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.     

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts

Intact chloroplasts from spinach showed a transient increasein Chl fluorescence after saturating illumination with actiniclight and its yield depended on the duration of illuminationand the intensity of the actinic light (AL). The increase waspartially suppressed when antimycin A was added immediatelyafter termination of the AL. The inhibited fluorescence increase,therefore, reflected the electron flow from the reductant(s)that had accumulated during the actinic illumination to theplastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitiveCyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743].Addition of dihydroxyacetone phosphate (DHAP) to chloroplastscaused the enhancement of the increase in fluorescence afterAL, which was inhibited by antimycin A. Decay of the transientlyraised fluorescence was retarded by 2-heptyl-4-hydroxyquinolineN-oxide and stigmatellin, suggesting that re-oxidation of thereduced PQ pool is coupled with the operation of Q-cycle. Althoughthe activity of the stromal enzyme system that supplies NADPHon addition of DHAP was constant irrespective of light or darkness,the capacity of the intact chloroplasts to show a DHAP-dependentfluorescence increase had a limited lifetime after AL was turnedoff. This result suggests that the antimycin A-sensitive Cytb-559 or ferredoxin-NADP reductase is activated by light anddeactivated in the dark. In ruptured chloroplasts, the additionof NADPH increased the dark fluorescence yield only in the presenceof Fd, which also was inhibited by antimycin A. Thus the photoregulatorymechanism of Cyt b-559 (Fd) in intact chloroplasts appearedto be lost when chloroplasts were ruptured. (Received June 21, 1995; Accepted September 25, 1995)     

Quantitation of plastoquinone photoreduction in spinach chloroplasts

The question of plastoquinone (PQ) concentration and its stoichiometry to photosystem I (PSI) and PSII in spinach chloroplasts is addressed here. The results from three different experimental approaches were compared. (a) Quantitation from the light-induced absorbance change at 263 nm (A₂₆₃) yielded the following ratios (mol:mol); Chl:PQ=70:1, PQ:PSI=9:1 and PQ:PSII=7:1. The kinetics of PQ photoreduction were a monophasic but non-exponential function of time. The deviation of the semilogarithmic plots from linearity reflects the cooperativity of several electron transport chains at the PQ pool level. (b) Estimates from the area over the fluorescence induction curve (A_fl) tend to exaggerate the PQ pool size because of electron transfer via PSI to molecular oxygen (Mehler reaction) resulting in the apparent increase of the pool of electron acceptors. The reliability of the A_fl method is increased substantially upon plastocyanin inhibition by KCN. (c) Quantitation of the number of electrons removed from PQH₂ by PSI, either under far-red excitation or after the addition of DCMU to preilluminated chloroplasts, is complicated due to the competitive loss of electrons from PQH₂ to molecular oxygen. The latter is biphasic reaction occurring with half-times of about 2 s (30–40% of PQH₂) and of about 60 s (60–70% of PQH₂).Abbreviations A_fl area over the fluorescence induction curve - Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PQ plastoquinone - PS photosystem - P700 reaction center of PSI - Q primary quinone acceptor of PSII - Tricine N-tris (hydroxymethyl) methyl glycine - Triton X-100 octyl phenoxy polyethoxyethanol     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Anaerobiosis Induced State Transition: A Non Photochemical Reduction of PQ Pool Mediated by NDH in Arabidopsis thaliana

Background

Non photochemical reduction of PQ pool and mobilization of LHCII between PSII and PSI are found to be linked under abiotic stress conditions. The interaction of non photochemical reduction of PQ pool and state transitions associated physiological changes are critically important under anaerobic condition in higher plants.

