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abolished by the addition of vitamin K or other naph- thoquinones (Arnon, 1955). This experiment marks the discovery of the cyclic phosphorylation process, (re-.

Chapter 37 Cyclic Electron Transfer Around Photosystem I Pierre Joliot∗ and Anne Joliot CNRS UMR 7141, Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, 75005 Paris, France

Giles Johnson University of Manchester, School of Biological Sciences, 3.614 Stopford Building, Oxford Road, Manchester, M13 9PT, UK

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639 I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640 II. Early Observations of Cyclic Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640 III. Possible Pathways of Electron Flow in Cyclic Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640 IV. Redox Poising of the Cyclic Electron Transfer Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641 V. Structural Organization of Thylakoid Membranes – Consequences for Cyclic Electron Transfer . . . . . . . . . . . 642 VI. Occurrence of Cyclic Flow in Higher Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643 VII. Pathway of Cyclic Flow in Higher Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647 A. NADPH-Dependent Cyclic Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647 B. Ferredoxin-Dependent Cyclic Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647 VIII. Cyclic Flow in Green Unicellular Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649 IX. Cyclic Flow in Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650 X. Functions and Regulation of Cyclic Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651 XI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652

Summary Cyclic electron transport around Photosystem I remains one of the last great enigmas in photosynthesis research. Although first described in 1955 by Arnon and coworkers, the molecular details of the pathway, its physiological role and even its very occurrence remain in question. Nevertheless, significant progress is starting to be made in our understanding of this process. At least two pathways of cyclic electron transport appear to operate, one involving the transfer of electrons from NADPH to plastoquinone and the other operating via the donation of electrons from ferredoxin to plastoquinone. The relative importance of these two pathways seems to vary between cyanobacteria, unicellular green algae and higher plants as do many details concerning the regulation of the pathway and its functional organization in the thylakoid membrane. Two distinct functions for cyclic electron transport can be defined — the generation of ATP and, in higher plants, the generation of pH to regulate light harvesting. These two functions give rise to the need for different regulatory processes to control the ratio of cyclic and linear electron flow. We discuss recent findings that cast new light on how cyclic electron transport is regulated under a range of physiological conditions.



Author for correspondence, email: [email protected]

John H. Golbeck (ed): Photosystem I: The Light-Driven Plastocyanin:Ferredoxin Oxidoreductase, 639–656.  C 2006 Springer.

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I. Introduction The concept of cyclic electron transport (ET) around Photosystem (PS) I is sufficiently established that it features in the diagrams of photosynthetic electron transport found in every undergraduate biochemistry textbook. In spite of this celebrity, the redox components involved, the functional importance, the regulation and, indeed, even the very occurrence of this pathway all remain unclear. Part of the problem in studying cyclic ET has been, that, by its very nature, it is difficult to quantify — as a cycle it involves no net flux and so we are forced to resort to use indirect means to deduce its existence. There is now growing evidence, however, that cyclic ET does indeed occur in a variety of organisms under a wide range of conditions, fulfilling at least two distinct functions. Here, we present a review of this evidence, focusing in particular on advances over the last decade and identifying the challenges that remain. For more detailed coverage of earlier work and alternative views on the subject, a number of good reviews are available (Heber and Walker, 1992; Fork and Herbert, 1993; Bendall and Manasse, 1995; Heber, 2002; Allen, 2003).

II. Early Observations of Cyclic Electron Transfer Photosynthetic phosphorylation (photophosphorylation) was discovered in 1954 by Arnon et al. (1954) who established that illumination of isolated chloroplasts in the presence of oxygen-induced ATP synthesis. As this process was not associated with oxygen formation or consumption, the contribution of a respiratory chain could be excluded. Later, it was established that the dependence of photophosphorylation on oxygen can be abolished by the addition of vitamin K or other naphthoquinones (Arnon, 1955). This experiment marks the discovery of the cyclic phosphorylation process, (reviewed in Arnon et al., 1961). The reaction previously characterized in 1954 by Arnon and coworkers appears now to be a pseudocyclic process in which electrons are transferred from water to O2 (Mehler, 1951) via the Abbreviations: cyt – cytochrome; DBMIB – 2,5-dibromo3-methyl-6-isopropyl- p-benzoquinone; DCMU – 3-(3,4dichloro-phenyl)-1,1-dimethylurea; ET – electron transport; ETC – electron transfer chain; Fd – ferredoxin; FNR – ferredoxin: NADP oxidoreductase; FQR – ferredoxin: plastoquinone reductase; HQNO – 2-heptyl-4-hydroxy-quinoline N-oxide; NDH – NADH dehydrogenase; PC – plastocyanin; PMS – Nmethylphenazonium-3-sulfonate; PQ – plastoquinone; PQH2 – plastoquinol; PS – Photosystem; RC – reaction center.

