Heterogeneity of Photosystem I reaction centers in barley leaves as related to the donation from stromal reductants

The light-response curves of P700 oxidation and time-resolved kinetics of P700⁺ dark re-reduction were studied in barley leaves using absorbance changes at 820 nm. Leaves were exposed to 45 °C and treated with either diuron or diuron plus methyl viologen (MV) to prevent linear electron flow from PS II to PSI and ferredoxin-dependent cyclic electron flow around PSI. Under those conditions, P700⁺ could accept electrons solely from soluble stromal reductants. P700 was oxidized under weak far-red light in leaves treated with diuron plus MV, while identical illumination was nearly ineffective in diuron-treated leaves in the absence of MV. When heat-exposed leaves were briefly illuminated with strong far-red light, which completely oxidized P700, the kinetics of P700⁺ dark reduction was fitted by a single exponential term with half-time of about 40 ms. However, two first-order kinetic components of electron flow to P700⁺ (fast and slow) were found after prolonged leaf irradiation. The light-induced modulation of the kinetics of P700⁺ dark reduction was reversed following dark adaptation. The fast component (half time of 80–90 ms) was 1.5 larger than the slow one (half time of about 1 s). No kinetic competition occurred between two pathways of electron donation to P700⁺ from stromal reductants. This suggests the presence of two different populations of PSI. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 5–11.Original Russian Text Copyright © 2005 by Bukhov, Egorova.     

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.     

Elevated Temperatures Inhibit Ferredoxin-Dependent Cyclic Electron Flow around Photosystem I

In experiments with barley (Hordeum vulgare L.) leaves, absorbance changes at 830 nm induced by far-red light were measured as indicator of redox conversions of primary electron donor (P700) of photosystem I (PSI). Using this method, the action of elevated temperature (45°C, 5 min) on PSI-driven electron transport through alternative pathways was examined. Thermally induced inactivation was found to transform nonmonotonic photooxidation of P700, induced by far-red light in untreated leaves, into a fast and monotonic process completed within 1-s illumination. The short-term heating of leaves fully eliminated the fast component in the kinetics of P700⁺ dark reduction, related to operation of ferredoxin-dependent cyclic electron transport around PSI. At the same time, thermoinactivation substantially accelerated the slow and middle components of dark P700⁺ reduction, i.e., the components determined by arrival of electrons to PSI from reductants located in the chloroplast stroma. The latter effect was also observed after heating of leaves pretreated with antimycin A or methyl viologen; both agents are known to inhibit the ferredoxin-dependent electron transport. It is concluded that the heat treatment of leaves inhibits the ferredoxin-dependent pathway of electron transport around PSI and activates electron transport through alternative routes providing reducing equivalents to PSI from stromal reductants.     

Monophasic kinetics of electron donation from stromal reductants in unicellular halophytic alga Tetraselmis viridis: Relation of the function to characteristic structure of chloroplast membrane system

Redox conversions of P700, the primary donor of photosystem I (PSI), were investigated in cells of a halophytic alga Tetraselmis viridis Rouch. under irradiation with white light pulses that excite both photosystems of the chloroplast and with far-red light initiating photochemical reactions in PSI only. The P700⁺ dark reduction after irradiation with 50-ms pulse of white light comprised three kinetic components. The half-decay times and relative contributions of the fast, middle, and slow components were 38 ms (49%), 295 ms (26%), and 1690 ms (23%), respectively. The treatment with diuron, known to block electron transport between the photosystems, eliminated the middle exponential term having the half-decay time of 295 ms. After irradiation with far-red light, the kinetics of P700⁺ dark reduction comprised only two components with half-deacy times of 980 ms (72%) and 78 ms (31%). The component with a decay halftime of about 100 ms was fully inhibited after treating the cells with antimycin A, a specific inhibitor of ferredoxin-dependent cyclic electron flow around PSI. In addition, this kinetic component was strongly suppressed by methyl viologen known to inhibit this alternative pathway of electron transport. Both aforementioned reagents had no effect on the slow component of P700⁺ reduction; this component remained monophasic. Unlike higher plant chloroplasts, the chloroplasts of Tetraselmis viridis contained no stacked grana. Based on inhibitor analysis and electron microscopy data, it was concluded that the slow component of P700⁺ reduction in the cells of halophytic microalga reflects the electron donation to PSI from reductants localized in the chloroplast stroma. The monophasic kinetics of this process in the halophytic microalga, compared to the biphasic kinetic pattern in higher plants, is related to the lack of stacked grana in Tetraselmis viridis cells.     

