCC7942. Photosynth Res 47: 131144 Canaani O, Schuster G and Ohad I (1989) Photoinhibition in Chlamydomonas reinhardtii: Effect on state transition, intersystem energy distribution and Photosystem I cyclic electron flow. Photosynth Res 20: 129146 Cao J, Vermaas WFJ and Govindjee (1991) Arginine residues in the D2 polypeptide may stabilize bicarbonate binding in Photosystem II of Synechocystis sp. PCC 6803. Biochim Biophys Acta l059: 171180 Carol P, Stevenson D, Bisanz C, Breitenbach J, Sandmann G, Mache R, Coupland G and Kuntz M (1999) Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation. Plant Cell 11: 5768 Casano LM, Zapata JM, Martn M and Sabater B (2000) Chlororespiration and poising of cyclic electron transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase. J Biol Chem 275: 942948 Charlebois D and Mauzerall D (1999) Energy storage and optical cross-section of PS I in the cyanobacterium Synechococcus PCC 7002 and a mutant. Photosynth Res 59: 2738 Cleland RE and Bendall DS (1992) Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity. Photosynth Res 34: 409418 Cornac L, Guedeney G, Jot T, Rumeau D, Latouche G, Cerovic Z, Redding K, Horvath E, Medgyesy P and Peltier G (1998) Non-photochemical reduction of intersystem electron carriers in chloroplasts of higher plants and algae. In: Garab G (ed) Photosynthesis: Mechanisms and Effects, Vol III, pp 1877 1882. Kluwer Academic Publishers, Dordrecht Coughlan SJ and Hind G (1987) A protein kinase that phosphorylates light-harvesting complex is autophosphorylated and is associated with Photosystem II. Biochemistry 26: 6515 6521 Davies EC and Bendall DS (1987) The antimycin-binding site of thylakoid membranes from chloroplasts. In Biggins J (ed) Progress in Photosynthesis Research, Vol II, pp 485488. Martinus Nijhoff, Dordrecht Delosme R, Olive J and Wollman F-A (1996) Changes in the light energy distribution upon state transitions: An in vivo photoacoustic study of the wild type and photosynthesis mutants from Chlamydomonas reinhardtii. Biochim Biophys Acta 1273: 150158 Delphin E, Duval J-C, Etienne A-L and Kirilovsky D (1996) State transitions or quenching of Photosystem II fluorescence in red algae. Biochemistry 35: 94359445 Demmig-Adams B (1990) Carotenoids and photoprotection in plants: A role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 124 Diner BA, Petrouleas V and Wendoloski JJ (199la) The ironquinone electron-acceptor complex of Photosystem II. Physiol Plant 81: 423436 Diner BA, Nixon PJ and Farchaus JW (1991b) Site-directed mutagenesis of photosynthetic reaction centers. Curr Opin Struct Biol 1: 546554 Elortza F, Asturias JA and Arizmendi JM (1999) Chloroplast NADH dehydrogenase from Pisum sativum: Characterization of its activity and cloning of ndhK gene. Plant Cell Physiol 40: 149154

