Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

Changes in the mode of electron flow to photosystem I following chilling-induced photoinhibition in a C3 plant, Cucumis sativus L.

This study provides evidence for enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700⁺ via the cytochrome b₆/f complex (Cyt b₆/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation–reduction kinetics studied in the absence of linear electron transport from water to NADP⁺, cyclic electron transfer mediated through the Q-cycle of Cyt b₆/f and charge recombination in photosystem I (PSI). The segregation of these pathways for P700⁺ rereduction were achieved by the use of a 50-ms multiple turnover white flash or a strong pulse of white or far-red illumination together with inhibitors. In cucumber leaves, chilling-induced photoinhibition resulted in ∼20% loss of photo-oxidizible P700. The measurement of P700⁺ was greatly limited by the turnover of cyclic processes in the absence of the linear mode of electron transport as electrons were rapidly transferred to the smaller pool of P700⁺. The above is explained by integrating the recent model of the cyclic electron flow in C₃ plants based on the Cyt b₆/f structural data [Joliot and Joliot (2006) Biochim Biophys Acta 1757:362–368] and a photoprotective function elicited by a low NADP⁺/NAD(P)H ratio [Rajagopal et al. (2003) Biochemistry 42:11839–11845]. Over-reduction of the photosynthetic apparatus results in the accumulation of NAD(P)H in vivo to prevent NADP⁺-induced reversible conformational changes in PSI and its extensive damage. As the ferredoxin:NADP reductase is fully reduced under these conditions, even in the absence of PSII electron transport, the reduced ferredoxin generated during illumination binds at the stromal openings in the Cyt b₆/f complex and activates cyclic electron flow. On the other hand, the excess electrons from the NAD(P)H pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow.     

Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts

Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559_HP, cytochrome b-559_LP, and cytochrome aa₃ by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa₃ in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559_HP but contain cytochrome b-559_LP (E_m7.5 = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559_LP in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559_LP indicating that this cytochrome is not directly involved in this pathway.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH₂) through the cytochrome b₆f complex (Cyt b₆f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700⁺ and PC⁺ was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC⁺ and P700⁺ components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b₆f to the PSI donors. A significant down-regulation of Cyt b₆f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b₆f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e⁻ transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.     

Upregulation of bundle sheath electron transport capacity under limiting light in C4 Setaria viridis

C₄ photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO₂ concentration at the site of CO₂ fixation. C₄ plants benefit from high irradiance but their efficiency decreases under shade, causing a loss of productivity in crop canopies. We investigated shade acclimation responses of Setaria viridis, a model monocot of NADP-dependent malic enzyme subtype, focussing on cell-specific electron transport capacity. Plants grown under low light (LL) maintained CO₂ assimilation rates similar to high light plants but had an increased chlorophyll and light-harvesting-protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen-evolving activity and the PSII/PSI ratio were enhanced in LL BS cells, indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b₆f and the chloroplast NAD(P)H dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were also increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts were associated with a more oxidised plastoquinone pool in LL plants and the formation of PSII – light-harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of PSII in BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to the understanding of the acclimation of PSII activity in C₄ plants and will support the development of strategies for crop improvement, including the engineering of C₄ photosynthesis into C₃ plants.     

Flash spectroscopic studies of cyclic electron flow in intact chloroplasts

The kinetic behaviours of cytochrome $\text{b}$-563 and cytochrome $\text{f}$ are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes $\text{b}$-563 and cytochrome $\text{f}$ is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes.     

Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L.

During daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b₆f complex, ATP synthase and several isoforms of ferredoxin‐NADP⁺ oxidoreductase (FNR), and ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b₆f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b₆f complex, FNR and redox‐inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone (PQ) and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd and cytochrome b_h of the cytochrome b₆f complex, and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both NADH and NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration that may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b₆f complex during cyclic electron transport in chloroplasts.     

Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP

The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b₆/f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP⁺-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting F_m. MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b₆/f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b₆/f complex and donate electrons directly to PC⁺ and P700⁺ with a donation time τ of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.     

Connectivity between electron transport complexes and modulation of photosystem II activity in chloroplasts

In chloroplasts, photosynthetic electron transport complexes interact with each other via the mobile electron carriers (plastoquinone and plastocyanin) which are in surplus amounts with respect to photosystem I and photosystem II (PSI and PSII), and the cytochrome b ₆ f complex. In this work, we analyze experimental data on the light-induced redox transients of photoreaction center P₇₀₀ in chloroplasts within the framework of our mathematical model. This analysis suggests that during the action of a strong actinic light, even significant attenuation of PSII [for instance, in the result of inhibition of a part of PSII complexes by DCMU or due to non-photochemical quenching (NPQ)] will not cause drastic shortage of electron flow through PSI. This can be explained by “electronic” and/or “excitonic” connectivity between different PSII units. At strong AL, the overall flux of electrons between PSII and PSI will maintain at a high level even with the attenuation of PSII activity, provided the rate-limiting step of electron transfer is beyond the stage of PQH₂ formation. Results of our study are briefly discussed in the context of NPQ-dependent mechanism of chloroplast protection against light stress.     