Modulating Effect of Far-Red Light on Activities of Alternative Electron Transport Pathways Related to Photosystem I

Barley (Hordeum vulgare L.) leaves were irradiated with far-red (FR) light of various intensities after different periods of dark adaptation in order to investigate activities of alternative electron transport pathways related to photosystem I (PSI). Photooxidation of P700, the primary electron donor of PSI, was saturated at FR light intensity of 0.15 μmol quanta/(m² s). As the photon flux density was raised in this range, the slow and middle components in the kinetics of P700⁺ dark reduction increased, whereas the fast component remained indiscernible. The amplitudes of the slow and middle components diminished upon further increase of FR photon flux density in the range 0.15–0.35 μmol quanta/(m² s) and remained constant at higher intensities. The fast component of P700⁺ reduction was only detected after FR irradiation with intensities above 0.15 μmol quanta/(m² s); the light-response curve for this component was clearly sigmoid. In dark-adapted barley leaves, three stages were distinguished in the kinetics of P700 photooxidation, with the steady state for P700⁺ achieved within about 3 min. In leaves predarkened for a short time, the onset of FR irradiation produced a very rapid photooxidation of P700. As the duration of dark exposure was prolonged, the amplitude of the first peak in the kinetic curve of photoinduced P700 photooxidation was diminished and the time for attaining the steady-state oxidation level was shortened. After a brief dark adaptation of leaves, ferredoxin-dependent electron flow did not appreciably contributed to the kinetics of P700⁺ dark reduction, whereas the components related to electron donation from stromal reductants were strongly retarded. It is concluded that FR light irradiation, selectively exciting PSI, suffices to modulate activities of alternative electron transport routes; this modulation reflects the depletion of stromal reductants due to continuous efflux of electrons from PSI to oxygen under the action of FR light. __________ Translated from Fiziologiya Rastenii, Vol. 52, No. 6, 2005, pp. 805–813. Original Russian Text Copyright ? 2005 by Egorova, Drozdova, Bukhov.     

Origin of Multiphase Reduction of P700<Superscript>+</Superscript> in Broad Bean Leaves after Irradiation with Far-Red Light

The kinetic curves of dark reduction of P700⁺ (oxidized primary donor of PSI) after far-red light irradiation were studied on broad bean (Vicia faba L.) leaves treated with antimycin A, methyl viologen, or diuron. Four components of P700⁺ reduction were found in untreated leaves, namely, an ultrafast component with a half-time of 25 ms, and fast (210 ms), middle (790 ms), and slow (6100 ms) components. The fast component disappeared in leaves treated with antimycin A or methyl viologen. At the same time, these substances did not affect other components of P700⁺ reduction. Treatment of leaves with diuron abolished both the ultrafast and fast components of P700⁺ reduction. As the length of far-red light exposure was increased, a lag phase appeared in the development of middle component in leaves treated with diuron, antimycin A, or methyl viologen. In thus treated leaves, an exponential pattern of the middle component was displayed with a certain delay after darkening. A conclusion was drawn that the minor ultrafast component of P700⁺ dark reduction in broad bean leaves was caused by electron donation to PSI from PSII, whereas the fast component of this process was determined by the operation of ferredoxin-dependent electron transport around PSI. The middle and slow components were supposed to be related to electron input to PSI from reductants localized in the chloroplast stroma.From Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 492–498.Original English Text Copyright © 2005 by Egorova, Nikolaeva, Bukhov.The article was translated by the authors.     