Pierre Joliot, Anne Joliot and Giles Johnson linear electron transfer chain. Subsequently, nonphysiological compounds, such as phenazine methosulfate (PMS), were shown to catalyze cyclic photophosphorylation more efficiently than vitamin K (Jagendorf and Avron, 1958). The anaerobic cyclic phosphorylation identified in chloroplasts appeared analogous to a cyclic phosphorylation process previously identified in chromatophores from photosynthetic bacteria (Frenkel, 1954). In Rhodospirillum rubrum, this cyclic process was observed to involve cytochrome (cyt) c (Smith and Baltscheffsky, 1959); in chloroplasts cyt f is involved (Arnon, 1959). In 1960, Hill and Bendall put forward a model describing the linear electron transfer chain involving both the photoreactions PS II and PS I working in series (Hill and Bendall, 1960). It became clear, however, that cyclic photophosphorylation involves only PS I, as it operates in the presence of specific inhibitors of oxygen evolution, such as 3-(3,4-dichloro-phenyl)-1,1dimethylurea (DCMU). Tagawa et al. (1963b) established that ferredoxin (Fd) was able to catalyze cyclic phosphorylation. This provided evidence for a physiological catalyst of cyclic ET, making it seems likely that this process was more than an artifact of in vitro conditions.

III. Possible Pathways of Electron Flow in Cyclic Electron Transfer There is a general agreement that both PS I and the cyt b/ f complex are obligatory involved in cyclic electron flow — cyclic ET is efficiently driven by farred light and is sensitive to inhibitors of the cyt b/ f complex, such as 2,5-dibromo-3-methyl-6-isopropylp-benzoquinone (DBMIB) and stigmatellin — however, the electron pathway from the acceptor side of PS I back to the cyt b/ f complex has not yet been clearly elucidated. Cyclic ET, as conventionally measured, leads to the generation of a trans-thylakoid pH gradient. Thus, whatever the pathway involved a protonpumping step involving the cyt b/ f complex must be postulated. Based mainly on studies of the effects of the inhibitor antimycin A, it has been postulated that at least two pathways exist. Experiments performed on isolated chloroplasts showed that antimycin inhibits cyclic flow in the presence of Fd (Tagawa et al., 1963b) but not in the presence of vitamin K or PMS (Whatley et al., 1959). Hosler and Yocum (1985) measured the ratio of P/O in the presence of Fd, using oxygen as a terminal electron acceptor and found this to be sensitive to antimycin. They explained this effect as being due to the

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Fig. 1. Possible pathways for cyclic electron flow.

inhibition of cyclic ET. By contrast, when the same experiment was performed using NADP as an electron acceptor, the P/O ratio was high and antimycin-insensitive (Hosler and Yocum, 1985). Scheller (1996) measured the rereduction of P700 following light flashes in the presence of DCMU and noted an antimycin-insensitive portion in Fd-mediated cyclic ET, possibly suggesting a third pathway (Scheller, 1996). However, the very slow rate of this, barely distinguishable from the DBMIBinhibited rate, makes it hard to separate this from background redox equilibration of the sample. Cleland and Bendall (1992) measured the oxidation of reduced Fd following illumination of DCMU-poisoned thylakoids and found this rate to be inhibited by antimycin A as completely as by stigmatellin (Cleland and Bendall, 1992). One can thus conclude that several mechanisms could be involved in a cyclic ET. In a conventional Q-cycle process (Mitchell, 1975; Crofts et al., 1983), electrons are transferred to the cyt b/ f complex via a reduced quinone that binds site Qo , on the lumenal side of this complex. Assuming this is the step generating pH in the cyclic process, we need to invoke an enzyme that is able to transfer electrons from NADP or Fd to plastoquinone (PQ), with the resultant plastoquinol (PQH2 ) being protonated on the stromal side of the membrane. In cyanobacteria, the presence of respiratory and photosynthetic electron transport chains in the same membrane means that this function could be fulfilled by a respiratory NADH dehydrogenase (NDH; complex I). Genes coding for such an enzyme have also been identified in the chloroplast genome of higher plants (Shinokazi et al., 1986), which

