Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F_o) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F_o quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.     

An Acid- and Heat-stable β-Fructofuranosidase from Corticium rolfsii

We reported previously that an ndhB gene disruptant, ΔndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as ΔndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.     

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts

Chlorophyll a fluorescence rise kinetics (from 50 μs to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O₂-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

Interplay between non-photochemical plastoquinone reduction and re-oxidation in pre-illuminated <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: a chlorophyll fluorescence study

In photosynthetic eukaryotes, the redox state of the plastoquinone (PQ) pool is an important sensor for mechanisms that regulate the photosynthetic electron transport. In higher plants, a multimeric nicotinamide adenine dinucleotide (phosphate) (NAD(P))H dehydrogenase (NDH) complex and a plastid terminal oxidase (PTOX) are involved in PQ redox homeostasis in the dark. We recently demonstrated that in the microalgae Chlamydomonas reinhardtii, which lacks the multimeric NDH complex of higher plants, non-photochemical PQ reduction is mediated by a monomeric type-II NDH (Nda2). In this study, we further explore the nature and the importance of non-photochemical PQ reduction and oxidation in relation to redox homeostasis in this alga by recording the ‘dark’ chlorophyll fluorescence transients of pre-illuminated algal samples. From the observation that this fluorescence transient is modified by addition of propyl gallate, a known inhibitor of PTOX, and in a Nda2-deficient strain we conclude that it reflects post-illumination changes in the redox state of PQ resulting from simultaneous PTOX and Nda2 activity. We show that the post-illumination fluorescence transient can be used to monitor changes in the relative rates of the non-photochemical PQ reduction and reoxidation in response to different physiological situations. We study this fluorescence transient in algae acclimated to high light and in a mutant deficient in mitochondrial respiration. Some of our observations indicate that the chlororespiratory pathway participates in redox homeostasis in C. reinhardtii.     

Effects of mutations and of ionophore on chlororespiration in Chlamydomonas reinhardtii

It is shown that electron-transport elements of the photosynthetic chain located between plastoquinone and photosystem I centers are not involved in oxidation of plastoquinone in the chlororespiratory pathway. The ionophore dicyclohexyl-18-crown-6 is shown to induce a reduction of the plastoquinone pool in the dark. This is interpreted as indicating the existence of a coupling site in the chlororespiratory pathway between NAD(P)H and plastoquinone.     

A novel nucleus-encoded chloroplast protein, PIFI, is involved in NAD(P)H dehydrogenase complex-mediated chlororespiratory electron transport in Arabidopsis

A transient rise in chlorophyll fluorescence after turning off actinic light reflects nonphotochemical reduction of the plastoquinone (PQ) pool. This process is dependent on the activity of the chloroplast NAD(P)H dehydrogenase (NDH) complex, which mediates electron flow from stromal reductants to the PQ pool. In this study, we characterized an Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant pifi (for postillumination chlorophyll fluorescence increase), which possesses an intact NDH complex, but lacks the NDH-dependent chlorophyll fluorescence increase after turning off actinic light. The nuclear gene PIFI (At3g15840) containing the T-DNA insertion encodes a chloroplast-targeted protein localized in the stroma and is annotated as a protein of unknown function. The pifi mutant exhibited a lower capacity for nonphotochemical quenching, but similar CO(2) assimilation rates, photosystem II (PSII) quantum efficiencies (PhiPSII), and reduction levels of the primary electron acceptor of PSII (1 - qL) as compared with the wild type. The pifi mutant grows normally under optimal conditions, but exhibits greater sensitivity to photoinhibition and long-term mild heat stress than wild-type plants, which is consistent with lower capacity of nonphotochemical quenching. We conclude that PIFI is a novel component essential for NDH-mediated nonphotochemical reduction of the PQ pool in chlororespiratory electron transport.     

Effects of anaerobiosis as probed by the polyphasic chlorophyll a fluorescence rise kinetic in pea (Pisum sativum L.)