Alternative pathways of photosystem I-dependent electron transport in two genetically different potato cultivars in vitro

Alternative pathways of electron transport involving photosystem I (PSI) only were studied in leaves of potato plants (Solanum tuberosum L., cv. Desiree), modified by yeast invertase gene, controlled by tuber-specific class I patatin B33 promoter with proteinase II signal peptide for apoplastic localization of the enzyme. Nontransformed (wild-type) potato cultivar Desiree was used as a source of control plants. Phototrophic cultures grown in vitro on the sucrose-free Murashige and Skoog medium, as well as plants grown on the medium with 4% sucrose were examined. Various PSI-dependent alternative pathways of electron transport were discriminated by quantitative analysis of kinetic curves of dark reduction of P700⁺, the primary electron donor of PSI, oxidized by far-red light known to excite selectively PSI. In potato plants with two different genotypes, four exponentially decaying kinetic components were found, which suggests the existence of multiple alternative routes for electron input to PSI. Inhibitor analysis (with diuron and antimycin A) allowed identification of each route. A minor ultra-fast component originated from weak residual excitation of PSII by far-red light and represented electron flow from PSII to PSI. Ferredoxin-dependent cyclic electron flow around PSI accounted for the middle component, and two slower components were assigned to donation of electrons to PSI from reductants localized in the chloroplast stroma. The rates of all components were somewhat higher in leaves of the transformed plants than in the wild-type plants. However, relative contributions of separate components to the kinetics of dark P700⁺ reduction in leaves of both potato genotypes were similar. Growing plants on the medium with sucrose dramatically increased the amplitude of absorbance change at 830 nm in the transformed (but not in wild type) plants, which indicated a drastic increase in P700 concentration in their leaves.     

Inhibition of photosystems I and II and enhanced back flow of photosystem I electrons in cucumber leaf discs chilled in the light

Pre-illumination of cucumber leaf discs at a chilling temperature in low-irradiance white light resulted in accelerated re-reduction of P700(+) [the special Chl pair in the photosystem I (PSI) reaction centre] when the far-red measuring light was turned off. Measurements (in +/- methyl viologen or +/- DCMU conditions) of the re-reduction half time suggest that accelerated re-reduction of P700(+) appeared to be predominantly due to charge recombination and only partly due to reductants sustained by previous cyclic electron flow around PSI. Apparently, charge recombination in PSI was greatly enhanced by inhibition of forward, linear electron flow. Inhibition of PSII electron transport was observed to occur to a lesser extent than that of PSI, but only if the measurement of PSII functionality was free from complications due to downstream accumulation of electrons in pools. We suggest that promotion of controlled charge recombination and cyclic electron flow round PSI during chilling of leaves in the light may partly prevent further damage to both photosystems.     

Redox states of photosystems I and II in irradiated leaves of wheat seedlings grown under different conditions of nitrogen nutrition

Changes in the redox states of photosystem I (PSI) and PSII in irradiated wheat leaves were studied after growing seedlings on a nitrogen-free medium or media containing either nitrate or ammonium. The content of P700, the primary electron donor of PSI was quantified using the maximum magnitude of absorbance changes at 830 nm induced by saturating white light. The highest content of P700 in leaves was found for seedlings grown on the ammonium-containing medium, whereas its lowest content was observed on seedlings grown in the presence of nitrate. At all irradiances of actinic light, the smallest accumulation of reduced Q_A was observed in leaves of ammonium-grown plants. Despite variations in light-response curves of P700 photooxidation and Q_A photoreduction, the leaves of all plants exposed to different treatments demonstrated similar relationships between steady-state levels of P700⁺ and Q_A ^–. The accumulation of oxidized P700 up to 40% of total P700 content was not accompanied by significant Q_A photoreduction. At higher extents of P700 photooxidation, a linear relationship was found between the steady-state levels of P700⁺ and Q_A ^–. The leaves of all treatments demonstrated biphasic patterns of the kinetics of P700⁺ dark reduction after irradiation by far-red light exciting specifically PSI. The halftimes of corresponding kinetic components were found to be 2.6–4 s (fast component) and 17–22 s (slow component). The two components of P700⁺ dark reduction were related to the existence of two PSI populations with different rates of electron input from stromal reductants. The magnitudes of these components differed for plants grown in the presence of nitrate, on the one hand, and plants grown either in the presence of ammonium or in the absence of nitrogen, on the other hand. This indicates the possible influence of nitrogen nutrition on synthesis of different populations of PSI in wheat leaves. The decrease in far-red light irradiance reduced the relative contribution of the fast component to P700⁺ reduction. The fast component completely disappeared at low irradiances. This finding indicates that the saturating far-red light must be applied to determine correctly the relative content of each PSI population in wheat leaves.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 165–171.Original Russian Text Copyright © 2005 by Dzhibladze, Polesskaya, Alekhina, Egorova, Bukhov.This revised version was published online in April 2005 with a corrected cover date.     

