Reduction Of P700 Research Articles

FesM, a membrane iron-sulfur protein, is required for cyclic electron flow around photosystem I and photoheterotrophic growth of the cyanobacterium Synechococcus sp. PCC 7002.

While it is known that cyclic electron flow around photosystem I is an important pathway of photosynthetic electron transfer for converting light energy to chemical energy, some components of cyclic electron flow remain to be revealed. Here, we show that fesM, encoding a novel membrane iron-sulfur protein, is essential to cyclic electron flow in the cyanobacterium Synechococcus sp. PCC 7002. The FesM protein is predicted to have a cAMP-binding domain, an NtrC-like domain, a redox sensor motif, and an iron-sulfur (4Fe-4S) motif. Deletion of fesM (fesM-D) led to an inability for Synechococcus cells to grow in the presences of glycerol and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Photoheterotrophic growth was restored by a complete fesM gene present on a replicable plasmid. A mutant fesM gene encoding a truncated FesM protein lacking the cAMP domain failed to restore the phenotype, suggesting this domain is important to the function of FesM. Measurements of reduction of P700(+) and the redox state of interphotosystem electron carriers showed that cells had a slower rate of respiratory electron donation to the interphotosystem electron transport chain, and cyclic electron flow around photosystem I in fesM-D was impaired, suggesting that FesM is a critical protein for respiratory and cyclic electron flow. Phosphatase fusion analysis showed that FesM contains nine membrane-spanning helices, and all functional domains of FesM are located on the cytoplasmic face of the thylakoid membranes.

Evidence for the operation of alternative electron transport routes through photosystem I in intact barley leaves under weak and moderate white light

The functioning of alternative routes of photosynthetic electron transport was analyzed from the kinetics of dark reduction of P700+ , an oxidized primary donor of PSI, in barley (Hordeum vulgare L.) leaves irradiated by white light of various intensities. Redox changes of P700 were monitored as absorbance changes at 830 nm using PAM 101 specialized device. Irradiation of dark-adapted leaves caused a gradual P700+ accumulation, and the steady-state level of oxidized P700 increased with intensity of actinic light. The kinetics of P700+ dark reduction after a pulse of strong actinic light, assayed from the absorbance changes at 830 nm, was fitted by a single exponential term with a halftime of 10–12 ms. Two slower components were observed in the kinetics of P700+ dark reduction after leaf irradiation by attenuated actinic light. The contribution of slow components to P700+ reduction increased with the decrease in actinic light intensity. Two slow components characterized by halftimes similar to those observed after leaf irradiation by weak white light were found in the kinetics of dark reduction of P700+ oxidized in leaves with far-red light specifically absorbed by PSI. The treatment of leaves with methyl viologen, an artificial PSI electron acceptor, significantly accelerated the accumulation of P700+ under light. At the same time, the presence of methyl viologen, which inhibits ferredoxin-dependent electron transport around PSI, did not affect three components of the kinetics of P700+ dark reduction obtained after irradiations with various actinic light intensities. It was concluded that some part of PSI reaction centers was not reduced by electron transfer from PSII under weak or moderate intensities of actinic light. In this population of PSI centers, P700+ was reduced via alternative electron transport routes. Insensitivity of the kinetics of P700+ dark reduction to methyl viologen evidences that the input of electrons to PSI from the reductants (NADPH or NADH) localized in the chloroplast stroma was effective under those light conditions.

Reduction of the thylakoid electron transport chain by stromal reductants--evidence for activation of cyclic electron transport upon dark adaptation or under drought.

The reduction of P700(+), the primary electron donor of photosystem I (PSI), following a saturating flash of white light in the presence of the photosystem II (PSII) inhibitor 3-(3.4-dichlorophenyl)-1,1-dimethylurea (DCMU), was examined in barley plants exposed to a variety of conditions. The decay kinetic fitted to a double exponential decay curve, implying the presence of two distinct pools of PSI. A fast component, with a rate constant for decay of around 0.03-0.04 ms(-1) was observed to be sensitive to the duration of illumination. This rate constant was slower than, but comparable to, that observed in non-inhibited samples (i.e. where linear flow was active). It was substantially faster than values typically reported for experiments where PSII activity is inhibited. The magnitude of this component rose in leaves that were dark-adapted or exposed to drought. This component was assigned to PSI centres involved in cyclic electron transport. The remaining slowly decaying P700(+) population (rate constant of around 0.001-0.002 ms(-1)) was assigned to centres normally involved in linear electron transport (but inhibited here because of the presence of DCMU), or inactivated centres involved in the cyclic pathway. Processes that might regulate the relative flux through cyclic electron transport are discussed.