is a likely candidate to be involved in the cyclic electron transfer chain (ETC) (Fig. 1, pathway 1). Moss and Bendall (1984) noted that, whilst both Fdmediated and artificially mediated cyclic ET are sensitive to the cyt b inhibitor 2-heptyl-4-hydroxy-quinoline N-oxide (HQNO), only Fd-mediated cyclic ET was sensitive to antimycin (Moss and Bendall, 1984). This led them to suggest an alternative site for antimycin inhibition on an enzyme distinct from the b/ f complex, termed ferredoxin:plastoquinone oxidoreductase (FQR) (Fig. 1, pathway 2). The involvement of ferredoxin:NADP oxidoreductase (FNR) in cyclic ET has been much discussed. Its involvement in the NADP-dependent pathway is presumed; however, a role in the Fd-dependent pathway has also been postulated. The observation that this enzyme is stoichiometrically bound to the cyt b/ f complex in spinach provides an intriguing indication of an additional role, other than that of linear electron transport (Zhang et al., 2001). It is suggested that FNR mediates the transfer of electrons from ferredoxin to site Qi (Fig. 1, pathway 3). The mechanisms involved in these different pathways will be discussed in more detail below.

IV. Redox Poising of the Cyclic Electron Transfer Chain Photochemical charge separation at the level of a reaction center (RC) requires the presence of a reduced primary donor and an oxidized electron acceptor.

642 Over-reduction or overoxidation of the cyclic chain will thus inhibit this process. The concept of redox poising of the cyclic ETC was introduced to explain the oxygen requirement of cyclic phosphorylation (Tagawa et al., 1963a; Whatley, 1963; reviewed in Allen, 1983). Later, Arnon and Chain (1977) established that a maximum efficiency of the cyclic ET in the presence of oxygen is observed in the presence of NADPH and subsaturating concentrations of DCMU that impose an optimal redox poise of the carriers involved in the cyclic chain (Arnon and Chain, 1977). Redox poising is determined by the relative rate of electron efflux or influx from or toward the carriers belonging to the cyclic electron transfer chain. Both efflux and influx will occur preferentially at the level of mobile carriers that are common to linear and cyclic chains. PS II will be the main source of reductive power (Fig. 1, pathways a and b) while electron efflux will occur at the level of PS I acceptors toward O2 and the Benson–Calvin cycle (Fig. 1, pathways c and d, respectively). If we assume that cyclic and linear pathways are connected, i.e., share the same mobile carriers, the redox poise of the cyclic chain will be controlled by the electron flow through the linear chain. Under strong illumination, given under condition where the Benson– Calvin cycle is inhibited (e.g., in isolated thylakoids or dark-adapted leaves), PS II will induce an overreduction of the cyclic chain, via pathway a or b (Fig. 1). Conversely, under conditions where PS II is inhibited or under far-red light, the electron efflux via pathway c or d will induce an overoxidation of the cyclic chain. On the other hand, if the cyclic and linear chains are structurally separated, the redox poise of the cyclic chain will be controlled by the rate of slow electron leaks that occur between carriers involved in both processes. We thus conclude that the structural organization of membrane proteins that controls the localization of the cyclic and linear chains within the membrane may play an essential role in the control of the efficiency of the cyclic process.

V. Structural Organization of Thylakoid Membranes – Consequences for Cyclic Electron Transfer Oxygenic photosynthetic organisms — higher plants, unicellular algae, and cyanobacteria — differ substantially in the supramolecular organization of their photosynthetic membranes and these differences may have important consequences for the pathway and regulation of cyclic ET.

