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Abstract. Photosystem I-dependent cyclic electron transport is shown to operate in intact spinach chloroplasts with ox- aloacetate, but not with nitrite or ...

Photosynthesis Research 57: 61–70, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.


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Photosystem I-dependent cyclic electron flow in intact spinach chloroplasts: Occurrence, dependence on redox conditions and electron acceptors and inhibition by antimycin A B. Ivanov1,∗, Y. Kobayashi2, N.G. Bukhov1,∗∗ & U. Heber1 1 Julius-von-Sachs-Institut

für Biowissenschaften der Universität, Julius-von-Sachs Platz 2, D-97082 Würzburg, Germany; 2 Department of Forestry, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812 Japan; ∗ Current address: Institute of Soil Sciences and Photosynthesis, Russian Academy of Sciences, Pushchino, Moscow Region, Russia; ∗∗ Current address: Timiriasev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia Received 8 December 1997; accepted in revised form 27 April 1998

Key words: electron transport, nitrite, oxaloacetate, photosynthesis, proton transport

Abstract Photosystem I-dependent cyclic electron transport is shown to operate in intact spinach chloroplasts with oxaloacetate, but not with nitrite or methylviologen as electron acceptors. It is regulated by the redox state of the chloroplast NADP system. Inhibition of cyclic electron transport by antimycin A occurs immediately on addition of this antibiotic in the light. It is unrelated to a different function of antimycin A, inhibition of nonphotochemical quenching of chlorophyll fluorescence, which requires prior dissipation of the transthylakoid proton gradient before antimycin A can become effective. Abbreviations: Ant A – antimycin A; Chl – chlorophyll; DCMU – 3-(3,4-dichlorophenyl)-1,1-dimethylurea; MV – methyviologen; OAA – oxaloacetate; PPFD – photosynthetically active photon flux density; PS I and PS II – Photosystems 1 and 2; 9AA – 9-aminoacridine Introduction For many years it appeared clear that cyclic electron transport around PS I is required for carbon assimilation of C3 plants which needs ATP and NADPH at a ratio of 3/2. The reason for this conviction was that linear electron flow from water to CO2 across PS II and PS I seemed to provide insufficient ATP at measured H+ /e− ratios close to two of linear electron transport and H+ /ATP ratios close to three for ATP synthesis (Portis and McCarty 1976). However, with the emergence of the Q-cycle (Mitchell 1977) the H+ /e− ratio of linear electron transport shifted slowly from two to three (Ivanov et al. 1985; Rich 1988). Unfortunately now, ATP and NADPH would be provided by linear electron transport at the ratio of 1/1 which, if correct, would be too high and therefore as inconvenient

in a stoichiometric sense as a ratio which is too low. The problem was solved by proposals that H+ /ATP is not three but four (Gräber et al. 1987; Rumberg et al. 1990; Kobayashi et al. 1995). If true, linear electron transport alone would provide ATP and NADPH exactly at the ratio that is required for carbon assimilation of C3 plants, and cyclic electron transport would have lost its function in providing ATP for photosynthesis. The question arose now whether it occurs at all in C3 plants (Hormann et al. 1994). In the classical experiments of DI Arnon with isolated thylakoids, proper redox poising decided on the occurrence of cyclic photophosphorylation (Arnon and Chain 1979). With an effective electron acceptor present and largely oxidized electron carriers in the electron transport chain, cyclic electron flow was suppressed in favor of linear electron flow. It was also