H2O2-induced inhibition of photosynthetic O2 evolution by Anabaena variabilis cells.

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.

Ascorbate in thylakoid lumen functions as an alternative electron donor to photosystem II and photosystem I

The roles of ascorbate (Asc) in the thylakoid lumen to support photosynthetic electron transport are investigated. Asc can be photooxidized in photosystem (PS) II and PSI. When the water oxidase complex (WOC) was inactivated by acidic pH or by UV-B, Asc was photooxidized in PSII with Asc replacing water as the electron donor. An apparent K m was 2.5 mM. At 20 mM Asc, the electron transport rate reached 50 μmol NADP +-reduced mg Chl −1 h −1. Asc was not oxidized by the PSII reaction center complex having an active WOC, and hence was suggested to function as an emergency donor only when WOC is inactivated. In the presence of 3-(3,4-dichlorophenyl)-1,1 ′-dimethylurea, Asc was photooxidized by PSI, with a lower affinity and an electron transport rate of 70 μmol NADP +-reduced mg Chl −1 h −1 in 50 mM Asc. Thus, Asc can support a PSI-mediated electron flow at a reasonably high rate at Asc concentrations in the physiologically relevant range. During the photooxidation of Asc by PSI, we observed the production of the monodehydroascorbate radical (MDA) that was unscavenged by the exogenously added MDA reductase. Reduction of P700 + by Asc was not affected by the inactivation of plastocyanin with cyanide. These results indicated that Asc was univalently oxidized on the lumenal side of PSI, directly by P700 +. The electron transport Asc → PSI → NADP + did not form a proton gradient across the thylakoid membrane, as determined by 9-aminoacridine fluorescence. Based on these results, we propose that the Asc-dependent cyclic electron flow around PSI, and that through both PSI and PSII can operate when the linear electron transport is partially impaired.

Photoaccumulation of two ascorbyl free radicals per photosystem I at 200 K

Illumination of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 at 200 K in the presence of ascorbate leads to the formation of two ascorbyl radicals per PSI, which are formed by P700 + reduction by ascorbate. During photoaccumulation, one half of the ascorbyl radicals is formed with a halftime of 1 min and the other half with a halftime of 7 min. Pulsed electron paramagnetic resonance (EPR) experiments with protonated/deuterated PSI show that a PSI proton/deuteron is strongly coupled to the ascorbyl radical. Our data indicate that reactive ascorbate molecules bind to PSI at two specific locations, which might be symmetrically located with respect to the pseudo-C 2 axis of symmetry of the heterodimeric core of PSI. Reduction of P700 + by ascorbate leads to multiple turnover of PSI photochemistry, resulting in partial photoaccumulation of the doubly reduced species (F A −, F B −). A modified form of F B −—in accordance with Chamorovsky and Cammack [Biochim. Biophys. Acta 679 (1982) 146–155], but not of F A −, is observed by EPR after illumination at 200 K, which indicates that reduction of F B at 200 K is followed by some relaxation process, in line with this cluster being the most exposed to the solvent.

Reduction of photosystem I reaction center in DrgA mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking soluble NAD(P)H:quinone oxidoreductase.

Photoautotrophically grown cells of the cyanobacterium Synechocystis sp. PCC 6803 wild type and the Ins2 mutant carrying an insertion in the drgA gene encoding soluble NAD(P)H:quinone oxidoreductase (NQR) did not differ in the rate of light-induced oxygen evolution and Photosystem I reaction center (P700+) reduction after its oxidation with a white light pulse. In the presence of DCMU, the rate of P700+ reduction was lower in mutant cells than in wild type cells. Depletion of respiratory substrates after 24 h dark-starvation caused more potent decrease in the rate of P700+ reduction in DrgA mutant cells than in wild type cells. The reduction of P700+ by electrons derived from exogenous glucose was slower in photoautotrophically grown DrgA mutant than in wild type cells. The mutation in the drgA gene did not impair the ability of Synechocystis sp. PCC 6803 cells to oxidize glucose under heterotrophic conditions and did not impair the NDH-1-dependent, rotenone-inhibited electron transfer from NADPH to P700+ in thylakoid membranes of the cyanobacterium. Under photoautotrophic growth conditions, NADPH-dehydrogenase activity in DrgA mutant cells was less than 30% from the level observed in wild type cells. The results suggest that NQR, encoded by the drgA gene, might participate in the regulation of cytoplasmic NADPH oxidation, supplying NADP+ for glucose oxidation in the pentose phosphate cycle of cyanobacteria.