Methodology/Findings

The present study focused on the effect of anaerobiosis on non-photochemical reduction of PQ pool which trigger state II transition in Arabidopsis thaliana. Upon exposure to dark-anaerobic condition the shape of the OJIP transient rise is completely altered where as in aerobic treated leaves the rise is unaltered. Rise in F _o and F _J was due to the loss of oxidized PQ pool as the PQ pool becomes more reduced. The increase in F_o′ was due to the non photochemical reduction of PQ pool which activated STN7 kinase and induced LHCII phosphorylation under anaerobic condition. Further, it was observed that the phosphorylated LHCII is migrated and associated with PSI supercomplex increasing its absorption cross-section. Furthermore, evidences from crr2-2 (NDH mutant) and pgr5 mutants (deficient in non NDH pathway of cyclic electron transport) have indicated that NDH is responsible for non photochemical reduction of the PQ pool. We propose that dark anaerobic condition accelerates production of reducing equivalents (such as NADPH by various metabolic pathways) which reduce PQ pool and is mediated by NDH leading to state II transition.

Conclusions/Significance

Anaerobic condition triggers non photochemical reduction of PQ pool mediated by NDH complex. The reduced PQ pool activates STN7 kinase leading to state II transition in A. thaliana.     

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - DLGA DL-glyceraldehyde - FNR ferredoxin-NADP reductase - FWHM full width at half maximum - LED light emitting diodes - OAA oxaloacetate - q_N non-photochemical quenching - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - q_P photochemical quenching - PPFD photosynthetic photon flux density - Q_A primary quinone acceptor of Photosystem II Preliminary results of this work were presented at the First Conference on the Physiology and Biochemistry of high Mountain Plants, 2–3 July 1992, Villar d'Arene, France.     

Dark Ammonium Assimilation Reduces the Plastoquinone Pool of Photosystem II in the Green Alga Selenastrum minutum

The impact of dark NH₄⁺ and NO₃⁻ assimilation on photosynthetic light harvesting capability of the green alga Selenastrum minutum was monitored by chlorophyll a fluorescence analysis. When cells assimilated NH₄⁺, they exhibited a large decline in the variable fluorescence/maximum fluorescence ratio, the fluorescence yield of photosystem II relative to that of photosystem I at 77 kelvin, and O₂ evolution rate. NH₄⁺ assimilation therefore poised the cells in a less efficient state for photosystem II. The analysis of complementary area of fluorescence induction curve and the pattern of fluorescence decay upon microsecond saturating flash, indicators of redox state of plastoquinone (PQ) pool and dark reoxidation of primary quinone electron acceptor (Q_A), respectively, revealed that the PQ pool became reduced during dark NH₄⁺ assimilation. NH₄⁺ assimilation also caused an increase in the NADPH/NADP⁺ ratio due to the NH₄⁺ induced increase in respiratory carbon oxidation. The change in cellular reductant is suggested to be responsible for the reduction of the PQ pool and provide a mechanism by which the metabolic demands of NH₄⁺ assimilation may alter the efficiency of photosynthetic light harvesting. NO₃⁻ assimilation did not cause a reduction in PQ and did not affect the efficiency of light harvesting. These results illustrate the role of cellular metabolism in the modulating photosynthetic processes.     

Contributions of the free oxidized and QB-bound plastoquinone molecules to the thermal phase of chlorophyll-a fluorescence

Variable chlorophyll a (Chl a) fluorescence is composed of a photochemical and a thermal phases of similar amplitudes. The photochemical phase can be induced by a saturating single turnover flash (STF) and reflects the reduction of the Photosystem II (PS II) Q_A primary electron acceptor. The thermal phase requires multiple turnover flash (MTF) and is somehow related to the reduction of the plastoquinone (PQ) molecules. This article aimed to determine the relative contributions of the Q_B-bound and the free oxidized PQ molecules to the thermal phase of Chl a fluorescence. We thus measured the interactive effects of exogenous PQ (PQex), of an inhibitor (DCMU) acting at the Q_B site of PS II and of an artificial quencher, 2-methyl-1,4-naphtoquinone, on Chl a fluorescence levels induced by STF (F_F) and MTF (F_M) in spinach thylakoids. We observed that: (1) the incorporation of PQex in thylakoids stimulated photosynthetic electron transport but barely affected F_F and F_M in the absence of DCMU; (2) DCMU significantly increased the amplitude of F_F but slightly quenched F_M; (3) 2-methyl-1,4-naphtoquinone quenched F_M to a larger-extent than F_F; (4) DCMU increased the quenching effects of PQex on F_F and F_M and also, of methyl-1,4-naphtoquinone on F_F. These results indicate that: (1) the Q_B-bound and the free PQ molecules contribute to about 56% and 25%, respectively, to the thermal phase Chl a fluorescence in dark-adapted thylakoids; and (2) the thermal phase of Chl a fluorescence is more susceptible than the photochemical phase to the non-photochemical quenching effect of oxidized quinones. This revised version was published online in June 2006 with corrections to the Cover Date.     