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62 suppressed when electron carriers between PS II and PS I were largely reduced and therefore incapable of accepting electrons from the cyclic pathway (ZiemHanck and Heber 1980). In this way the activity of PS II controlled PS I activity. The question whether PS I can, in turn, control PS II was answered in the affirmative when it was observed that in intact leaves water stress which caused stomata to close shifted the quantum efficiency of electron flow through PS I and PS II from a 1/1 ratio to higher ratios clearly indicating higher electron flow through PS I than PS II (Heber et al. 1993; Gerst et al. 1995). When stomata close in the light, or when the CO2 concentration of air is reduced, light scattering by the leaves, non-photochemical quenching of chlorophyll fluorescence and oxidation of P700 in the reaction center of PS I increase (Heber et al. 1992) indicating increased control of PS II activity. Such control is known to be exerted by a protonation reaction in the thylakoid interior which requires an increased transthylakoid proton gradient 1pH (Horton et al. 1996). Obviously, increased PS I activity relative to that of PS II and a simultaneously decreased intrathylakoid pH suggest a role of PS I-dependent cyclic electron flow in the control of PS II (Heber and Walker 1992). Since the problem of how cyclic electron flow is regulated in vivo is difficult to solve in experiments with intact leaves, we used isolated intact chloroplasts of spinach to gain more insight into its regulation. Since effective electron acceptors are known to suppress cyclic electron flow, it was of particular interest to know whether an electron acceptor such as OAA which, like CO2 , takes electrons from NADPH, permits cyclic electron flow to occur.

Materials and methods Chloroplasts were isolated essentially as described by Jensen and Bassham (1966). Further purification was obtained by Percoll centrifugation (Asada et al. 1990). Except for the experiments shown in Figures 1–3, 10 mM ascorbate was present in the isolation media and during the Percoll centrifugation. The intactness of chloroplasts was at least 80% as judged by the ferricyanide method (Heber and Santarius 1970). The chloroplasts were capable of photoreducing CO2 at rates between 80 and 150 µmol (mg Chl × h)−1 . Oxygen exchange was measured with a Clark-type electrode, modulated Chl fluorescence with a PAM

Figure 1. The influence of additions of OAA (A) or nitrite (B) on light-induced 9AA fluorescence quenching under anaerobic conditions. 1.8 µM DCMU was added to inhibit linear electron flow from PS II (Ziem-Hanck and Heber 1980). Numbers show final concentration of OAA or nitrite in µM (produced by sequential additions of the electron acceptors). PPFD was 300 µmol m−2 s−1 .

101 fluorometer (Walz, Effeltrich, Germany), and 9AA fluorescence either by a photomultiplier or by the Xenon-PAM described by Schreiber et al. (1993). These measurements were performed simultaneously in a square quartz cuvette thermostated at 20 ◦ C. 1 s pulses of high intensity light (PPFD 7000 µmol m−2 s−1 ) were applied on top of actinic illumination to produce transient increases in Chl fluorescence that indicated full reduction of the primary quinone acceptor QA in the reaction center of PS II. 9AA concentration was 0.5 or 1 µM. Its fluorescence was excited by xenon flashes (4 Hz) which were passed through a 5874 filter of Corning, NY, USA, a UG 11 filter of Schott, Mainz, Germany, and a gray filter. It was recorded by a photodiode which was protected by BG 28, BG 38 and GG 19 filters of Schott against actinic light. Actinic illumination was provided by a 250 W halogen lamp the light of which was filtered through a RG 610 cutoff filter, a 675 nm interference filter (half-

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Figure 2. (A) Extent of 9AA fluorescence quenching (in percent of total 9AA fluorescence) versus rates of oxygen uptake with 0.5 mM MV (0.5 mM KCN also present), or of oxygen evolution with 1 mM OAA (catalase present). Oxygen exchange was varied by changing the intensity of red light. (B) Percentage of proton transport during the OAA reduction which is not coupled to linear electron flow (left ordinate, open circles) and corresponding rates of this proton transport (right ordinate, closed circles) versus linear electron flow to OAA. For explanation, see text.

Figure 3. Dependence of light-induced 9AA fluorescence quenching on rates of electron transport as determined by the product of quantum yield of charge separation in PS II and intensity of incident light (1F/Fm 0 × PFD; Genty et al. 1989) (A) or on rates of net oxygen uptake with 0.3 mM MV (1 mM KCN also present), (E ), or of net oxygen evolution with 0.2 mM ( ) or 0.5 mM ( ) nitrite (B). Electron transport rates were changed by changing the intensity of 675 nm light, the maximal intensity being 60 µmol quanta m−2 s−1 . In experiments with nitrite the suspensions also contained catalase (40 µg ml−1 ).