Effect of exogenous glucose on electron flow to photosystem I and respiration in cyanobacterial cells

The effects of exogenous glucose on the rates of alternative pathways of photosystem II (PSII)-independent electron flow to PSI and of dark respiration in Synechocystis sp. 6803 cells were studied. The presence of glucose was shown to accelerate the electron flow to P700⁺, the PSI primary electron donor oxidized with Far-red light (FRL), which excites specifically only PSI. An increase in the glucose concentration was accompanied by a further activation of electron flow to PSI, which was supported by the dark donation of reducing equivalents to the electron transport chain. An increase in the external glucose concentration resulted also in the disappearance of lag-phase in the kinetics of P700⁺ reduction, which was observed in the cells incubated without glucose after FRL switching off. A similarity of nonphotochemical processes of electron transfer to PSI in cyanobacteria and higher plants was supposed, basing on the earlier observed fact of the occurrence of such lagphase in higher plants and its dependence on the exhausting of stromal reductants in the light. Acceleration of dark electron flow to PSI in the presence of glucose, a major respiratory substrate, may indicate the coupling between nonphotochemical processes in the photosynthetic and respiratory chains of electron transport in cyanobacterial cells. A close correlation between photosynthesis and respiration in cyanobacterial cells is also confirmed by a sharp acceleration of respiration with an increase in the glucose concentration in medium.     

Nonmonotonic Redox Conversions of P700 in Leaves Irradiated with Far-Red Light Originate from Variable Contributions of Several Alternative Electron Transport Pathways during the Induction Period

The origin of nonmonotonic changes in the redox state of P700, the primary electron donor of PSI, was investigated on predarkened barley (Hordeum vulgare L.) leaves exposed to far-red light. To accomplish this, the relaxation kinetics of absorbance changes at 830 nm, reflecting the dark reduction of P700⁺, were measured at different stages of the induction curve. The onset of far-red light resulted in rapid oxidation of P700, which was followed by its partial reduction and subsequent slow oxidation of P700 to a steady-state level. This steady-state level was usually attained within 10 s under far-red light. The relative contribution of the slow kinetic component of P700⁺ reduction decreased in parallel with the transient photoreduction of P700⁺ and increased upon a subsequent stage of P700 photooxidation. The contribution of the middle component to the dark reduction of P700⁺ increased monotonically with the length of far-red light irradiation. The relative amplitude of the fast component of P700⁺ reduction increased sharply during the first 3 s of irradiation and decreased upon longer light exposures. The rates of fast and slow components of dark reduction of P700⁺ remained constant upon illumination of dark-adapted leaves with far-red light for 1 s and longer periods. Thus, nonmonotonic changes in the redox state of P700 in barley leaves exposed to far-red light reflect variable contributions of few alternative electron transport pathways characterized by different rates of electron donation to PSI. The results show the principle possibility of switching-over between alternative pathways of PSI-related electron transfer within one complex of this photosystem. Such switching may occur irrespective of active operation or inhibition of ferredoxin-dependent electron transport.     