552
Funk E, Schfer E and Steinmller K (1999) Characterization of the Complex I-homologous NAD(P)H-PlastoquinoneOxidoreductase (NDH-complex) of Maize Chloroplasts. J Plant Physiol 154: 1623 Gal A, Herrmann RG, Lottspeich F and Ohad I (1992) Phosphorylation of cytochrome by the LHCII kinase associated with the cytochrome complex. FEBS Lett 298: 33 35 Gans P and Rebeille F (1990) Control in the dark of the plastoquinone redox state by mitochondrial activity in Chlamydomonas reinhardtii. Biochim Biophys Acta 1015: 150155 Genty B and Harbinson J (1996) Regulation of light utilization for photosynthetic electron transport. In: Baker NR (ed) Photosynthesis and the Environment, pp 6799. Kluwer Academic Publishers, Dordrecht Gilmore A, Shinkarev VP, Hazlett TL and Govindjee (1998) Quantitative analysis of the effects of intrathylakoid pH and the xanthophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids. Biochemistry 37: 1358213593 Greenbaum E, Lee JW, Tevault CV, Blankinship SL and Mets LJ (1995) fixation and photoevolution of and in a mutant of Chlamydomonas lacking Photosystem I. Nature 376: 438441 Groom QJ, Kramer DM, Crofts AR and Ort DR (1993) The nonphotochemical reduction of plastoquinone in leaves. Photosynth Res 36: 205215 Guedeney G, Corneille S, Cuin S and Peltier G (1996) Evidence for an association of ndhB, ndhJ gene products and ferredoxinNADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex. FEBS Lett 378: 277280 Hagemann M, Jeanjean R, Fulda S, Havaux M, Joset F and Erdmann N (1999) Flavodoxin accumulation contributes to enhanced cyclic electron flow around Photosystem I in saltstressed cells of Synechocystis sp. strain PCC 6803. Physiol Plant 105: 670678 Havaux M (1996) Short-term responses of Photosystem I to heat stress. Induction of a PS II-independent electron transport through PS I fed by stromal components. Photosynth Res 47: 8597 Heber U and Walker D (1992) Concerning a dual function of coupled cyclic electron transport in leaves. Plant Physiol 100: 16211626 Heber U, Gerst U, Krieger A, Neimanis S and Kobayashi Y (1995) Coupled cyclic electron transport in intact chloroplasts and leaves of C3 plants: Does it exist ? If so, what is its function? Photosynth Res 46: 269275 Heineke D, Riens B, Grosse H, Hoferichter P, Peter U, Flgge UI and Heldt HW (1991) Redox transfer across the inner chloroplast envelope membrane. Plant Physiol 95: 11311137 Herbert SK, Fork DC and Malkin S (1990) Photoacoustic measurements in vivo of energy storage by cyclic electron flow in algae and higher plants. Plant Physiol 94: 926934 Hinerwadel R and Berthomieu C (1995) Bicarbonate binding to the non-haem iron of Photosystem II investigated by Fourier transform infrared difference spectroscopy and bicarbonate. Biochemistry 34: 1628816297 Horton P, Ruban AV and Walters RG (1996) Regulation of lightharvesting in green plants. Annu Rev Plant Physiol Plant Mol Biol 47: 655684 Hosier JP and Yocum CF (1985) Evidence for two cyclic

Peter J. Nixon and Conrad W. Mullineaux

photophosphorylation reactions concurrent with ferredoxincatalyzed non-cyclic electron transport. Biochim Biophys Acta 808:2131 Howitt CA and Vermaas WFJ (1998) Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp. PCC 6803. Biochemistry 37: 1794417951 Howitt CA, Udall PK and Vermaas WFJ (1999) Type 2 NADH dehydrogenases in the cyanobacterium Synechocystis sp. strain PCC 6803 are involved in regulation rather than respiration. J Bacteriol 181: 39944003 Johnson GN, Scholes JD, Horton P and Young AJ (1993) The dissipation of excitation energy in British plant species. Plant, Cell and Environment 16: 673679 Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M and Tabata S (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. 2. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3: 109136 Katona E, Neimanis S, Schnknecht G and Heber U (1992) Photosystem I-dependent cyclic electron transport is important in controlling Photosystem II activity in leaves under conditions of water stress. Photosynth Res 34: 449464 Klimov VV and Baranov S V (2001) Bicarbonate requirement for the water-oxidizing complex of Photosystem II. Biochim Biophys Acta 1503: 187196 Klimov VV, Baranov SV and Allakhverdiev SI (1997) Bicarbonate protects the donor side of Photosystem II against photoinhibition and thermoinactivation. FEBS Lett 418: 243 246 Klughammer B, Sultemeyer D, Badger MR and Price GD (1999) The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria. Mol Microbiol 32: 13051315 Klukas O, Schubert W-D, Jordan P, Krau N, Fromme P, Witt HT and Saenger W (1999) Photosystem I, an improved model of the stromal subunits PsaC, PsaD and PsaE. J Biol Chem 274: 73517360 Kofer W, Koop H-U, Wanner G and Steinmller K (1998) Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation. Mol Gen Genet 258: 166173 Koop H-U, Kofer W and Steinmller K (1998) Judging the homoplastomic state of plastid transformantsa reply. Trends Plant Sci 3: 377 Kramer DM and Crofts AR (1993) The concerted reduction of the low and high potential chains of the bf complex by plastoquinol. Biochim Biophys Acta 1183: 7284 Kramer DM and Crofts AR (1996) Control and measurement of photosynthetic electron transport in vivo. In: Baker NR (ed) Photosynthesis and the Environment, pp 2566. Kluwer Academic Publishers, Dordrecht Krause GH, Briantais J-M and Vernotte C (1983) Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K. Biochim Biophys Acta 723: 169175 Krieger A, Moya I and Weis E (1992) Energy-dependent