Pierre Joliot, Anne Joliot and Giles Johnson In higher plants, PS II and PS I are localized in different membrane regions (Andersson and Anderson, 1980). Most of PS II is localized in the appressed regions of the grana stacks while PS I is localized in the stroma lamellae and in the granal end membranes. Unlike PS II and PS I, cyt b/ f complex is distributed across all membrane regions (Cox and Andersson, 1981). An open question concerns a possible localization of PS I centers in the margin of the grana stacks (Webber et al., 1988; Anderson, 1989; Albertsson, 1995). Albertsson (2001) put forward the hypothesis that PS I localized in the margin and ends of the grana stacks contributes to the linear pathway while PS I localized in the stroma lamellae contributes to the cyclic pathway (Albertsson, 2001). The localization of PS I and PS II in different membrane regions requires long-range diffusion of the mobile carriers PQ or plastocyanin (PC). A detailed analysis of the kinetics of electron transfer reaction between PS II and the PQ pool has shown that diffusion of PQ is restricted to small heterogeneous domains including an average of three to four RC, a membrane surface much smaller than the size of a grana disk (Joliot et al., 1992; Lavergne et al., 1992; Kirchhoff et al., 2000). It is assumed that the membrane proteins, which occupy more than half of the membrane surface, limit the diffusion of PQ. This implies that PQ is not involved in long distance transfer and that the linear process exclusively involves cyt b/ f complexes localized close to PS II, i.e., in the appressed regions. Conversely, the sole role of the cyt b/ f complexes localized in the stroma region might be to participate in the cyclic process (“cyclic cyt b/ f ”). It worth pointing out that, if the photosynthetic apparatus were exclusively devoted to a linear process, one would expect that its optimization during evolution would have led to a random distribution of RCs within the membrane. Such a distribution would minimize the distance between membrane proteins, leading to faster electron exchanges mediated by PQ or PC. Thus, the segregation of PS I and PS II centers in different membrane regions can be taken as a way to separate the carriers involved in the cyclic and the linear flows, which limits redox cross-talk between the cyclic and linear chains. Structural separation between the linear and cyclic processes is pushed to an extreme in the case of C4 plants, in which only a cyclic process operates in bundle sheath cells that mainly include PS I centers (Bassi et al., 1985). In green unicellular algae, the supramolecular organization of thylakoid membranes significantly differs from that in higher plants. Thylakoid membranes

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consist of long flat vesicles (disks) that are generally stacked in groups of 2–4, a much smaller number than that seen in higher plants. Freeze-fracture images suggest that appressed and nonappressed regions are more widely connected than is the case in chloroplasts of higher plants, which are connected by narrow fret junctions. This membrane organization suggests that cyclic and linear pathways could interact more in green algae than in higher plants. In cyanobacteria, thylakoids membranes are unstacked and appear as isolated flat vesicles. The organization of these membranes differs between species but concentric arrangements of thylakoids are often seen in rod-shaped cells as Synechococcus sp. PCC 6803 or filamentous species such as Phormidium laminosum. There is evidence from freeze-fracture images of cyanobacterial thylakoid membranes that PS I and PS II are typically found in the same membrane regions — see Mullineaux (1999), although Sherman et al. (1994) noted a slight asymmetry in the distribution of complexes in Synechococcus sp. PCC 6803, suggesting a concentration of PS I near to the plasma membranes. Spatial segregation of linear and cyclic chains is thus very unlikely and one expects these pathways to share the same electron carriers. In contrast to photosynthetic eukaryotes, thylakoids in cyanobacteria include a respiratory chain that shares the PQ pool and the cyt b/ f complex with the photosynthetic chain (for a review see Schmetteter, 1994). Thus, redox poising of a putative cyclic chain could be controlled by interaction with the linear photosynthetic chain as well as with the respiratory chain. An open question is the nature of the substrate of the NDH enzymes of the respiratory chain—NADH or NADPH— localized in the thylakoids. NADPH-PQ reductase, present at high concentration in the chloroplasts, could itself contribute efficiently to a cyclic process (Fig. 1, pathway 1).