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m²·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll <Emphasis Type="Italic">b</Emphasis>-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 μmol m⁻² s⁻¹). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (F_v/F_m), quantum efficiency of PS2 electron transport (Φ_PS2), photochemical quenching (q_P), as well as rate of photochemistry (P_rate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (D_rate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–q_P (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 μmol m⁻² s⁻¹) and high, HI (700 μmol m⁻² s⁻¹) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.     

Analysis of Qualitative Contribution of Assimilatory and Non-Assimilatory De-Excitation Processes to Adaptation of Photosynthetic Apparatus of Barley Plants to High Irradiance

The adaptation of barley (Hordeum vulgare L. cv. Akcent) plants to low (LI, 50 µmol m^–2 s^–1) and high (HI, 1000 µmol m^–2 s^–1) growth irradiances was studied using the simultaneous measurements of the photosynthetic oxygen evolution and chlorophyll a (Chl a) fluorescence at room temperature. If measured under ambient CO₂ concentration, neither increase of the oxygen evolution rate (P) nor enhancement of non-radiative dissipation of the absorbed excitation energy within photosystem 2 (PS2) (determined as non-photochemical quenching of Chl a fluorescence, NPQ) were observed for HI plants compared with LI plants. Nevertheless, the HI plants exhibited a significantly higher proportion of Q_A in oxidised state (estimated from photochemical quenching of Chl a fluorescence, q_P), by 49–102 % at irradiances above 200 µmol m^–2 s^–1 and an about 1.5 fold increase of irradiance-saturated PS2 electron transport rate (ETR) as compared to LI plants. At high CO₂ concentration the degree of P stimulation was approximately three times higher for HI than for LI plants, and the irradiance-saturated P values at irradiances of 2 440 and 2 900 µmol m^–2 s^–1 were by 130 and 150 % higher for HI plants than for LI plants. We suggest that non-assimilatory electron transport dominates in the adaptation of the photosynthetic apparatus of barley grown at high irradiances under ambient CO₂ rather than an increased NPQ or an enhancement of irradiance-saturated photosynthesis.     

Characterization of factors affecting the activity of photosystem I cyclic electron transport in chloroplasts

PSI cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the antimycin A-sensitive pathway is the main route of electrons in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, its function is still unclear, and there are numerous debates on the rate of electron transport in vivo and its regulation. To assess how PGR5-dependent PSI cyclic electron transport is regulated in vivo, we characterized its activity in ruptured chloroplasts isolated from Arabidopsis thaliana. The activity of ferredoxin (Fd)-dependent plastoquinone (PQ) reduction in the dark is impaired in the pgr5 mutant. Alkalinization of the reaction medium enhanced the activity of Fd-dependent PQ reduction in the wild type. Even weak actinic light (AL) illumination also markedly activated PGR5-dependent PSI cyclic electron transport in ruptured chloroplasts. Even in the presence of linear electron transport [11 mumol O2 (mg Chl)(-1) h(-1)], PGR5-dependent PSI electron transport was detected as a difference in Chl fluorescence levels in ruptured chloroplasts. In the wild type, PGR5-dependent PSI cyclic electron transport competed with NADP+ photoreduction. These results suggest that the rate of PGR5-dependent PSI cyclic electron transport is high enough to balance the production ratio of ATP and NADPH during steady-state photosynthesis, consistently with the pgr5 mutant phenotype. Our results also suggest that the activity of PGR5-dependent PSI cyclic electron transport is regulated by the redox state of the NADPH pool.     

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.     