We analysed the changes of the chlorophyll (Chl)a fluorescence rise kinetic (from 50 s to 1 s) that occur when leaves or chloroplasts of pea ( Pisum sativum L.) are incubated under anaerobic conditions in the dark. In control leaves, Chl a fluorescence followed a typical O-J-I-P polyphasic rise [Strasser et al. (1995) Photochem Photobiol 61: 32–42]. Anaerobiosis modified the shape of the transient with the main effect being a time-dependent increase in the fluorescence yield at the J-step (2 ms). Upon prolongation of the anaerobic treatment (> 60 min), the O-J-I-P fluorescence rise was eventually transformed to an O-J (J = P) rise. A similar transformation was observed when pea leaves were treated with DCMU or sodium dithionite. Anaerobiosis resulted in a 10–20% reduction in the maximum quantum yield of the primary photochemistry of Photosystem II, as measured by the ratio of the maximal values of variable and total fluorescence (F_V/F_M). When the leaves were returned to the air in the dark, the shape of the fluorescence transient showed a time-dependent recovery from the anaerobiosis-induced change. The original O-J-I-P shape could also be restored by illuminating the anaerobically treated samples with far-red light but not with blue or white light. Osmotically broken chloroplasts displayed under anaerobic conditions fluorescence transients similar to those observed in anaerobically treated leaves, but only when they were incubated in a medium comprising reduced pyridine nucleotides (NADPH or NADH). As in intact leaves, illumination of the anaerobically treated chloroplasts by far-red light restored the original O-J-I-P transient, although only in the presence of methyl viologen. The results provide additional evidence for the existence of a chlororespiratory pathway in higher plant cells. Furthermore, they suggest that the J-level of the fluorescence transient is strongly determined by the redox state of the electron carriers at the PS II acceptor side.     

An Investigation of the Sustained Component of Nonphotochemical Quenching of Chlorophyll Fluorescence in Isolated Chloroplasts and Leaves of Spinach

The slowly reversible component of nonphotochemical quenching of Chl fluorescence, ql, has been investigated in intact leaves and chloroplasts of spinach (Spinacia oleracea). In leaves, between 50 and 100% of ql (defined as the quenching that remained after at least 10 min of dark adaptation of a previously illuminated leaf) is instantly reversible when leaves were infiltrated with nigericin. Chloroplasts isolated from leaves in which ql had been induced by prior illumination retained the same level of quenching. No pH gradient, as measured by quenching of 9-aminoacridine fluorescence, was present. However, addition of nigericin caused a partial removal of ql, as observed in whole leaves. It is concluded that ql is not related to a persistence of a bulk phase pH gradient in darkness but to a structural change in the thylakoid that can be reversed by addition of nigericin. The relationship between these observations and the hypothesis that nonphotochemical quenching of chlorophyll fluorescence results from protonation of light-harvesting complex of photosystem II components is discussed.     

The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.     

Luminescence characteristics of strawberry leaves at early stages of injury by spider mite

In this work, we have investigated how the luminescence characteristics and the total content of antioxidants in leaves of strawberry (Fragaria × ananassa Duch.) change after infestation of plants by spider mites (Tetranychus atlanticus McGregor). At early stages of plant injury, the chlorophyll concentration remained unchanged. However, the total amount of antioxidants decreased and the intensity of high-temperature thermoluminescence band (60–100°C) increased, indicating degradation of membrane lipids caused by oxidative stress. In infested leaves, the light-induced rise of nonphotochemical quenching of chlorophyll fluorescence was slower, while the relaxation of nonphotochemical quenching in the dark was faster than in control leaf samples. These data may indicate that the thylakoid membranes in infested leaves were more permeable to ions than in control leaves.     

Photosynthetic characterization of flavodoxin-expressing tobacco plants reveals a high light acclimation-like phenotype