Results and discussion bandwidth 11.1 nm) of Schott and a heat absorbing filter Calflex X of Balzers. PPFDs were measured with a LiCor quantum meter (Licor, Lincoln, NE, USA). The reaction medium was the same as that used by Jensen and Bassham (1966), but contained in addition 10 mM glyceraldehyde in all experiments to inhibit Calvin cyle activity. Additions of ascorbate, catalase, KCN and electron acceptors are indicated in the figure legends. The pH of stock solutions of KCN was adjusted to 7.6. Anaerobic conditions were established by adding glucose, glucose oxidase and catalase to chloroplast suspensions as described by Ziem-Hanck and Heber (1980).

Cyclic electron transport in the presence of linear electron transport and its redox regulation In the two experiments of Figure 1, intact chloroplasts were made anaerobic by adding glucose, glucose oxidase and catalase. PS II activity was drastically curtailed by the addition of 1.8 µM DCMU which largely inhibits electron flow through the QB site of the PS II reaction center. In this situation, illumination decreased the fluorescence of 9AA. Light-dependent quenching of 9AA fluorescence is known to indicate the formation of a transthylakoid proton gradient (Fiolet et al. 1974; De Benedetti and Garlaschi 1977; Ha-

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64 raux and de Kouchkovsky 1980; Vu Van and Rumberg 1986). Without DCMU, quenching was insignificant (not shown) because an active PS II rapidly reduced the electron carriers of the electron transport chain thereby blocking, in the absence of electron acceptors, cyclic electron transport (Ziem-Hanck and Heber 1980; Kobayashi and Heber 1994). By inhibiting PS II activity, DCMU made the formation of a transthylakoid pH gradient by cyclic electron flow possible. Addition of 1 µM OAA and then of another 1 µM increased the proton gradient, but further additions decreased it by inhibiting cyclic electron flow. The extent of quenching maintained after the final addition of 284 µM indicates the magnitude of the proton gradient that is a result of linear electron flow to OAA in the presence of DCMU. Owing to the inhibition of PS II activity, it is very small. Whereas OAA was effective to stimulate cyclic electron flow at low concentrations, another electron acceptor, nitrite, was not (Figure 1). The main difference between the two electron acceptors is that nitrite receives electrons from ferredoxin and OAA from NADPH. The enzyme mediating OAA reduction, NADP-dependent malic dehydrogenase, is lightregulated. The enzyme is inactive in the dark. It is activated in the light by the chloroplast thioredoxin system (Buchanan 1980), and it is also regulated by the chloroplast NADPH/NADP ratio (Scheibe 1987). The data of Figure 1 suggest that, at low concentrations, OAA stimulated cyclic electron transport by relieving excessive reduction of electron carriers which was caused by residual PS II activity. At high concentrations, OAA inhibited cyclic electron flow. The experiment illustrates redox poising of cyclic electron transport in intact chloroplasts. Similar redox posing has been observed in intact chloroplasts with oxygen as electron acceptor in a Mehler-type reaction (Kobayashi and Heber 1994; Heber et al. 1995). In the aerobic experiment of Figure 2A, net O2 exchange (evolution or uptake) is plotted against the extent of 9AA quenching with either OAA or MV as electron acceptors. In the absence of ascorbate, and with KCN present, one molecule of oxygen taken up in the light corresponds to four electrons transferred from water to oxygen (Kobayashi et al. 1995). Likewise, during oxygen evolution with OAA as electron acceptor, four electrons are transferred to OAA when two molecules of water are oxidized and one molecule of oxygen is released. Whereas 9 AA fluorescence quenching increased linearly with electron flow at low light intensities when MV was electron ac-