State transitions at the crossroad of thylakoid signalling pathways

In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b ₆ f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (–196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 mol O₂ per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable fluorescence - LHC light-harvesting complex - PpBQ phenyl-p-benzoquinone - PQ plastoquinone pool - P700 reaction center of PS I - PS I, PS II Photosystem I, II - Q_A first bound plastoquinone accepter - RC reaction centre     

The state of the photosynthetic apparatus in leaves as analyzed by rapid gas exchange and optical methods: the pH of the chloroplast stroma and activation of enzymes in vivo

The exchange of CO₂ and O₂ was measured in leaves using specially constructed equipment capable of responding to rapid transients. Optical measurements provided information on cytochrome f and P 700 oxidation in the light. The following results were obtained: i) The solubilization of CO₂ was used to calculate the pH of the chloroplast stroma in darkened leaves. Values ranged from pH 7.8 to pH 8.0 in different C₃ plants. ii) Illumination of predarkened leaves of Helianthus annuus L. resulted in three distinct phases of O₂ evolution that illustrate the complexity of light activation of the photosynthetic apparatus. A first burst of O₂ is attributed to the reduction of electron carriers of the electron-transport chain. While plastoquinone was reduced, cytochrome f was oxidized. Appreciable oxidation of P 700 became possible only during the second O₂ burst, which indicates the reduction of the phosphoglycerate pool. Extensive oxidation required the opening of an electron gate on the reducing side of photosystem I. The subsequent slow rise in O₂ evolution towards a steady state reflects activation of the Calvin cycle and is the result of CO₂ assimilation. iii) Light-dependent CO₂ uptake by predarkened leaves occurred in four phases, three of them based on pH changes in the chloroplast stroma. Initial CO₂ uptake was small and probably caused by protonation of reduced plastoquinone. In the second phase, which coincided with the reduction of the pool of phosphoglycerate, the initial alkalization of the chloroplast stroma was substantially increased. In the third phase, the stroma alkalization decreased, and the fourth phase was dominated by CO₂ assimilation. iv) Respiratory CO₂ production was partially suppressed in the light during the second phase of O₂ evolution while phosphoglycerate was being reduced.Abbreviations DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - PSI(II) photosystem I(II) Dedicated to Professor Stacy French, Dept. of Plant Biology, Carnegie Institution of Washington, on the occasion of his 80th birthday     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

Maximal cyclic electron flow rate is independent of PGRL1 in Chlamydomonas

Cyclic electron flow (CEF) is defined as a return of the reductants from the acceptor side of Photosystem I (PSI) to the pool of its donors via the cytochrome b₆f. It is described to be complementary to the linear electron flow and essential for photosynthesis. However, despite many efforts aimed to characterize CEF, its pathway and its regulation modes remain equivocal, and its physiological significance is still not clear. Here we use novel spectroscopic to measure the rate of CEF at the onset of light in the green alga Chlamydomonas reinhardtii. The initial redox state of the photosynthetic chain or the oxygen concentration do not modify the initial maximal rate of CEF (60 electrons per second per PSI) but rather strongly influence its duration. Neither the maximal rate nor the duration of CEF are different in the pgrl1 mutant compared to the wild type, disqualifying PGRL1 as the ferredoxin-plastoquinone oxidoreductase involved in the CEF mechanism.     

Inhibition of plastoquinol oxidation at the cytochrome b6f complex by dinitrophenyl ether of iodonitrothymol (DNP-INT) depends on irradiance and H+ uptake by thylakoid membranes

Inhibitory analysis is a useful tool for studying reactions in the photosynthetic apparatus. After introducing by Aachim Trebst in 1978, dinitrophenylether of iodonitrothymol (DNP-INT), a competitive inhibitor of plastoquinol oxidation at the cytochrome (cyt.) b₆f complex, has been widely applied to study reactions occurring in the plastoquinone pool and the cyt. b₆f complex. Here we examine the inhibitory efficiency of DNP-INT by implementing three approaches to estimate the extent of blockage of electron flow from the plastoquinone pool to photosystem I in isolated thylakoids from spinach (Spinacia oleracea). We confirm that DNP-INT is a potent inhibitor of electron flow to photosystem I and demonstrate that inhibitory action of DNP-INT depends on irradiance and H⁺ uptake by thylakoid membranes. Based on these findings, we infer that affinity of the quinol-oxidizing site of the cyt. b₆f complex to DNP-INT is increased in the light due to hydrogen bonding between DNP-INT molecules and acidic amino acid residue(s), which is (are) protonated in the light.