Chapter 30 Photosynthetic Electron Transport

quenching of chlorophyll a fluorescence: Effect of pH on stationary fluorescence and picosecond-relaxation kinetics in thylakoid membranes and Photosystem II preparations. Biochim Biophys Acta 1102: 167176 Krmer S, Stitt M and Heldt HW (1988) Mitochondrial oxidative phosphorylation participating in photosynthetic metabolism of a leaf cell. FEBS Lett 226: 352356 Kruse O, Nixon PJ, Schmid GH and Mullineaux CW (1999) Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging. Photosynth Res 61: 4351 Kubicki A, Funk E, Westhoff P and Steinmller K (1996) Differential expression of plastome-encoded ndh genes in mesophyll and bundle-sheath chloroplasts of the C4 plant Sorghum bicolor indicates that the complex I-homologous NAD(P)H-plastoquinone oxidoreductase is involved in cyclic electron transport. Planta 199: 276281 Lemaire C, Wollman F-A and Bennoun P (1988) Restoration of photoautotrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplastic atpB gene of the ATP synthase has a deletion: An example of mitochondrial dependent photosynthesis. Proc Natl Acad Sci USA 85: 13441348 Li X-P, Bjrkman O, Shih C, Grossman AR, Rosenquist M, Jansson S and Niyogi KK (2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403: 391395 Maliga P and Nixon PJ (1998) Judging the homoplastomic state of plastid transformants. Trends Plant Sci 3: 376377 Martn M, Casano LM and Sabater B (1996) Identification of the product of ndhA gene as a thylakoid protein synthesised in response to photooxidative treatment. Plant Cell Physiol 37: 293298 Matsuo M, Endo T and Asada K (1998) Properties of the respiratory NAD(P)H dehydrogenase isolated from the cyanobacterium Synechocystis PCC6803. Plant Cell Physiol 39: 263267 Mehler AH (1951) Studies on the reaction of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents. Arch Biochem Biophys 33: 6577 Mi H, Endo T, Schreiber U, Ogawa T and Asada K (1992) Electron donation from cyclic and respiratory flows to the photosynthetic intersystem chain is mediated by pyridine nucleotide dehydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 33: 12331237 Mi H, Endo T, Ogawa T and Asada K (1995) Thylakoid membrane-bound, NADPH-specific pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 36: 661668 Mills JD, Crowther D, Slovacek RE, Hind G and McCarty RE (1979) Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark. Biochim Biophys Acta 547: 127137 Miyake C, Schreiber U and Asada K (1995) Ferredoxin-dependent and antimycin A-sensitive reduction of cytochrome b-559 by far-red light in maize thylakoids: participation of a menadiolreducible cytochrome b-559 in cyclic electron flow. Plant Cell Physiol 36: 743748 Mizuno T, Kaneko T and Tabata S (1996) Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of the cyanobacterium Synechocystis