Role of NAD(P)H:quinone oxidoreductase encoded by drgA gene in reduction of exogenous quinones in cyanobacterium Synechocystis sp. PCC 6803 cells.

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700(+)) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700(+) reduction in WT cells, whereas in Ins2 mutant the reduction of P700(+) was strongly inhibited. In the presence of menadiol the reduction of P700(+) was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).

Recovery of photosystem I and II activities during re-hydration of lichen Hypogymnia physodes thalli.

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700(+) and the reduced acceptor F(X) of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700(+) reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q(A)(-), the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.

Enhanced rates of P700(+) dark-reduction in leaves of Cucumis sativus L photoinhibited at chilling temperature.

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 micromol photons m(-2) s(-1)) at 4 degrees C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (deltaA(820)), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of deltaA(820) changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700(+) reduction after far-red light excitation as the half-times of the two exponential components of P700(+) decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700(+) reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700(+) re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.

Effect of the Pool Size of Stromal Reductants on the Alternative Pathway of Electron Transfer to Photosystem I in Chloroplasts of Intact Leaves

The effect of elevated temperature on electron flow to plastoquinone pool and to PSI from sources alternative to PSII was studied in barley (Hordeum vulgare L.) and maize (Zea mays L.) leaves. Alternative electron flow was characterized by measuring variable fluorescence of chlorophyll and absorption changes at 830 nm that reflect redox changes of P700, the primary electron donor of PSI. The treatment of leaves with elevated temperature resulted in a transient increase in variable fluorescence after cessation of actinic light. This increase was absent in leaves treated with methyl viologen (MV). The kinetics of P700+ reduction in barley and maize leaves treated with DCMU and MV exhibited two exponential components. The rate of both components markedly increased with temperature of the heat pretreatment of leaves when the reduction of P700+ was measured after short (1 s) illumination of leaves. The acceleration of both kinetic components of P700+ reduction by high-temperature treatment was much less pronounced when P700+ reduction rate was measured after illumination of leaves for 1 min. Since the treatment of leaves with DCMU and MV inhibited both the electron flow to PSI from PSII and ferredoxin-dependent cycling of electrons around PSI, the accelerated reduction of P700+ indicated that high temperature treatment activated electron flow to PSII from reductants localized in the chloroplast stroma. We conclude that the lesser extent of activation of this process by elevated temperature after prolonged illumination of heat-inhibited leaves is caused by depletion of the pool stromal reductants in light due to photoinduced electron transfer from these reductants to oxygen.

Modeling of the P700 + Charge Recombination Kinetics with Phylloquinone and Plastoquinone-9 in the A 1 Site of Photosystem I

Light activation of photosystem I (PS I) induces electron transfer from the excited primary electron donor P700 (a special pair of chlorophyll a/ a′ molecules) to three iron–sulfur clusters, F X, F A, and F B via acceptors A 0 (a monomeric chlorophyll a) and A 1 (phylloquinone). PS I complexes isolated from menA and menB mutants contain plastoquinone-9 rather than phylloquinone in the A 1 site and show altered rates of forward electron transfer from A 1 − to [F A/F B] and altered rates of back electron transfer from [F A/F B] − to P700 + (Semenov, A. Y., et al., J. Biol. Chem. 275:23429–23438, 2000). To identify the modified electron transfer steps, we studied the kinetics of flash-induced P700 + reduction in PS I that contains either an intact set or a subset of iron–sulfur clusters F X, F A, and F B and with the A 1 binding site occupied by phylloquinone or plastoquinone-9. A modeling of the forward and backward electron transfer kinetics in P700–F A/F B complexes, P700–F X cores, and P700–A 1 cores shows that the replacement of phylloquinone by plastoquinone-9 induces a decrease in the free energy gap between A 1 and F A/F B from ∼−205 mV in wild-type PS I to ∼−70 mV in menA PS I. The +135 mV increase in the midpoint potential of A 1 explains the acceleration in the rate of P700 + dark reduction in menA PS I, and the resulting uphill electron transfer from A 1 to F X in menA PS I explains the absence of a contribution from F X − to the reduction of P700 +. This fully quantitative description of PS I relates electron transfer rates, equilibrium constants, and redox potentials, and can be used to predict changes in these parameters upon substitution of electron transfer cofactors.

Electron flow to photosystem I from stromal reductants in vivo: the size of the pool of stromal reductants controls the rate of electron donation to both rapidly and slowly reducing photosystem I units.