High Irradiance Induced Pigment Degradation and Loss of Photochemical Activity of Wheat Chloroplasts

Loss of chlorophyll (Chl) and carotenoids (Car) of leaves and changes in Chl fluorescence emission and polarisation, malondialdehyde (MDA) accumulation, and 2,6-dichlorophenol indophenol (DCPIP) photoreduction in chloroplasts of wheat seedlings grown under different irradiance and subsequently exposed to high irradiance stress (HIS; 250 W m^–2) were studied in mature and senescent primary wheat leaves. Faster rate of pigment loss was observed in leaves of moderate irradiance (MI; 15 W m^–2) grown plants, compared to high irradiance (HI-1 and HI-2; 30 and 45 W m^–2) ones when exposed to HIS. A relatively lower loss of Car in the plants grown in HI-1 and HI-2 exposed to HIS suggests HI adaptation of these seedlings. The slower rate of increase in the ratio of Chl fluorescence emission (F₆₈₅/F₇₃₅) also may suggest photoprotective strategy of HI grown seedlings. There was a positive correlation between MDA accumulation and Chl fluorescence polarisation. The DCPIP photoreduction activity in chloroplasts isolated from HI-1 and HI-2 grown plants exposed to HIS showed slower loss of electron transport activity compared to MI grown plants. These observations suggest that plants grown under higher irradiance have capacity to manage the excess quanta better than those grown under lower irradiance.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

Redox of Plastoquinone Pool Regulates the Expression and Activity of NADPH Dehydrogenase Supercomplex in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. Strain PCC 6803

A highly active NADPH dehydrogenase supercomplex, which is essential for cyclic electron transport around photosystem I (cyclic PSI) and respiration, was newly identified in cyanobacteria. Synechocystis sp. strain PCC 6803 cells were treated with exogenous glucose (Glc) or 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); subsequently, active staining of NADPH-nitroblue tetrazolium oxidoreductase, western blot, and the initial rate of P700⁺ dark reduction were assessed in the cyanobacterium at several time points. The expression and enzyme activity levels of NADPH dehydrogenase supercomplex were gradually inhibited and closely associated with the decrease in the rate of cyclic PSI accompanying the addition of exogenous Glc to the cultures. In contrast, the activity levels were significantly stimulated but did not cause an increase in the rate of cyclic PSI as expected in the presence of DCMU. Since Glc results in the partial reduction of the plastoquinone (PQ) pool while DCMU results in the overoxidation of the PQ pool, the present results demonstrate that the expression and activity of NADPH dehydrogenase supercomplex are under the influence of the redox control of the PQ pool while the operation of cyclic PSI as mediated by this supercomplex requires an appropriate redox poise of the PQ pool.     

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b₆f complex is active in plastoquinone reduction: implications for cyclic electron transport

In this study, we have compared three isolation methods of cytochrome b₆f complex, obtained from spinach (Spinacia oleracea), differing in the preservation of the cytochrome b₆f‐associated ferredoxin:NADP⁺ oxidoreductase (FNR). Although the complexes isolated by all the methods showed the presence of the FNR peptide(s), when incorporated into liposome membranes, the NADPH‐PQ (plastoquinone) oxidoreductase activity was not detected for the cytochrome b₆f complex isolated with the original method including a NaBr wash. Some activity was found for the complex isolated with the omission of the wash, but the highest activity was detected for the complex isolated with the use of digitonin. The reaction rate of PQ reduction of the investigated complexes in liposomes was not significantly influenced by the addition of free FNR or ferredoxin. The reaction was inhibited by about 60% in the presence of 2 µM 2‐n‐nonyl‐4‐hydroxyquinoline N‐oxide, an inhibitor of the cytochrome b₆f complex at the Q_i site, while it was not affected by triphenyltin or isobutyl cyanide that interacts with the recently identified heme c_i. The obtained data indicate that FNR associated with the cytochrome b₆f complex can participate in the cyclic electron transport as PSI‐PQ or NADPH‐PQ oxidoreductase. Moreover, we have shown that PQ can be non‐enzymatically reduced by ascorbate in liposomes and this reaction might be involved in non‐photochemical reduction pathways of the PQ‐pool in chloroplasts.     