ceptor, its initial slope was much increased during the OAA reduction compared to that observed with MV. During linear electron flow, the H+ /e− ratio is three in intact chloroplasts (Kobayashi and Heber 1995). Since MV is a very effective electron acceptor its reduction is not accompanied by cyclic electron flow. Thus, by comparing 9 AA fluorescence quenching during MV reduction with that during linear electron flow to OAA in Figure 2A, it is possible to calculate the contribution of proton transport attributable to cyclic electron transport with OAA as electron acceptor. The applied procedure is identical to that published earlier for cyclic electron transport during oxygen reduction (Kobayashi and Heber 1994). It is assumed that equal 9AA fluorescence quenching with different electron acceptors indicates comparable proton pumping and that the H+ /e− ratio is three during linear electron flow to OAA as it is with MV. Protons pumped into the thylakoids in addition to those derived from linear electron flow must then be attributed to cyclic electron flow. Figure 2B shows corresponding calculations from the data of Figure 2A. Proton transport into the thylakoids coupled to cyclic electron transport increased steeply with increasing linear electron transport to OAA until an optimum was reached at a rate of linear electron transport of about 30 µeq (mg Chl × h)−1 . At low light intensities and correspondingly slow linear electron transport, cyclic electron transport contributed more than linear electron transport to the formation of 1pH. As linear electron flow increased, the contribution of cyclic electron transport decreased although it remained high. The nitrite experiment of Figure 1 suggests that nitrite inhibited cyclic electron transport very efficiently. This is confirmed by the data presented in Figure 3A where 9AA fluorescence quenching is plotted against relative rates of linear electron transport as measured by the chlorophyll fluorescence and light parameters 1F/Fm 0 × PPFD according to Genty et al. (1989) with 0.2 and 0.5 mM nitrite and with 0.3 mM MV. In all instances, 9AA fluorescence quenching with nitrite was comparable to that observed with MV when electron fluxes were comparable. However, when the same data were plotted not against linear electron transport as in Figure 3A but against oxygen evolution which was measured simultaneously with modulated chlorophyll fluorescence and 9AA fluorescence, a different picture emerged. In this case, 9AA fluorescence quenching increased steeply with 0.2 mM nitrite, less steeply with 0.5 mM

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65 nitrite and linearly with electron flow to MV (Figure 3B). The Km of nitrite reductase for nitrite is 0.1 mM (Losada and Paneque 1971). Thus, in the experiment of Figure 3 nitrite reductase was not saturated in respect to its substrate. Apparently, in this situation, oxygen served as a secondary substrate. Oxygen reduction contributes to the formation of the proton gradient, but does not give rise to oxygen evolution. Only the fluorescence data of Figure 3A indicate the real extent of linear electron transport. Oxygen reduction is known to be accompanied by cyclic electron transport (Kobayashi and Heber 1994; Heber et al. 1995). However, the data of Figure 3A give no evidence of the presence of cyclic electron transport in the presence of limiting nitrite concentrations. If such electron transport had occurred, it should have increased 9AA fluorescence quenching when compared with that caused by MV. When the nitrite concentration was much higher than in the experiments of Figure 3, 9AA fluorescence quenching was frequently somewhat larger than even with 2 mM OAA (Figure 4). At first sight, this does not agree with the observations documented in Figures 1 and 3, as it might suggest that cyclic electron transport operates not only in the presence of OAA but also in that of nitrite. However, there is evidence that in the presence of high concentrations of nitrite the chloroplast stroma is acidified (Purczeld et al. 1978). When this occurs, the intrathylakoid pH also decreases. Since 9AA fluorescence quenching indicates the 1pH between the outside medium and the intrathylakoid pH and not the 1pH between the chloroplast stroma and the intrathylakiod space, this explains larger fluorescence quenching with nitrite than with MV or, occasionally, even OAA when nitrite concentrations are sufficiently high to decrease the stroma pH. Antimycin A as inhibitor of cyclic electron transport and of energy-dependent quenching of chlorophyll fluorescence Ant A is known to be a potent inhibitor of cyclic electron flow (Moss and Bendall 1984). Figure 4A shows 9AA fluorescence quenching as a function of the intensity of 675 nm light with 2 mM OAA or 2 mM nitrite as electron acceptors. In both cases, the proton gradient decreased in the presence of 1µM Ant A but the decrease was larger with OAA than with nitrite. Electron transport was stimulated by Ant in both cases,