VI. Occurrence of Cyclic Flow in Higher Plants Work establishing the existence of pathways for cyclic ET has largely been performed in isolated systems, usually thylakoid membranes (broken chloroplasts), with addition of natural or artificial mediators. While such studies are essential in characterizing the pathway of cyclic ET, they leave open the question; does this pathway actually operate under in vivo conditions? More specifically, if we are to understand the function of cyclic ET it must be established whether it occurs un-

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der normal physiological conditions, where linear ET is also possible. Many of the studies that have indicated the presence of cyclic ET in intact leaves have used rather indirect means, typically involving conditions that largely or totally suppress PS II turnover. While such studies are valuable, especially in trying to understand the regulation of the cyclic pathway, they do not, in themselves, show that this pathway is able to compete with linear ET. One commonly used assay taken as evidence for cyclic flux is the measurement of the relaxation of P700+ following illumination of a leaf with a period of far-red light ( > 695 nm) or in the presence of a PS II inhibitor, such as DCMU (Maxwell and Biggins, 1976). When a leaf is exposed to far-red light to oxidize P700 and then that light is abruptly cut, typically at least two phases of P700+ reduction can be detected. The half time for the fast phase is typically of the order of 200–1,000 msec (Jo¨et et al., 2002). By comparison, in white light, under conditions where PS II is turning over normally, the half time for P700+ reduction is of the order of 10–20 msec. Thus, it seems immediately unlikely that cyclic ET could ever compete effectively with a linear flow that is 10–50 times faster. However, measurements made in the absence of PS II turnover will tend to result the accumulation of electron transfer components (including Fd and NADP) in the oxidized state and so, the half times measured probably represent a gross underestimate of the maximum rate of electron flow from the stroma to P700+ . The rate of P700+ reduction varies between different groups of organisms, being slow in plants and especially slow in C3 plants (Herbert et al., 1990; Jo¨et et al., 2002). The rate can, however, be accelerated under certain conditions, for example under anaerobiosis (Jo¨et et al., 2002) or following heat stress (Maxwell and Biggins, 1976; Burrows et al., 1998; Bukhov et al., 1999). The former probably reflects an increase in the reduction state of the chloroplast in the dark, increasing the supply of reductant to re-reduce P700+ . In the latter case, it is less clear what gives rise to the effect, it may relate to a temperature induced shift in redox poise or to an “opening up” of redox components to the surrounding medium accelerating their reduction. Recently, Golding et al. (2004), examining the relaxation of P700+ in the presence of the PS II inhibitor DCMU, observed that preillumination of leaves eliminates the fast component of P700+ reduction but that this effect is removed if the leaves are experiencing drought stress. It is suggested that a transient “cyclic-enabled” state existing in the dark is stabilized under drought conditions (Johnson, 2005).

644 Another parameter that has been taken to indicate cyclic ET is the presence of a transient rise in fluorescence following illumination (Asada et al., 1993; Burrows et al., 1998). When actinic light is removed, the fluorescence yield falls, due to the oxidation of Q− A. In some conditions, the yield of fluorescence is seen to rise transiently and then fall again. This effect is attributed to the reduction of the PQ pool by electrons originating from the stroma, indicating that a pathway exists between the stroma and the electron transport chain that could participate in cyclic ET. Such measurements can be made following conditions of steady-state white light, so may reflect better the normal physiological state of the leaf but, as with the decay of P700+ , involve processes that are far too slow (several tens of seconds) for them to be supposed to be involved in efficient cyclic ET in competition with linear ET. As with the decay of P700+ following far-red light, this effect is enhanced by exposing plants to heat stress (Sazanov et al., 1998). Although cyclic ET involves no net flux that can be measured, it does nonetheless have “products” that are measurable, notably the formation of a pH gradient across the thylakoid membrane, which can be evidenced using optical spectroscopy and the storage of light energy, which is measured using the technique of photoacoustics. Two absorbance signals have been used as indicators of the pumping of protons across the thylakoid membrane during cyclic ET: Proton translocation results in the formation of a transmembrane electrical potential, which can be measured as an apparent absorbance change around 515 nm; and the swelling of the thylakoid membrane that occurs when a pH is generated induces a change in its light scattering properties, giving an apparent absorbance change in the region of 535 nm. Both of these absorbance changes can be observed when leaves are illuminated with far-red light, indicating that cyclic ET is occurring and is generating pH. However, the conditions needed to observe such changes are often quite specific, so again it is difficult to be certain whether the cycling observed is relevant to conditions of steady-state photosynthesis. Photoacoustics measures pressure waves produced in samples in response to light, which can be interpreted to provide information on energy storage and gas exchange. Photoacoustic signals are complex, with a number of different processes contributing, so it is necessary to design experiments carefully to give any information on cyclic ET. As with most other methods, this often means using far-red light to avoid any contribution from PS II photochemistry. A large number