Reduction of the primary donor P700 of photosystem I during steady-state photosynthesis under low light in <Emphasis Type="Italic">Arabidopsis</Emphasis>

During steady-state photosynthesis in low-light, 830-nm absorption (A₈₃₀) by leaves was close to that in darkness in Arabidopsis, indicating that the primary donor P700 in the reaction center of photosystem I (PSI) was in reduced form. However, P700 was not fully oxidized by a saturating light pulse, suggesting the presence of a population of PSI centers with reduced P700 that remains thermodynamically stable during the application of the saturating light pulse (i.e., reduced-inactive P700). To substantiate this, the effects of methyl viologen (MV) and far-red light on P700 oxidation by the saturating light pulse were analyzed, and the cumulative effects of repetitive application of the saturating light pulse on photosynthesis were analyzed using a mutant crr2-2 with impaired PSI cyclic electron flow. We concluded that the reduced-inactive P700 in low-light as revealed by saturating light pulse indicates limitations of electron flow at the PSI acceptor side.     

Populations of photosystem 1 units rapidly and slowly reduced by stromal reductants represent photosystem 1α and photosystem 1β complexes: Evidence from irradiance-response curves of P700 photooxidation in intact barley leaves

Photon-induced absorbance changes at 830 nm (A₈₃₀) related to redox transformations of P700, primary electron donor of photosystem 1 (PS1), were examined in barley leaves treated with diuron and methyl viologen. In such leaves, only soluble reductants localized in chloroplast stroma could serve as electron donors for P700⁺. A₈₃₀ were induced by 1-min irradiation of leaves with actinic light (AL, 700±6 nm) of various irradiances. Two exponentially decaying components with half-times of 2.75 (fast component, relative magnitude of 62 % of A₈₃₀) and 11.90 s (slow one, 38 % of A₈₃₀) were distinguished in the kinetics of dark relaxation of A₈₃₀ after leaf irradiation with saturating AL. The components reflecting P700⁺ dark reduction in two units of PS1 differed in the rate of electron input from stromal reductants. The decline in AL irradiance reduced steady state A₈₃₀ magnitude, which was also accompanied by a decrease in the contribution of fast component to the overall P700⁺ dark reduction kinetics. The photon-response curves were obtained separately for rapidly and slowly decaying A₈₃₀. The values of half-saturating irradiance were 0.106 and 0.035 mol m^–2 s^–1 for rapidly and slowly reduced PS1 units, respectively. The ratio of rate constants of P700⁺ dark reduction for rapidly and slowly reduced PS1 units was 1.4 times higher than the ratio of their half-saturating irradiances thus indicating higher relative antenna size in rapidly reduced PS1 units. The latter finding, taken together with higher relative amount of P700, favours the view that rapidly and slowly reduced PS1 units reflect P700⁺ reduction by stromal reductants in spatially separated PS1 and PS1 complexes.     

Identification of Ferredoxin-Dependent Cyclic Electron Transport around Photosystem I Using the Kinetics of Dark P700<Superscript>+</Superscript> Reduction

Kinetic curves of absorbance changes induced by far-red light (FR, 830 nm) (A ₈₃₀), which reflect redox transformations of PSI primary electron donor, P700, were examined in intact barley (Hordeum vulgare L.) leaves. In intact leaves, FR induced the biphasic increase in absorbance related to P700 photooxidation. Leaf treatment with methyl viologen or antimycin A suppressed the slow phase of P700 photooxidation, which was attained in such leaves within the first second of light exposure. With FR turned off, the previously increased absorbance at 830 nm dropped down to its initial level, thus reflecting P700⁺ reduction. In the control leaves, the kinetics of P700⁺ reduction consisted of three exponentially decaying components, with the corresponding half-times of 8.8 s (the slow component, with its magnitude comprising 24% of the total A ₈₃₀ signal), 0.73 s (the middle component, 49% of A ₈₃₀), and 0.092 s (the fast component, 26% of A ₈₃₀). The rate of the fast component of P700⁺ reduction, following FR irradiation of leaves, was about ten times lower than that of the noncyclic electron transfer from PSII to PSI computed from A ₈₃₀ relaxation after the abrupt offset of white light. The treatment of leaves with methyl viologen or antimycin A completely abolished the fast component of A ₈₃₀ relaxation after FR exposure. It was concluded that the fast component is determined by the operation of ferredoxin-dependent cyclic electron transport around PSI. This study represents the first report on the identification of this pathway of electron transport in vivo and the estimation of its rate.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 325–330.Original Russian Text Copyright © 2005 by Bukhov, Egorova.     