Electron donation from stromal reductants to photosystem I (PSI) was studied using the kinetics of P700(+) (the oxidized primary donor of PSI) reduction in the dark after irradiation of barley ( Hordeum vulgare L.) leaves. The leaves were treated with diuron and methyl viologen to abolish both the electron flow from PSII and PSI-driven cyclic electron transport. The redox state of P700 was monitored using the absorbance changes at 830 nm (Delta A(830)). Two exponentially decaying components with half-times of about 3 s (the slow component) and about 0.6 s (the fast one) were distinguished in the kinetic curves of Delta A(830) relaxation after a 1-s pulse of far-red light. The complex kinetics of P700(+) reduction thus manifested two types of PSI unit differing in the rate of electron input from stromal reductants. The rates of both kinetic components assayed after 1-s pulses were increased about 20-fold by a short (2-5 min) heat-pretreatment of leaves, indicating the accelerated input of electrons to both types of PSI unit. The increased rates of electron flow to P700(+) were even observed 1.5 h after the action of heat had been completed. Both kinetic components were dramatically slowed down upon irradiation of heat-treated leaves for 20-30 s. Their rates were restored after a short (20-30 s) period of darkness. A 5-min leaf exposure at 38 degrees C was sufficient to stimulate by severalfold the reduction of P700(+) pre-oxidized by a brief light pulse. In contrast, the acceleration of P700(+) reduction after a 1-min irradiation was observed only if leaves were subjected to temperatures above 40 degrees C. Neither heat treatment of leaves nor light-dark modulations in the rates of the fast and the slow components of P700(+) dark reduction influenced the relative magnitudes of the two kinetic components, providing strong additional evidence in favor of two distinct types of PSI existing per se in barley leaves. The key role in the control of the activity of electron donation to P700(+) in both rapidly and slowly reducing PSI units was attributed to the amount of stromal reductants available for P700(+) reduction. The latter was expected to be reduced under illumination in the presence of methyl viologen, while increased again in the dark. The regeneration of the pool of stromal reductants in the dark was likely provided by starch breakdown within the chloroplast stroma, but not by import of reducing equivalents from the cytosol. This was evidenced by much lower rates, compared with 1-h dark-adapted leaves, of dark reduction of both components of P700(+) in leaves stored for 24 h in the dark and thus depleted of starch but containing large amounts of glucose, the respiratory substrate.

Single point mutation in the Rieske iron–sulfur subunit of cytochrome b6/ f leads to an altered pH dependence of plastoquinol oxidation in Arabidopsis

The pgr1 mutant of Arabidopsis thaliana carries a single point mutation (P194L) in the Rieske subunit of the cytochrome b 6/ f (cyt b 6/ f) complex and is characterised by a reduced electron transport activity at saturating light intensities in vivo. We have investigated the electron transport in this mutant under in vitro conditions. Measurements of P700 reduction kinetics and of photosynthetic electron transport rates indicated that electron transfer from cyt b 6/ f to photosystem I is not generally reduced in the mutant, but that the pH dependence of this reaction is altered. The data imply that the pH-dependent inactivation of electron transport through cyt b 6/ f is shifted by about 1 pH unit to more alkaline pH values in pgr1 thylakoids in comparison with wild-type thylakoids. This interpretation was confirmed by determination of the transmembrane ΔpH at different stromal pH values showing that the lumen pH in pgr1 mutant plants cannot drop below pH 6 reflecting most likely a shift of the p K and/or the redox potential of the oxidised Rieske protein.

Temperature acclimation of photosynthesis and related changes in photosystem II electron transport in winter wheat.

Winter wheat (Triticum aestivum L. cv Norin No. 61) was grown at 25 degrees C until the third leaves reached about 10 cm in length and then at 15 degrees C, 25 degrees C, or 35 degrees C until full development of the third leaves (about 1 week at 25 degrees C, but 2-3 weeks at 15 degrees C or 35 degrees C). In the leaves developed at 15 degrees C, 25 degrees C, and 35 degrees C, the optimum temperature for CO(2)-saturated photosynthesis was 15 degrees C to 20 degrees C, 25 degrees C to 30 degrees C, and 35 degrees C, respectively. The photosystem II (PS II) electron transport, determined either polarographically with isolated thylakoids or by measuring the modulated chlorophyll a fluorescence in leaves, also showed the maximum rate near the temperature at which the leaves had developed. Maximum rates of CO(2)-saturated photosynthesis and PS II electron transport determined at respective optimum temperatures were the highest in the leaves developed at 25 degrees C and lowest in the leaves developed at 35 degrees C. So were the levels of chlorophyll, photosystem I and PS II, whereas the level of Rubisco decreased with increasing temperature at which the leaves had developed. Kinetic analyses of chlorophyll a fluorescence changes and P700 reduction showed that the temperature dependence of electron transport at the plastoquinone and water-oxidation sites was modulated by the temperature at which the leaves had developed. These results indicate that the major factor that contributes to thermal acclimation of photosynthesis in winter wheat is the plastic response of PS II electron transport to environmental temperature.