Pierre Joliot, Anne Joliot and Giles Johnson of studies have been published using this approach, often in combination with other approaches. For example (Jo¨et et al., 2002), recently combined measurements of photoacoustics with relaxation of P700+ to investigate the functioning of cyclic ET in tobacco. In spite of the array of different methods that have been applied in an attempt to determine whether cyclic ET occurs in higher plants, the question still remains controversial. A clear case where cyclic ET is thought to be the norm is in the bundle sheath cells of certain C4 plants, including maize (Herbert et al., 1990; Asada et al., 1993; Jo¨et et al., 2002). In such cells, PS II is largely absent, yet these cells are responsible for the fixation of CO2 through normal Benson–Calvin cycle activity. By contrast, photosynthesis (and specifically PS II) is not responsible for generating the reducing potential required to drive CO2 fixation, since this is generated through the oxidation and decarboxylation of malate imported from the mesophyll. Cyclic ET is thought to provide the ATP. Studies using photoacoustics and P700+ reduction kinetics have both provided support for the occurrence of cyclic ET in maize bundle sheath chloroplasts (Herbert et al., 1990; Jo¨et et al., 2002) as have combined measurements of P700 and chlorophyll fluorescence under combinations of far-red and red light (Asada et al., 1993). In C3 plants, the situation is less clear. As early as 1978, Heber et al. (1978) observed the presence of far red-induced light scattering changes in spinach leaves. This scattering was slow to form however and was inhibited by oxygen. The latter observation can easily be explained in terms of the ability of oxygen to oxidize the acceptor side of PS I. If PS II activity is suppressed, then electrons taking part in the cyclic pathway that are leaked to oxygen cannot be replaced. The cyclic ETC will rapidly become completely oxidized and cyclic ET will stop. This contrasts with the situation in C4 bundle sheath cells, where reductant is available from the decarboxylation of malate, such that, even in the absence of PS II activity, electrons can be reinjected in to the cyclic pathway. A number of studies have, however, indicated that efficient far red-induced cyclic ET can occur in C3 leaves, however, this requires careful selection of conditions. For example, Katona et al. (1992) observed that, under conditions of CO2 free air, far-red light was able to efficiently energize chloroplasts in cabbage leaves, as indicated by light scattering changes. This effect was however suppressed by O2 or CO2 . Heber et al. (1992) performed similar experiments on ivy leaves and reached similar conclusions, with the CO2 concentration again being crucial. By contrast, the combination of red light

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(rather than far-red) and low CO2 and O2 did not result in chloroplast energization. In other words, conditions giving rise to PS II turnover in the absence of any terminal electron acceptor result in the rapid total reduction of the ETC. Under such conditions, no further electron transport, linear or cyclic, is possible (see section on redox poising above). Taking an alternative approach, Cornic and colleagues observed the effects of periods of actinic illumination on the ability of limiting levels of far-red light to oxidize P700. Even short periods of high light were found to be sufficient to reduce the effectiveness of far-red light in oxidizing P700. This was interpreted as being due to an activation of cyclic ET during high light, feeding electrons back into the ETC via an efficient cyclic pathway (Cornic et al., 2000). While the above discussion leads to the conclusion that cyclic ET can be induced in the leaves of higher plants, it does not tell us whether it does occur under conditions of steady-state photosynthesis. Given the low rates of ET that have been measured and the careful poising that is often needed to observe cyclic ET, it is not at all clear that this pathway can compete under conditions where linear ET is feeding electrons into the ETC. To determine whether or not cyclic is a real physiological phenomenon, we need to be able to measure it under conditions of normal photosynthesis. The most common approach evidencing cyclic ET under conditions of steady-state photosynthesis is to examine the relationship between PS I and PS II electron transport. Given that cyclic ET involves only PS I and linear both PS I and PS II, any change in cyclic ET, relative to linear ET will give a change in the ratio of the flux of the two photosystems. Measurements of PS II flux are usually made using analysis of chlorophyll fluorescence. The quantum efficiency of PS II is measured as the parameter PS II (Genty et al., 1989). Multiplying this parameter by the light intensity gives a measure of relative flux. Provided light interception by the PS II antenna remains constant (which might not be the case, e.g., due to state transitions) this parameter is thought to give a robust relative measure of the PS II electron transport rate. In vivo measurements of PS I electron transport under conditions of steady-state photosynthesis have proved more controversial. A commonly used approach has been to measure the redox state of the P700 pool and to take the extent of reduction of this pool as a measure of the quantum efficiency of PS I. Comparisons of PS I turnover measured in this way with PS II turnover measured by fluorescence have been made at a variety of irradiances and CO2 concentrations (Harbinson and Foyer, 1991; Harbinson, 1994). These studies have