553
sp. strain PCC6803. DNA Res 3: 407414 Moss DA and Bendall DS (1984) Cyclic electron transport in chloroplasts: The Q cycle and the site of action of antimycin. Biochim Biophys Acta 767: 389395 Mullineaux CW (1992) Excitation energy transfer from phycobilisomes to Photosystem I in a cyanobacterium. Biochim Biophys Acta 1100: 285292 Mullineaux CW (1994) Excitation energy transfer from phycobilisomes to Photosystem I in a cyanobacterial mutant lacking Photosystem II. Biochim Biophys Acta 1184: 7177 Mullineaux CW and Allen JF (1990) State 1 -state 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II. Photosynth Res 22: 157166 Mullineaux CW, Tobin, MJ and Jones GR (1997) Mobility of photosynthetic complexes in thylakoid membranes. Nature 390: 421424 Nilsson A, Stys D, Drakenberg T, Spangfort MD, Forsen S and Allen JF (1997) Phosphorylation controls the three-dimensional structure of plant light harvesting complex II. J Biol Chem 272: 1835018357 Nixon PJ (2000) Chlororespiration. Phil Trans R Soc Lond B 355: 15411547 Nixon PJ, Gounaris K, Coomber SA, Hunter CN, Dyer TA and Barber J (1989) psbG is not a Photosystem two gene but may be an ndh gene. J Biol Chem 264: 1412914135 Niyogi KK (1999) Photoprotection revisited: Genetic and molecular approaches. Annu Rev Plant Physiol Plant Mol Biol 50: 333359 Norling B, Zak E, Andersson B and Pakrasi H (1998) 2Disolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett 436: 189192 Ohkawa H, Sonoda M, Katoh H and Ogawa T (1998) The use of mutants in the analysis of the mechanism in cyanobacteria. Can J Bot 76: 10351042 Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H and Ozeki H (1986) Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572574 Olive J, Ajlani G, Astier C, Recouvreur M and Vernotte C (1997) Ultrastructure and light adaptation of phycobilisome mutants of Synechocystis PCC6803. Biochim Biophys Acta 1319: 275282 Osmond CB (1981) Photorespiration and photoinhibition: some implications for the energetics of photosynthesis. Biochim Biophys Acta 639: 7798 Peltier G and Thibault P (1988) Oxygen-exchange studies in Chlamydomonas mutants deficient in photosynthetic electron transport: evidence for a Photosystem II-dependent oxygen uptake in vivo. Biochim Biophys Acta 936: 319324 Petrouleas V, Deligiannakis Y and Diner BA (1994) Binding of carboxylate anions at the non-haem Fe(II) of PS II. 2. Competition with bicarbonate and effects on the QA/QB electron transfer rate. Biochim Biophys Acta 1188: 271277 Phillip D, Ruban AV, Horton P, Asato A and Young AJ (1996) Quenching of chlorophyll fluorescence in the major lightharvesting complex of Photosystem II: A systematic study of the effect of carotenoid structure. Proc Natl Acad Sci USA 93: 14921497 Quiles MJ and Cuello J (1998) Association of ferredoxin-NADP