but once again the increase was larger with OAA than with nitrite (Figure 4B). A large stimulation of electron transport by Ant A was also observed with intact maize mesophyll chloroplasts during OAA reduction (Ivanov and Edwards 1995). Increased electron transport as a consequence of a decreased proton gradient is reminiscent of uncoupling. However, at the applied concentration (1 µM) which ensures a low ratio between Ant A and chlorophyll (Ivanov and Edwards 1995), Ant A is not an uncoupler. Therefore, increased electron flow and decreased 9AA fluorescence quenching suggest decreased proton pumping. This suggestion is in full accord with the data of Figure 2 for OAA, but not necessarily with the data of Figure 3A for nitrite. When Ant A was added in the dark to a chloroplast suspension which contained 1 µM 9AA, 9AA fluorescence increased (data not shown). This increase was Ant A-dependent. It indicated competition of Ant A and 9AA for chloroplast binding sites. It is known that 9AA not only distributes between the suspending medium, the chloroplast stroma and the intrathylakoid space but is also bound to thylakoid membranes (Fiolet et al. 1974). Unbinding of 9AA by Ant A explains the small decrease in 9AA fluorescence quenching shown in Figure 4A for nitrite. In chloroplasts uncoupled by nigericin which is an effective K+ /H+ exchanger, maximum lightdependent oxygen evolution with nitrite was about 100 µmol (mg Chl × h)−1 and with OAA about 30 µmol (mg Chl × h)−1 (data not shown). The differences are attributable to different activities of ferredoxin-dependent nitrite reductase and NADPdependent malic dehydrogenase which limit electron flow at different maximum levels. When compared with maximum electron flow in uncoupled chloroplasts, the data of Figure 4B show strict photosynthetic control both with nitrite and OAA in fully coupled chloroplasts. This control was relieved appreciably by Ant A only in the case of OAA. Ant A is known not only to inhibit PS I-dependent cyclic electron transport but also the radiationless dissipation of excess activation energy that is indicated by nonphotochemical fluorescence quenching (Oxborough and Horton 1987). By decreasing radiationless dissipation in the pigment bed (Horton et al. 1996), it increases excitation pressure on PS II. This can explain slightly increased electron flow with nitrite in the presence of Ant A as shown in Figure 4B. To clarify the situation, modulated chlorophyll fluorescence was measured simultaneously with oxygen

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Figure 4. Effects of Ant A (1 µM) on light-induced 9AA fluorescence quenching (A) and on rates of oxygen evolution (B) during the reduction of OAA (2 mM) and nitrite (2 mM) at different intensities of 675 nm light. The suspensions contained also ascorbate (10 mM) and catalase (40 µg ml−1 ).

exchange. Electron acceptors were OAA, nitrite, MV and oxygen. Results are shown in Figure 5. When Ant A was added in the light, oxygen evolution increased immediately by 25% with OAA as electron acceptor, but less than 5% with nitrite and MV. Accordingly, 1F/Fm 0 , as an expression of effective charge sepa-

ration in PS II, increased by about 25% with OAA, not or less with the other electrons acceptors. Modulated chlorophyll fluorescence Fs increased slightly with OAA and MV, more with oxygen and not at all with nitrite. This increase was related to the pH of the intrathylakoid space. It was suppressed when the pH

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Figure 5. Effects of Ant A (1 µM) on rates of oxygen exchange (upper recordings) and on modulated chlorophyll fluorescence (lower recordings). Electron acceptors were 2 mM OAA, 2 mM nitrite, 0.3 mM MV (1 mM KCN also present) and 280 µM oxygen. Numbers near the oxygen traces are rates of oxygen evolution or uptake in µmol O2 (mg Chl)−1 h−1 ; numbers near fluorescence spikes which are caused by 1 s saturating light pulses are values of 1F/Fm 0 (quantum yield of charge separation in PS II; Genty et al. ( 1989)). PFD of 675 nm light was 200 µmol m−2 s−1 . In experiments with OAA, nitrite, and without added acceptors the suspensions contained 10 mM ascorbate.

