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.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

The Effect of Sulfur Re-Addition on H<Subscript>2</Subscript> Photoproduction by Sulfur-Deprived Green Algae

Sulfur deprivation of algal cultures selectively and partially inactivates photosystem II (PSII)-catalyzed O₂ evolution, induces anaerobiosis and hydrogenase expression, and results in sustained H₂ photoproduction for several days. We show that re-addition of limiting amounts of sulfate (1–10 μM final concentration) to the cultures during the H₂-production phase temporarily reactivates PSII photochemical and O₂-evolution activity and re-establishes higher rates of electron transport through the photosynthetic electron transport chain. The reactivation of PSII occurs by de novo D1 protein synthesis, but does not result in the re-establishment of aerobic conditions in the reactor, detectable by dissolved-O₂ sensors. However, concomitant H₂ photoproduction is inhibited, possibly due to excessive intra-cellular levels of photosynthetically-evolved O₂. The partial recovery of electron transport rates correlates with the re-oxidation of the plastoquinone (PQ) pool, as observed by pulse-amplitude modulated (PAM) and fluorescence-induction measurements. These results show that the presence of a more oxidized PQ pool releases some of the down-regulation of electron transport caused by the anaerobic conditions.     

Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga)

Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Effects of extracellular pH on the metabolic pathways in sulfur-deprived,H2-producing Chlamydomonas reinhardtii cultures

Sustained photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, can be obtained by incubating cells in sulfur-deprived medium [Ghirardi et al. (2000b) Trends Biotechnol. 18: 506; Melis et al. (2000) Plant Physiol. 122: 127]. The current work focuses on (a) the effects of different initial extracellular pHs on the inactivation of photosystem II (PSII) and O(2)-sensitive H(2)-production activity in sulfur-deprived algal cells and (b) the relationships among H(2)-production, photosynthetic, aerobic and anaerobic metabolisms under different pH regimens. The maximum rate and yield of H(2) production occur when the pH at the start of the sulfur deprivation period is 7.7 and decrease when the initial pH is lowered to 6.5 or increased to 8.2. The pH profile of hydrogen photoproduction correlates with that of the residual PSII activity (optimum pH 7.3-7.9), but not with the pH profiles of photosynthetic electron transport through photosystem I or of starch and protein degradation. In vitro hydrogenase activity over this pH range is much higher than the actual in situ rates of H(2) production, indicating that hydrogenase activity per se is not limiting. Starch and protein catabolisms generate formate, acetate and ethanol; contribute some reductant for H(2) photoproduction, as indicated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone inhibition results; and are the primary sources of reductant for respiratory processes that remove photosynthetically generated O(2). Carbon balances demonstrate that alternative metabolic pathways predominate at different pHs, and these depend on whether residual photosynthetic activity is present or not.     

Role of carbonyl cyanide m-chlorophenylhydrazone in enhancing photobiological hydrogen production by marine green alga Platymonas subcordiformis

We demonstrated that a significant volume of H(2) gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H(2) gas was produced without CCCP. The role of CCCP in enhancing photobiological H(2) production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water-splitting enzyme system Y) rapidly inhibited the photosystem II (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O(2) in the light. As a result, anaerobiosis during the stage of photobiological H(2) evolution was established, preventing severe O(2) inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of DeltapH across the thylakoid membrane and thus enhances the accessibility of electron and H(+) to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light-harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H(2) evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H(2) evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells.     

Absolute quantification of selected photosynthetic electron transfer proteins in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> in the presence and absence of oxygen

The absolute amount of plastocyanin (PC), ferredoxin-NADP⁺-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382?±?96.5 μmolH₂ min^?1 mg^?1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.     

The photochemical activity of photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells depends on the redox state of the quinone pool during the transition to anaerobiosis

Measurements with a PAM fluorometer showed that the photochemical activity of photosystem II (PS II) in sulfur-deprived Chlamydomonas reinhardtii cells (media TAP-S) decreases slowly under aerobic conditions. In a closed cultivator, when the rate of O2 photosynthetic evolution declines below the rate of respiration, the cell culture is under anaerobic conditions in which the activation of hydrogenase and the production of hydrogen take place. We found that the slow decrease in PS II activity is followed by an abrupt inactivation of PS II centers just after the onset of anaerobiosis. This fast PS II inactivation is reversed by aeration of the media and is accompanied by an increase in the fluorescence parameter Ft. Moreover, the rate of the abrupt PS II inactivation diminished after the addition into the medium of electron acceptors such as CO2 (carbonate-bicarbonate buffer), NO3- and SO4(2-) , the assimilation of which in chloroplasts requires a lot of reductants. We suggest that the PS II inactivation is due to the overreduction of the plastoquinone pool after the onset of anaerobiosis.     

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme.     

Photoproduction of hydrogen by sulfur-deprived <Emphasis Type="Italic">C. reinhardtii</Emphasis> mutants with impaired Photosystem II photochemical activity

Photoproduction of H₂ was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O₂-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H₂-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H₂-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H₂-production, a shorter duration of H₂-production, lower H₂ yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H₂-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.     

Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii

The work describes a novel approach for sustained photobiological production of H(2) gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H(2) production method circumvents the severe O(2) sensitivity of the reversible hydrogenase by temporally separating photosynthetic O(2) evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H(2) production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O(2) evolution. Under these conditions, oxidative respiration by the cells in the light depleted O(2) and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H(2) gas production was observed in the light but not in the dark. The mechanism of H(2) production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H(2) evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H(2) in the chloroplast stroma.     

An alternate photosynthetic electron donor system for PSI supports light dependent nitrogen fixation in a non-heterocystous cyanobacterium,Plectonema boryanum

Plectonema boryanum exhibits temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. During nitrogen fixation, the photosynthetic electron transport chain becomes impaired, which leads to the uncoupling of the PSII and PSI activities. A 30-40% increase in PSI activity and continuous generation of ATP through light-dependent processes seem to support the nitrogen fixation. The use of an artificial electron carrier that shuttles electrons between the plastoquinone pool and plastocyanin, bypassing cytochrome b/f complex, enhanced the photosynthetic electron transport activity five to six fold during nitrogen fixation. Measuring of full photosynthetic electron transport activity using methyl voilogen as a terminal acceptor revealed that the photosynthetic electron transport components beyond plastocyanin might be functional. Further, glycolate can act as a source of electrons for PSI for the nitrogen fixing cells, which have residual PSII activity. Under conditions when PSI becomes largely independent of PSII and glycolate provides electrons for PSI activity, the light-dependent nitrogen fixation also was stimulated by glycolate. These results suggest that during nitrogen fixation, when the photosynthetic electron transport from PSII is inhibited at the level of cytochrome b/f complex, an alternate electron donor system for PSI may be required for the cells to carry out light dependent nitrogen fixation.     

Characterization of the wave phenomenon of flash-induced chlorophyll fluorescence in Chlamydomonas reinhardtii

Flash-induced chlorophyll fluorescence relaxation is a powerful tool to monitor the reoxidation reactions of the reduced primary quinone acceptor, Q_A^? by Q_B and the plastoquinone (PQ) pool, as well as the charge recombination reactions between the donor and acceptor side components of Photosystem II (PSII). Under certain conditions, when the PQ pool is highly reduced (e.g. in microaerobic conditions), a wave phenomenon appears in the fluorescence relaxation kinetics, which reflects the transient reoxidation and re-reduction of Q_A^? by various electron transfer processes, which in cyanobacteria is mediated by NAD(P)H dehydrogenase (NDH-1). The wave phenomenon was also observed and assigned to the operation of type 2 NAD(P)H dehydrogenase (NDH-2) in the green alga Chlamydomonas reinhardtii under hydrogen-producing conditions, which required a long incubation of algae under sulphur deprivation (Krishna et al. J Exp Bot 70 (21):6321–6336, 2019). However, the conditions that induce the wave remained largely uncharacterized so far in microalgae. In this work, we investigated the wave phenomenon in Chlamydomonas reinhardtii under conditions that lead to a decrease of PSII activity by applying hydroxylamine treatment, which impacts the donor side of PSII in combination with a strongly reducing environment of the PQ pool (microaerobic conditions). A similar wave phenomenon could be induced by photoinhibitory conditions (illumination with strong light in the presence of the protein synthesis inhibitor lincomycin). These results indicate that the fluorescence wave phenomenon is activated in green algae when the PSII activity decreases relative to Photosystem I (PS I) activity and the PQ pool is strongly reduced. Therefore, the fluorescence wave could be used as a sensitive indicator of altered intersystem electron transfer processes, e.g. under stress conditions.

     

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.     

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     

Action Spectra of Photosystems I and II in State 1 and State 2 in Intact Sugar Maple Leaves

Photochemical activity, measured as energy storage of photosystems I (PSI) and II (PSII) together and individually, is studied in sugar maple (Acer saccharum Marsh.) leaves in the spectral range between 400 and 700 nm in state 1 and state 2. Total photochemical activity remains the same in both state 1 and state 2 between 580 and 700 nm, but it is lower in state 2 between 400 and 580 nm. Both PSI and PSII activities change significantly during the state transition due to the migration of light-harvesting chlorophyll a/b protein complex of PSII (LHCII). In the action spectra of PSI and PSII, peak positions vary depending on the association or dissociation of LHCII, except for the peak at 470 nm in the PSII spectrum. PSII activity is about 3 times higher than or equal to PSI in state 1 or state 2, respectively, over most of the spectrum except in the blue and far-red regions. At 470 nm, PSII activity is 8 or 1.6 times higher than PSI in state 1 or state 2, respectively. The amplitude of LHCII coupling-induced change is the same in both PSI and PSII between 580 and 700 nm, but it is less in PSI than in PSII between 400 and 580 nm, which explains the lower photochemical activity of the leaf in state 2 than in state 1. This may be due to a decrease in energy transfer efficiency of carotenoids to chlorophylls in LHCII when it is associated with PSI.     

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.