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found the relationship between these two parameters to be linear. Thus, it has been concluded that, under most physiological conditions, cyclic ET is either absent in the presence of light or forms a constant proportion of the linear flux. More recent data have however found clear evidence for cyclic electron transport using this approach (Clark and Johnson, 2001; Miyake et al., 2004; Miyake et al., 2005a,b). The contradictions between the above studies have not yet been fully explained however probably relate to the measuring conditions or the physiological status of the plants concerned. Observations of cyclic ET have been made under conditions where the supply of CO2 to the leaf is restricted. For example, Harbinson and Foyer (1991) observed that the relationship between PS I and PS II quantum efficiency (using light as a variable) was different in CO2 free air to that seen in the presence of CO2 . Gerst et al. (1995) observed that, upon imposing drought stress upon a leaf in the light, PS II was more sensitive to inhibition than PS I. By contrast, however, in experiments where a leaf was exposed to varying CO2 , the relationship between PS I and PS II efficiency was found to be linear, extrapolating to the origin (Harbinson, 1994). Thus any cyclic flow that occurs must be in proportion to linear flow. In a similar experiment, Golding and Johnson (2003) noted that, although the total amount of reduced P700 was proportional to PS II efficiency, this relationship did not extrapolate to the origin, giving space for a constant rate of cyclic ET (Golding and Johnson, 2003). In addition, however, these authors applied the method of Klughammer and Schreiber (1994), to estimate the proportion of PS I in an “active” state. This measurement is performed by superimposing a saturating flash of white light on top of background actinic illumination and then transferring the sample directly to darkness, taking the total signal following the flash-to-dark transition as a measure of active centers. In the study of Klughammer and Schreiber (1994), the loss of active centers was supposed to be related to a limitation on the acceptor side of PS I, preventing centers from turning over. Surprisingly, Golding and Johnson (2003) noted that active PS I rose at low CO2 (i.e., under conditions where PS I is most likely to be acceptor-side limited). They suggested that a distinct population of PS I centers exists that is largely or wholly involved in cyclic ET. This “cyclic-only” pool is activated at low concentrations of CO2 and under high light. Thus, the contradictions in earlier measurements might be related to the way in which CO2 limitation was applied and whether these “cyclic” centers were already activated or not.