of the suspending medium was decreased from pH 7.6 to pH 7.2 (data not shown). During darkening, dissipation of the proton gradient relieved non-photochemical quenching of chlorophyll fluorescence. Upon renewed illumination nonphotochemical quenching, which was not or not much affected when Ant A was added in the light, was much decreased. Apparently, dissipation of the proton gradient during darkening permitted Ant A to occupy sites which in the light had been blocked by protonation. Once occupied, they were inaccessible to Ant A. Darkening was required for binding which then pre-

vented the protonation in the light which is needed for effective nonphotochemical fluorescence quenching. Thus, Ant A has two effects which can be separated kinetically. When added in the light, it inhibits cyclic electron transport immediately. In contrast, inhibition of non-photochemical fluorescence quenching occurs only after binding sites have become available in the dark which were protonated in the light. Binding of Ant A in the dark is slower than the dissipation of the proton gradient after darkening. This is demonstrated in Figure 6 which shows the kinetics of the decrease in non-photochemical fluorescence quenching as influenced by different periods of darkening.

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Figure 6. Effects of the length of dark intervals between periods of illumination on the level of steady state chlorophyll fluorescence yield (Fs ) after 1 µM Ant A was added during a first illumination period. 2 mM nitrite was electron acceptor. (A) Recordings of chlorophyll fluorescence. (B) Increase in steady state fluorescence Fs as a function of individual dark times. Other conditions as for Figure 5.

Nitrite was electron acceptor. During the illumination periods, binding of Ant A did not occur or was negligible. This was seen when a fresh chloroplast sample was used for every dark time experiment. The half time of darkening producing increased steady state fluorescence Fs during subsequent illumination periods was 52 s compared to a half time of 26 s of 1pH dissipation. The latter was measured as increased 9AA fluorescence after darkening. In fact, 1pH dissipation is very probably considerably faster than indicated by the 9AA fluorescence method because what is actually measured is the diffusion of 9AA from the intrathylakoid space into the medium, not proton efflux from the thylakoids into the chloroplast stroma.

Conclusions Oxaloacetate reduction in chloroplasts requires reductive activation of NADP malic dehydrogenase by thioredoxin which itself is reduced when reduced ferredoxin becomes available in the light (Buchanan 1980). NADP is an allosteric inhibitor of activity.

For these reasons, effective reduction of OAA proceeds only at elevated NADPH/NADP ratios (Scheibe 1987). This explains why at low rates of linear electron transport, proton transport into the thylakoids is dominated by cyclic electron transport (Figure 2). However, even when 1pH was almost saturated and linear electron transport to OAA was partially controlled by the proton gradient (Figures 2 and 4), proton transport attributable to cyclic electron flow was still 50% of total proton deposition into the intrathylakoid space (Figure 2). This shows that the range of redox regulation of cyclic electron transport is broad in intact chloroplasts. Cyclic electron transport is inhibited when electrons are effectively intercepted by electron acceptors such as MV or nitrite (Figures 2 and 3). The data suggesting cyclic electron transport in the presence of high concentrations of nitrite (Figure 4) are not convincing since nitrite reduction was stimulated by Ant A only when Ant A was added in the dark, not when it was added during illumination (Figure 5). Since the same was observed with MV, which is a highly effective electron acceptor, we conclude that nitrite, like MV, inhibits cyclic electron transport. Whereas nitrite reductase accepts electrons from reduced ferredoxin, ferredoxin-NADP reductase donates electrons to NADP. Shahak et al. (1981) have proposed that this enzyme is also capable of directing electrons into the cyclic pathway. The absence of cyclic electron flow with nitrite in our experiments also suggests that ferredoxin-NADP reductase is a component of the cyclic electron transport pathway. Apparently, NADP and the cyclic pathway compete for electrons. For the outcome of this competition, the redox state of the chloroplast NADP system is decisive. In photosynthesizing leaves of C3 plants, quantum efficiencies of electron transport through PS II and PS I are very similar under many conditions (Foyer et al. 1992). As long as NADPH is efficiently oxidized by glyceraldehydephosphate dehydrogenase, cyclic electron transport cannot operate. However, when stomata close under strong light, cyclic electron transport is activated (Gerst et al. 1995). This indicates that stromal NADP/NADP ratios rise sufficiently for making cyclic electron transport possible when entry of external CO2 into leaves becomes severely restricted. By decreasing the intrathylakoid pH, cyclic electron transport permits radiationless dissipation of excess activation energy thereby contributing to photoprotection.

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