Activities of Noncyclic and Alternative Pathways of Photosynthetic Electron Transport in Leaves of Broad Bean Plants Grown at Various Light Irradiances

Activities of noncyclic and alternative pathways of photosynthetic electron transport were studied in intact leaves of broad been (Vicia faba L.) seedlings grown under white light at irradiances of 176, 36, and 18 µmol quanta/(m² s). Electron flows were followed from light-induced absorbance changes at 830 nm related to redox transformations of P700, the photoactive PSI pigment. The largest absorbance changes at 830 nm, induced by either white or far-red light, were observed in leaves of seedlings grown at irradiance of 176 µmol quanta/(m² s), which provides evidence for the highest concentration of PSI reaction centers per unit leaf area in these seedlings. When actinic white light of 1800 µmol quanta/(m² s) was turned on, the P700 oxidation proceeded most rapidly in leaves of seedlings grown at irradiance of 176 µmol quanta/(m² s). The rates of electron transfer from PSII to PSI were measured from the kinetics of dark P700⁺ reduction after turning off white light. These rates were similar in leaves of all light treatments studied, and their characteristic reaction times were found to range from 9.2 to 9.5 ms. Four exponentially decaying components were resolved in the kinetics of dark P700⁺ reduction after leaf exposure to far-red light. A minor but the fastest component of P700⁺ reduction with a halftime of 30–60 ms was determined by electron transfer from PSII, while the three other slow components were related to the operation of alternative electron transport pathways. Their halftimes and relative magnitudes were almost independent on irradiance during plant cultivation. It is concluded that irradiance during plant growth affects the absolute content of PSI reaction centers in leaves but did not influence the rates of noncyclic and alternative electron transport.From Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 485–491.Original English Text Copyright © 2005 by Nikolaeva, Bukhov, Egorova.The article was translated by the authors.     

Variable fluorescence of photosystem I particles and its application to the study of the structure and function of photosystem I

Chlorophyll a fluorescence in Photosystem I (PSI) particles isolated according to the method of Bengis and Nelson [J. Biol. Chem.252, 4564–4569 (1977)]was found to be dependent on the redox state of both P700 and X (an acceptor on the reducing side of PSI). Addition of dithionite plus neutral red to PSI caused an increase in fluorescence intensity and a shift of the main fluorescence peak from 689 to 674 nm. Addition of electron acceptors such as ferredoxin and methyl viologen decreased the fluorescence yield when added to PSI incubated under anaerobic conditions in the presence of excess dichlorophenol indophenol (DCIPH₂). The K_m for ferredoxin agreed with that determined from direct measurements of ferredoxin reduction, showing that X is a quencher of fluorescence. P700 was also found to be a quencher of fluorescence, since electron donors such as DCIPH₂, TMPD, and plastocyanin decreased fluorescence with K_m's nearly identical to those observed for P700⁺ reduction. Chemical modification of PSI (with ethylene diamine + a water-soluble carbodiimide) to make it positively charged increased the fluorescence yield and shifted the 689-nm peak to 674 nm. The K_m's for DCIPH₂ and ferredoxin were decreased. In contrast, modification of PSI with succinic anhydride, which increased the net negative charge, increased the K_m for ferredoxin. Salts affected the interaction of methyl viologen with PSI. Both anion and cation selectivity were observed. Limited proteolysis increased the K_m for both methyl viologen and ferredoxin, indicating that their binding site on PSI was altered. These results suggest that the binding site for ferredoxin is on either the 70- or the 20-kDa subunit of PSI.     