646 In measurements of cyclic ET in far-red light it has been common to take the rate of P700+ reduction as an indicator of PS I turnover (Maxwell and Biggins, 1976). Essentially, the same approach can be applied in white light conditions. This relies on the limiting step in electron transport lying prior to PS I, which is usually the case under physiological conditions, such that the flow of electrons to P700 can be measured and reflects the overall flux through PS I. A potential problem with this method was noted by Sacksteder and Kramer (2000), who noted that a net reduction of cyt f , measured at the time the light is switched off, might have to be taken into account to determine the electron flux toward P700 (Sacksteder and Kramer, 2000). Strictly, there should be no net reduction of cyt f at the moment when the light is switched off, as at steady-state the rate of oxidation and reduction are identical and neither of these are instantly affected by cutting the light. Practically, given the time resolution and sensitivity of most instruments, this may be a problem but only under low light conditions where P700 is largely reduced but cyt f oxidized, giving rise to a short time lag in the reduction of cyt f . Sacksteder and Kramer (2000) have compared the turnover of PS I and PS II in greenhouse grown sunflowers. They observed a linear relationship between PS I and PS II ET, implying no (or a constant proportion of) cyclic ET, the same conclusion as reached by Harbinson and colleagues using P700 redox state to measure PS I turnover (Harbinson et al., 1990; Harbinson, 1994). In contrast, Clarke and Johnson compared the rates of PS I and PS II turnover across a range of temperatures and light intensities in barley grown in a growth cabinet. They observed that PS II photochemistry saturated at lower irradiances and was more sensitive to low temperature than PS I. Thus, they concluded that high light and low temperatures lead to enhancement of cyclic ET (Clarke and Johnson, 2001). The contradiction between these two studies may reflect a species difference, but is more likely explained by the range of light intensities used in each case. In the experiments of Sacksteder and Kramer, the highest light intensities used were just saturating, whereas for Clarke and Johnson light intensities were used that went well above saturating for PS II electron transport (though not necessarily for PS I). Thus it appears that cyclic ET is a characteristic of saturating light, although a low rate at subsaturating light cannot be excluded, if this forms a constant proportion of the linear flux. A similar approach by Golding and Johnson (2003) drew the same conclusion concerning responses to low CO2 and drought. A new technical approach, based on membrane potential measurements (Joliot and Joliot, 2002, 2004)

Pierre Joliot, Anne Joliot and Giles Johnson has been developed to determine the absolute rate of the cyclic and linear pathways whatever the intensity of illumination. In this method, the sum of the rates of photochemical reactions I and II is measured by the difference in the rate of membrane potential changes determined immediately before or after switching off the light. Experiments were performed under strong light excitation in the presence of air, with dark-adapted spinach (Joliot and Joliot, 2002) or Arabidopsis thaliana leaves (Joliot and Joliot, 2004), i.e., in conditions where the Benson–Calvin cycle and thus, the linear ET, is mainly inactive. Under saturating illumination, rate of the cyclic flow is estimated to ∼130 sec−1 and remains roughly constant during the first 10 sec of illumination. Unexpectedly, this cyclic process is not inhibited by antimycin (Joliot and Joliot, 2002). Under the same conditions, fluorescence induction kinetics shows that a pool of soluble PS I acceptors (Fd, FNR and NADP) of approximately nine electron equivalents is reduced in less than 100 msec via the linear pathway. This implies that, even in dark-adapted leaves, PS I remains able to transfer electrons to a pool of oxidized PS I acceptors. In the presence of DCMU, where the linear flow is fully inhibited, a similar rate of the cyclic flow is measured during the first seconds of illumination. This rate progressively drops to zero in ∼7 sec, due to slow electron leaks that lead to the oxidation of the carriers involved in the cyclic chain. After 100 msec of illumination at an intensity that is saturating for the cyclic process, most of P700 is reduced (Harbinson and Hedley, 1993; Strasser et al., 2001; Joliot and Joliot, 2002; Schankser et al., 2003) implying that a fast charge recombination between P700+ and reduced acceptors will occur. In agreement with this assumption, it is observed that the kinetics of the membrane potential displays a fast decaying phase of small amplitude, which is completed in ∼ 500 μsec. The amplitude of this phase is roughly proportional to the light intensity. This phase correlates with a reduction phase of P700+ (measured by the absorption changes at 810 nm) and has been ascribed to a charge recombination between P700+ and most probably the iron-sulfur carrier FX (Joliot and Joliot, 2002). It can be thus concluded that most of the carriers of the linear and cyclic chains are poised in their reduced state. A small fraction of “cyclic PS I” centers includes P700+ and a single negative charge on the FA /FB cluster. For these RCs, the rate of P700+ reduction via the cyclic pathway is faster than the rate of charge recombination between P700+ and (FA /FB )− (t1/2 ∼ 45 msec; Hiyama and Ke, 1971). A charge recombination process is also observed in the presence of DCMU, the amplitude of which is half

Chapter 37

Cyclic Electron Transfer Around Photosystem I

that measured in its absence. This is explained by the fact that illumination induces the oxidation of all the carriers of the linear chain, while the carriers involved in the cyclic pathway are transitorily poised at a potential able to induce the reduction of most of the FA /FB acceptors (

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