Effects of Nanoanatase TiO2 on Photosynthesis of Spinach Chloroplasts Under Different Light Illumination

With a photocatalyzed characteristic, nanoanatase TiO₂ under light could cause an oxidation–reduction reaction. Our studies had proved that nano-TiO₂ could promote photosynthesis and greatly improve spinach growth. However, the mechanism of nano-TiO₂ on promoting conversion from light energy to electron energy and from electron energy to active chemistry energy remains largely unclear. In this study, we report that the electron transfer, oxygen evolution, and photophosphorylation of chloroplast (Chl) from nanoanatase-TiO₂-treated spinach were greatly increased under visible light and ultraviolet light illumination. It was demonstrated that nanoanatase TiO₂ could greatly improve whole chain electron transport, photoreduction activity of photosystem II, O₂-evolving and photophosphorylation activity of spinach Chl not only under visible light, but also energy-enriched electron from nanoanatase TiO₂, which entered Chl under ultraviolet light and was transferred in photosynthetic electron transport chain and made NADP⁺ be reduced into NADPH, and coupled to photophosphorylation and made electron energy be transformed to ATP. Moreover, nanoanatase h⁺, which photogenerated electron holes, captured an electron from water, which accelerated water photolysis and O₂ evolution.     

Regulation of energy dissipation in photosystem I by the redox state of the plastoquinone pool

The effect of exogenous plastoquinone (PQ) on the different deexcitation pathways of photosystem I (PSI) was investigated. Addition of oxidized decyl-plastoquinone (dPQ) and PQ-2 strongly quenched the chlorophyll (Chl) emission spectra of PSI submembrane fractions over all wavelengths. This quenching increased with the concentration of exogenous PQ added and followed the modified Stern-Volmer law. The Stern-Volmer constants found for dPQ and PQ-2 were 1.25 x 10(6) M-1 and 0.55 x 10(6) M-1, respectively, and the fraction of fluorescence accessible to the quencher was 0.7 for both exogenous PQ. dPQ and PQ-2 also retarded the P700 photooxidation measured under limiting actinic light irradiances. Photoacoustic measurements showed that addition of dPQ increased the heat dissipation and decreased the photochemical capacity of PSI. From these results, exogenous oxidized PQ were shown to efficiently quench the Chl excited state in the PSI antenna and change the balance between Chl deexcitation pathways. Moreover, reduction of the endogenous PQ pool in whole thylakoid membranes by NADPH increased PSI fluorescence by 65%, indicating the importance of the redox state of the PQ pool on PSI energy dissipation.     

The interrelationship between the lower oxygen limit,chlorophyll fluorescence and the xanthophyll cycle in plants

The lower oxygen limit (LOL) in plants may be identified through the measure of respiratory gases [i.e. the anaerobic compensation point (ACP) or the respiratory quotient breakpoint (RQB)], but recent work shows it may also be identified by a sudden rise in dark minimum fluorescence (F _o). The interrelationship between aerobic respiration and fermentative metabolism, which occur in the mitochondria and cytosol, respectively, and fluorescence, which emanates from the chloroplasts, is not well documented in the literature. Using spinach (Spinacia oleracea), this study showed that F_o and photochemical quenching (q _P) remained relatively unchanged until O₂ levels dropped below the LOL. An over-reduction of the plastoquinone (PQ) pool is believed to increase F _o under dark + anoxic conditions. It is proposed that excess cytosolic reductant due to inhibition of the mitochondria’s cytochrome oxidase under low-O₂, may be the primary reductant source. The maximum fluorescence (F _m) is largely unaffected by low-O₂ in the dark, but was severely quenched, mirroring changes to the xanthophyll de-epoxidation state (DEPS), under even low-intensity light (≈4 μmol m⁻² s⁻¹). In low light, the low-O₂-induced increase in F _o was also quenched, likely by non-photochemical and photochemical means. The degree of quenching in the light was negatively correlated with the level of ethanol fermentation in the dark. A discussion detailing the possible roles of cyclic electron flow, the xanthophyll cycle, chlororespiration and a pathway we termed ‘chlorofermentation’ were used to interpret fluorescence phenomena of both spinach and apple (Malus domestica) over a range of atmospheric conditions under both dark and low-light.