554
oxidoreductase with the chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves. Plant Physiol 117: 235244 Ravenel J, Peltier G and Havaux M (1994) The cyclic electron pathways around Photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage. Planta 193: 251259 Redding K, Cournac L, Vassiliev IR, Golbeck JH, Peltier G and Rochaix J-D (1999) Photosystem I is indispensable for photoautotrophic growth, fixation, and photoproduction in Chlamydomonas reinhardtii. J Biol Chem 274: 10466 10473 Rich PR (1988) A critical examination of the supposed variable proton stoichiometry at the chloroplast cytochrome b/f complex. Biochim Biophys Acta 932: 3342 Rich PR, Hoefnagel MHN and Wiskich JT (1998) Possible chlororespiratory reactions ofthylakoid membranes. In: Mller IM, Gardestrm P, Glimelius K and Glaser E (eds) Plant Mitochondria: From Gene to Function, pp 1723. Backhuys Publishers, Leiden Rintamki E, Salonen M, Suoranta U-M, Carlberg I, Andersson B and Aro E-M (1997) Phosphorylation of light-harvesting complex II and Photosystem II core proteins shows different irradiance-dependent regulation in vivo. J Biol Chem 272: 3047630482 Sazanov LA, Burrows PA and Nixon PJ (1998) The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes. Proc Natl Acad Sci USA 95: 13191324 Scheibe R (1987) -malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme. Physiol Plant 71: 393400 Scherer S, Almon H and Bger P (1988) Interaction of photosynthesis, respiration and nitrogen fixation in cyanobacteria. Photosynth Res 15: 95114 Schmetterer G (1994) Cyanobacterial respiration. In: Bryant DA (ed) The Molecular Biology of Cyanobacteria, pp 409435. Kluwer Academic Publishers, Dordrecht Scholes JD and Rolfe SA (1996) Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): Quantitative imaging of chlorophyll fluorescence. Planta 199: 573582 Schreiber U, Endo T, Mi H and Asada K (1995) Quenching analysis of chlorophyll fluorescence by the saturation pulse method: particular aspects relating to the study of eukaryotic algae and cyanobacteria. Plant Cell Physiol 36: 873882 Schweitzer RH and Brudvig GW (1997) Fluorescence quenching by chlorophyll cations in Photosystem II. Biochemistry 36: 1135111359 Shikanai T, Endo T, Hashimoto T, Yamada Y, Asada K and Yokota A. (1998) Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around Photosystem I. Proc Natl Acad Sci USA 95: 97059709 Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H and Sugiura M (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 20432049

Peter J. Nixon and Conrad W. Mullineaux

Snel JFH and van Rensen JJS (1984) Reevaluation of the role of bicarbonate and formate in the regulation of photosynthetic electron flow in broken chloroplasts. Plant Physiol 75: 146 150 Snyders S and Kohorn BD (1999) TAKS, thylakoid membrane protein kinases associated with energy transduction. J Biol Chem 274: 91379140 Stemler AJ (1998) Bicarbonate and photosynthetic oxygen evolution: An unwelcome legacy of Otto Warburg. Indian J Exp Biol 36: 841848 Stumpp MT, Motohashi K and Hisabori T (1999) Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the submit of chloroplast ATP synthase. Biochem J 341: 157163 Sundby C (1990) Bicarbonate effects on photoinhibition. Including an explanation for the sensitivity to photo-inhibition under anaerobic conditions. FEBS Lett 274: 7781 Tagawa K, Tsujimoto H Y and Arnon DI (1963) Role of chloroplast ferredoxin in the energy conversion process of photosynthesis. Proc Natl Acad Sci USA 49: 567572 Takahama U, Shimidzu-Takahama M and Heber U (1981) The redox state of NADP in illuminated chloroplasts. Biochim Biophys Acta 637: 530539 Teicher HB and Scheller HV (1998) The NAD(P)H dehydrogenase in barley thylakoids is photoactivatable and uses NADPH as well as NADH. Plant Physiol 117: 525532 Turmel C, Otis C and Lemieux C (1999) The complete chloroplast DNA sequence of the alga Nephroselmis olivacea: Insights into the architecture of ancestral chloroplast genomes. Proc Natl Acad Sci USA 96: 1024810253 Turpin DH and Bruce D (1990) Regulation of photosynthetic light-harvesting by nitrogen assimilation in the green alga Selenastrum minutum. FEBS Lett 263: 99103 Vallon O, Bult L, Dainese P, Olive J, Bassi R and Wollman FA (1991) Lateral redistribution of cytochrome complexes along thylakoid membranes upon state transitions. Proc Natl Acad Sci USA 88: 82628266 van Rensen JJS, Xu C and Govindjee (1999) Role of bicarbonate in Photosystem II, the water-plastoquinone oxido-reductase of plant photosynthesis. Physiol Plant 105: 585592 van Thor JJ, Geerlings TH, Matthijs HCP and Hellingwerf KJ (1999a) Kinetic evidence for the PsaE-dependent transient ternary complex Photosystem I/ferredoxin/ferredoxin: reductase in a cyanobacterium. Biochemistry 38: 1273512746 van Thor JJ, Gruters OWM, Matthijs HCP and Hellingwerf KJ (1999b) Localization and function of ferredoxin: reductase bound to the phycobilisomes of Synechocystis. EMBO J 18: 41284136 van Walraven HS, Strotmann H, Schwarz O and Rumberg B (1996) The coupling ratio of the ATP synthase from thiol-modulated chloroplasts and two cyanobacterial strains is four. FEBS Lett 379: 309313 Vener AV, van Kan PJM, Rich PR, Ohad I and Andersson B (1997) Plastoquinol at the quinol oxidation site of reduced cytochrome bf mediates signal transduction between light and protein phosphorylation: Thylakoid protein kinase deactivation by a single-turnover flash. Proc Natl Acad Sci USA 94: 1585 1590 Vermaas W (1994) Molecular genetic approaches to study photosynthetic and respiratory electron transport in thylakoids from cyanobacteria. Biochim Biophys Acta 1187: 181186