Characterization of photosynthetic electron transport in bundle sheath cells of maize. I. Ascorbate effectively stimulates cyclic electron flow around PSI

Redox changes of the reaction-center chlorophyll of photosystem I (P700) and chlorophyll fluorescence yield were measured in bundle sheath strands (BSS) isolated from maize (Zea mays L.) leaves. Oxidation of P700 in BSS by actinic light was suppressed by nigericin, indicating the generation of a proton gradient across the thylakoid membranes of BSS chloroplasts. Methyl viologen, which transfers electrons from photosystem I (PSI) to O₂, caused a considerable decrease in the reduction rate of P700⁺ in BSS after turning off actinic light, showing that electron flow from the acceptor side of PSI to stromal components is critical for this reduction. Ascorbate (Asc), and to a lesser extent malate (Mal), caused a lower level of P700⁺ in BSS under aerobic conditions in far-red light, implying electron donation from these substances to the intersystem carriers. When Asc or Mal was added to BSS during pre-illumination under anaerobic conditions in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), the far-red-induced level of P700⁺ was lowered. The results suggest Asc and Mal can cause reduction of stromal donors, which in turn establishes conditions for rapid PSI-driven P700⁺ reduction. Addition of these metabolites also strongly stimulated the development of a proton gradient in thylakoids under aerobic conditions in the absence of DCMU, i.e. under conditions analogous to those in vivo. Ascorbate was a much more effective electron donor than Mal, suggesting it has a physiological role in activation of cyclic electron flow around PSI.     

Electron Fluxes through Photosystem I in Cucumber Leaf Discs Probed by far-red Light

Cucumber leaf discs were illuminated at room-temperature with far-red light to photo-oxidise P700, the chlorophyll dimer in Photosystem (PS) I. The post-illumination kinetics of P700(+) re-reduction were studied in the presence of inhibitors or cofactors of photosynthetic electron transport. The re-reduction kinetics of P700(+) were well fitted as the sum of three exponentials, each with its amplitude and rate coefficient, and an initial flux (at the instant of turning off far-red light) given as the product of the two. Each initial flux is assumed equal to a steady state flux during far-red illumination. The fast phase of re-reduction, with rate coefficient k (1) approximately 10 s(-1), was completely abolished by a saturating concentration of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); it is attributed to electron flow to P700(+) from PS II, which was stimulated to some extent by far-red light. The intermediate phase, with rate coefficient k (1) approximately 1 s(-1), was only partly diminished by methyl viologen (MV) which diverts electron flow to oxygen. The intermediate phase is attributed to electron donation from reduced ferredoxin to the intersystem pool; reduced ferredoxin could be formed: (1) directly by electron donation on the acceptor of PS I; and/or (2) indirectly by stromal reductants, in line with only a partial inhibition of the intermediate phase by MV. Duroquinol enhanced the intermediate phase in the presence of DCMU, presumably through its interaction with thylakoid membrane components leading to the partial reduction of plastoquinone. The slow phase of P700(+) re-reduction, with rate coefficient k (1) approximately 0.1 s(-1), was unaffected by DCMU and only slightly affected by MV; it could be associated with electron donation to either: (1) the intersystem chain by stromal reductants catalysed by NAD(P)H dehydrogenase slowly; or (2) plastocyanin/P700(+) by ascorbate diffusing across the thylakoid membrane to the lumen. It is concluded that a post-illumination analysis of the fluxes to P700(+) can be used to probe the pathways of electron flow to PS I in steady state illumination.     

Genetic decrease in fatty acid unsaturation of phosphatidylglycerol increased photoinhibition of photosystem I at low temperature in tobacco leaves

Leaves of transgenic tobacco plants with decreased levels of fatty acid unsaturation in phosphatidylglycerol (PG) exhibited a slightly lower level of the steady state oxidation of the photosystem I (PSI) reaction center P700 (P700(+)) than wild-type plants. The PSI photochemistry of wild-type plants was only marginally affected by high light treatments. Surprisingly, all plants of transgenic lines exhibited much higher susceptibility to photoinhibition of PSI than wild-type plants. This was accompanied by a 2.5-fold faster re-reduction rate of P700(+) in the dark, indicating a higher capacity for cyclic electron flow around PSI in high light treated transgenic leaves. This was associated with a much higher intersystem electron pool size suggesting over-reduction of the PQ pool in tobacco transgenic lines with altered PG unsaturation compared to wild-type plants. The physiological role of PG unsaturation in PSI down-regulation and modulation of the capacity of PSI-dependent cyclic electron flows and distribution of excitation light energy in tobacco plants under photoinhibitory conditions at low temperatures is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.