Single and Double Knockouts of the Genes for Photosystem I Subunits G, K, and H of Arabidopsis

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.

Metabolic control of photosynthetic electron transport in crassulacean acid metabolism-induced Mesembryanthemum crystallinum.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. We investigated photosynthetic electron transport in leaves of the facultative crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum L. After CAM induction, electron transport exhibited variable redox kinetics during the diurnal CAM cycle. In CAM Phase IV, most of PSI (P700) and chlorophyll a fluorescence relaxed with a halftime of 20 ms after a saturating light pulse. This time-constant may reflect the overall linear electron flux from PSII to PSI in saturating light. Comparable relaxation kinetics were also determined for C3 plants. At the end of CAM Phase I and during Phase II, slow components (> 50% of signal amplitude) appeared in both P700 reduction and fluorescence relaxation. They displayed halftimes > 250 ms and > 1 s, suggesting a strong restriction of the linear electron flux from H2O to NADP. The appearance of the slow redox components was accompanied by a decrease in the Fv/Fm ratio of chlorophyll a fluorescence, suggesting a reversible detachment of light-harvesting complex (LHC) II from PSII. The slow redox fractions and the depression of Fv/Fm disappeared again in parallel to malate decarboxylation during CAM Phase III. We discuss a reversible downregulation of linear electron flux during CAM Phases II and III, due to a reversible deprivation of cytochrome-b6f complexes (cyt-bfs) and PSI from the linear system. In parallel, a redistribution of some LHCIIs could also occur. This could be an adaptive response to a reduced metabolic demand for NADPH due to a limited carbon flux through the Calvin cycle, resulting from low Rubisco activation. Furthermore, the cyt-bfs and PSIs deprived of linear electron transport could support cyclic electron flux to cover an increased ATP demand during gluconeogenesis in CAM Phase III.

Characterization of P700 as a photochemical quencher in isolated Photosystem I particles using simultaneous measurements of absorbance changes at 830 nm and photoacoustic signal.

The relationship between the redox state of P700, the primary donor of PS I, monitored using absorbance changes at 830 nm and photochemical energy storage in PS I reaction centers assayed with the photoacoustic method (PA) was studied in isolated PS I submembrane particles aspirated onto nitrocellulose filters. Several donors have been used to support the electron transport through PS I. NADPH and NADH demonstrated low rates of electron donation to PS I, while ascorbate and ascorbate plus 2,6-dichlorophenolindophenol (DCIP) couple have been found more effective in both P700(+) reduction and stimulation of the variable component of the PA signal. A linear relationship was found in isolated PS I particles between the (A(830,max) - A(830,steady))/A(830,max) and (PA(max) - PA(steady))/PA(max) ratios, which characterized the relative amount of P700 in the reduced state and the relative magnitude of the variable PA component, respectively. That linear relationship was obtained independently from the nature of electron donor used for the reduction of P700(+). Such linear relationship was also obtained at various wavelengths of modulated light in the range of 660 to 720 nm, only the slope of the linear fits varied with wavelength. It is concluded that reduced P700 act as a photochemical quencher of absorbed energy. Variable thermal dissipation in PS I reaction centers of isolated submembrane particles linearly depends on the amount of reduced P700 and thus constitutes an appropriate indicator of the redox pressure applied to PS I.

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.

Optical Study of Cytochrome cM Formation in Synechocystis

The expression of the cytM gene, which encodes cytochrome cM in Synechocystis sp. PCC 6803, is induced under stress conditions such as low temperature. Results of spectrophotometric studies revealed that cytochrome cM was oxidized by light absorbed by photosystem I in cells that had acclimatized to a low temperature. However, no similar light-induced oxidation of cytochrome cM was observed in delta cytM mutant cells. The kinetics of the oxidation and reduction of P700 before and after acclimation of Synechocystis cells to low temperature suggested that cytochrome cM might donate electrons to photooxidized P700. Our observations indicate that cytochrome cM is synthesized under low-temperature stress and that it might carry electrons directly to P700+ in photosystem I.