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Photosynthetic Electron Transport

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Xiong J, Minagawa J, Crofts A and Govindjee (1998) Loss of inhibition by formate in newly constructed Photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii. Biochim Biophys Acta 1365: 473491 Xue X, Gauthier DA, Turpin DH and Weger HG (1996) Interactions between photosynthesis and respiration in the green alga Chlamydomonas reinhardtii. Plant Physiol. 112: 10051014 Yruela I, Allakhverdiev SI, Ibarra JV and Klimov VV (1998) Bicarbonate binding to the water-oxidizing complex in the Photosystem II. A Fourier transform infrared spectroscopy study. FEBS Lett 425: 396400 Yu L, Zhao J, Mhlenhoff U, Bryant DA and Golbeck JH (1993) PsaE is required for in vivo cyclic electron flow around Photosystem I in the cyanobacterium Synechococcus sp. PCC 7002. Plant Physiol 103: 171180 Zer H, Vink M, Keren N, Dilly-Hartwig HG, Paulsen H, Herrmann RG, Andersson B and Ohad I (1999) Regulation of thylakoid protein phosphorylation at the substrate level: Reversible lightinduced conformational changes expose the phosphorylation site of the light-harvesting complex II. Proc Natl Acad Sci USA 96: 82778282

Wakasugi T, Tsudzuki J, Ito S, Nakashima K, Tsudzuki T and Sugiura M (1994) Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii. Proc Natl Acad Sci USA 91: 97949798 Walters RG, Ruban AV and Horton P (1996) Identification of proton-active residues in a higher plant light-harvesting complex. Proc Natl Acad Sci USA 93: 1420414209 Weis E (1981) Reversible heat-inactivation of the Calvin cycle: A possible mechanism of the temperature regulation of photosynthesis. Planta 151: 3339 Wollman F-A and Lemaire C (1988) Studies on kinase-controlled state transitions in Photosystem II and mutants from Chlamydomonas reinhardtii which lack quinone-binding proteins. Biochim Biophys Acta 933: 8594 Wu D, Wright DA, Wetzel C, Voytas DF and Rodermel S (1999) The IMMUTANS variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast biogenesis. Plant Cell 11: 4355 Xiong J, Hutchinson RS, Sayre RT and Govindjee (1997) Modification of the Photosystem II acceptor side function in a D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardtii. Biochim Biophys Acta 1322: 6076