Flavodoxins are electron carrier flavoproteins present in bacteria and photosynthetic microorganisms which duplicate the functional properties of iron-sulphur containing ferredoxins and replace them under adverse environmental situations that lead to ferredoxin decline. When expressed in plant chloroplasts, flavodoxin complemented ferredoxin deficiency and improved tolerance to multiple sources of biotic, abiotic and xenobiotic stress. Analysis of flavodoxin-expressing plants grown under normal conditions, in which the two carriers are present, revealed phenotypic effects unrelated to ferredoxin replacement. Flavodoxin thus provided a tool to alter the chloroplast redox poise in a customized way and to investigate its consequences on plant physiology and development. We describe herein the effects exerted by the flavoprotein on the function of the photosynthetic machinery. Pigment analysis revealed significant increases in chlorophyll a, carotenoids and chlorophyll a/b ratio in flavodoxin-expressing tobacco lines. Results suggest smaller antenna size in these plants, supported by lower relative contents of light-harvesting complex proteins. Chlorophyll a fluorescence and P700 spectroscopy measurements indicated that transgenic plants displayed higher quantum yields for both photosystems, a more oxidized plastoquinone pool under steady-state conditions and faster plastoquinone dark oxidation after a pulse of saturating light. Many of these effects resemble the phenotypes exhibited by leaves adapted to high irradiation, a most common environmental hardship faced by plants growing in the field. The results suggest that flavodoxin-expressing plants would be better prepared to cope with this adverse situation, and concur with earlier observations reporting that hundreds of stress-responsive genes were induced in the absence of stress in these lines.     

N,N,N′,N′-tetramethyl-p-phenylenediamine initiates the appearance of a well-resolved I peak in the kinetics of chlorophyll fluorescence rise in isolated thylakoids

Addition of N,N,N′,N′-tetramethyl-p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J–I–P phase of fluorescence rise and greatly retarded the I–P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence (F_v) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.     

Effect of Elevated Temperatures on the Activity of Alternative Pathways of Photosynthetic Electron Transport in Intact Barley and Maize Leaves

The light-induced rise in chlorophyll fluorescence and the subsequent decay of fluorescence in darkness were measured in barley and maize leaves exposed to heat treatment. The redox conversions of the photosystem I primary donor P700, induced by far-red light, were also monitored from the absorbance changes at 830 nm. After heating of leaves at temperatures above 40°C, the ratio of variable and maximum fluorescence decreased for leaves of both plant species, indicating the inhibition of photosystem II (PSII) activity. A twofold reduction of this ratio in barley and maize leaves was observed after heating at 45.3 and 48.1°C, respectively, which suggests the higher functional resistance of PSII in maize. The amplitude of the slow phase in the dark relaxation of variable fluorescence did not change after the treatment of barley and maize leaves at temperatures up to 48°C. In leaves treated at 42 and 46°C, the slow phase of dark relaxation deviated from an exponential curve. The relaxation kinetics included a temporary increase in fluorescence to a peak about 1 s after turning off the actinic light. Unlike the slow component, the fast and intermediate phases in the dark relaxation of variable fluorescence disappeared fully or partly after the treatment of leaves at 46°C. The photooxidation of P700 in heat-treated leaves was saturated at much higher irradiances of far-red light than in untreated leaves. At the same time, the dark reduction of P700⁺ was substantially accelerated after heat treatment. The data provide evidence that the heating of leaves stimulated the alternative pathways of electron transport, i.e., cyclic transport around photosystem I and/or the donation of electrons to the plastoquinone pool from the reduced compounds located in the chloroplast stroma. The rate of alternative electron transport after the heat treatment was higher in maize leaves than in barley leaves. It is supposed that the stimulation of alternative electron transport, associated with proton pumping into the thylakoid, represents a protective mechanism that prevents the photoinhibition of PSII in leaves upon a strong suppression of linear electron transport in chloroplasts exposed to heat treatment.     

Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the Photosystem II acceptor side

Iron deficiency was found to affect the redox state of the Photosystem II acceptor side in dark-adapted, attached leaves of sugar beet (Beta vulgaris L.). Dark-adapted iron-deficient leaves exhibited relatively high F_o and F_pl levels in the Kautsky chlorophyll fluorescence induction curve when compared to the iron-sufficient controls. However, far-red illumination led to marked decreases in the apparent F_o and F_pl levels. Modulated fluorescence showed that far-red light decreased the fluorescence yield to the true F_o levels by increasing photochemical quenching, without inducing changes in the level of non-photochemical quenching. In dark-adapted, iron-deficient leaves, far-red illumination induced a faster fluorescence decay in the µs-ms time domain, indicating an improvement in the electron transport after the primary quinone acceptor in the reducing side of Photosystem II. All these data indicate that in iron-deficient leaves the plastoquinone pool was reduced in the dark. The extent of the plastoquinone reduction in sugar beet depended on the chlorophyll concentration of the leaf, on the time of preillumination and on the duration of dark adaptation. The dark reduction of plastoquinone was observed not only in sugar beet but also in other plant species affected by iron deficiency both in controlled conditions and in the field.     

Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures

When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de-epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O₂, 0% CO₂ at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5°C leaf temperature) in air and then held under control conditions in low light in air at 25°C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5°C did not recover at all. During a dark/light transient in 2% O₂, 0% CO₂ in low light at 5°C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content.     

Activation of Diadinoxanthin De-Epoxidase Due to a Chiororespiratory Proton Gradient in the Dark in the Diatom Phaeodactylum tricornutum

Abstract: An exponential dynamic light regime with prolonged dark periods (light/dark cycle 8/40 h) was used to simulate deep mixing of algae in natural waters and to investigate the adaptation of the diatom Phaeodactylum tricornutum to these extreme light conditions. After prolonged dark periods Phaeo dactylum cells showed surprisingly high contents of diatoxan-thin, low photosynthetic efficiency and high non-photochemical quenching (NPQ) of chlorophyll fluorescence. Diatoxanthin con centrations and NPQ were low at the beginning of the dark peri od and increased with the duration of the dark incubation. Addi tion of the diadinoxanthin de-epoxidase inhibitor, DTT, prevent ed the formation of diatoxanthin, thereby excluding de novo synthesis of diatoxanthin during the prolonged dark period. Evi dence of chlororespiratory electron flow and the establishment of a diadinoxanthin de-epoxidase activating proton gradient in the dark was derived from two observations. First, uncoupling of electron transport with NH₄CI at the beginning of the dark period prevented the development of non-photochemical quenching of chlorophyll fluorescence and the formation of diatoxanthin during the dark period. Second, inhibition of the electron and proton consuming terminal redox component of chlororespiratory electron transport, cytochrome oxidase, by addition of KCN induced stronger NPQ and a higher de-epoxidation state of the xanthophyll cycle. These results strongly indi cate that the activation of diadinoxanthin de-epoxidase in the dark is the consequence of a chlororespiratory proton gradient. We furthermore present evidence that diatoxanthin formed by the chlororespiratory proton gradient has the same efficiency in the mechanism of enhanced heat dissipation as diatoxanthin induced by a light-driven ApH.     

Post-illumination reduction of the plastoquinone pool in chloroplast transformants in which chloroplastic NAD(P)H dehydrogenase was inactivated

We reported previously that an ndhB gene disruptant, delta ndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as delta ndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.     

Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes.

The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.     

Stromal over-reduction by high-light stress as measured by decreases in P700 oxidation by far-red light and its physiological relevance

The oxidation level of P700 induced by far-red light (DeltaA(FR)) in briefly dark-treated leaves of some sun plants decreased during the daytime and recovered at night. The dark recovery of decreased DeltaA(FR) proceeded slowly, with a half-time of about 5 h. We propose that stromal over-reduction induced by sunlight was the direct cause of the depression of DeltaA(FR). The depression of DeltaA(FR) found during the daytime was reproduced by controlled illumination with saturating light of fully dark-treated leaves. Simultaneous measurement of P700 redox and chlorophyll fluorescence showed that the depression of DeltaA(FR) was associated with dark reduction of the plastoquinone pool, which represented cyclic electron transport activity. The decrease of DeltaA(FR) in the light-stressed chloroplasts was partly reversed by treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of electron transport at the cytochrome b6/f complex, and the subsequent addition of methyl viologen, an efficient electron acceptor from photosystem I (PSI), stimulated further recovery, showing that both cyclic electron flow around PSI and the charge recombination within PSI were responsible for the light-induced depression of DeltaA(FR). The dark level of blue-green fluorescence, an indicator of NAD(P)H concentration, from intact chloroplasts was increased by high-light stress, suggesting that NADPH accumulated in stroma as a result of the high-light treatment. Possible effects on photosynthetic activity of over-reduction and its physiological relevance are discussed.