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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping

The role of cyclic electron transport has been re-examined in leaves of C₃ plants because the bioenergetics of chloroplasts (H⁺/e = 3 in the presence of a Q-cycle; H⁺/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C₃ photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large increase in the number of electrons available to reduce P700⁺ in the dark. At low CO₂ and O₂ molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, F_v/F_m) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in F_v/F_m brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO₂-dependent O₂ evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease F_v/F_m. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H⁺/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. Received: 23 February 1999 / Accepted: 19 August 1999     

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.     

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.     

Photochemical activities of two photosystems in Bratonia orchid protocorms cryopreserved by vitrification method

Functional activities of two photosystems in orchid-specific embryos (protocorms) of a tropical hybrid orchid Bratonia were investigated before and after their cryopreservation by vitrification method. The kinetics of light-induced absorbance changes at 830 nm was analyzed as indicator of P700 redox conversions; changes in the variable chlorophyll fluorescence served to indicate the oxidation-reduction changes of the primary acceptor Q_A. Untreated protocorms exhibited low photochemical activity of photosystem II (PSII). In freeze-treated Bratonia protocorms, examined immediately after thawing, photosynthetic electron transport was strongly inhibited. Nevertheless, the cells retained activities of noncyclic electron flow and of alternative electron transport pathways related solely to PSI. However, Bratonia protocorms subjected to deep-freezing lost the capability of P700 photooxidation during the first day of reculturing. Deep freezing of protocorms had virtually no effect on the kinetics of dark relaxation of chlorophyll variable fluorescence, when measurements were made immediately after thawing. Unlike chlorophyll fluorescence, the kinetics of dark reduction of P700⁺ in protocorms exposed to freezing-thawing was substantially modified compared to untreated protocorms. Two exponential components with half-decay times of 27 and 310 ms were distinguished in the kinetics of P700⁺ reduction in treated samples, whereas the absorbance relaxation attributed to P700⁺ reduction in untreated samples followed an exponential decay with a half-decay time of 24 ms. Despite the appearance of additional slow component in the kinetics of P700⁺ reduction, the dark relaxation of variable fluorescence remained unaltered after deep freezing of protocorms. This observation indicates that the freezing-thawing procedure caused partial disorders in linear electron transport between PSII and PSI. Apparently, the functional interactions among carriers in the electron-transport chain were disturbed between the plastoquinone pool and the PSI reaction center. It is concluded that the vitrification method applied to protocorm cryopreservation did not cause their immediate death, but the protocorms died later, on the first day after reculturing.     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700 induction kinetics

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA₈₁₀, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700⁻ oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700⁺ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700⁺ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q_r site of the cytb₆f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.     

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.     

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.     

Photoinduction of electron transport on the acceptor side of PSI in Synechocystis PCC 6803 mutant deficient in flavodiiron proteins Flv1 and Flv3

After transferring the dark-acclimated cyanobacteria to light, flavodiiron proteins Flv1/Flv3 serve as a main electron acceptor for PSI within the first seconds because Calvin cycle enzymes are inactive in the dark. Synechocystis PCC 6803 mutant Δflv1/Δflv3 devoid of Flv1 and Flv3 retained the PSI chlorophyll P700 in the reduced state over 10?s (Helman et al., 2003; Allahverdiyeva et al., 2013). Study of P700 oxidoreduction transients in dark-acclimated Δflv1/Δflv3 mutant under the action of successive white light pulses separated by dark intervals of various durations indicated that the delayed oxidation of P700 was determined by light activation of electron transport on the acceptor side of PSI. We show that the light-induced redox transients of chlorophyll P700 in dark-acclimated Δflv1/Δflv3 proceed within 2?min, as opposed to 1–3?s in the wild type, and comprise a series of kinetic stages. The release of rate-limiting steps was eliminated by iodoacetamide, an inhibitor of Calvin cycle enzymes. Conversely, the creation with methyl viologen of a bypass electron flow to O₂ accelerated P700 oxidation and made its extent comparable to that in the wild-type cells. The lack of major sinks for linear electron flow in iodoacetamide-treated Δflv1/Δflv3 mutant, in which O₂- and CO₂-dependent electron flows were impaired, facilitated cyclic electron flow, which was evident from the decreased steady-state oxidation of P700 and from rapid dark reduction of P700 during and after illumination with far-red light. The results show that the photosynthetic induction in wild-type Synechocystis PCC 6803 is largely hidden due to the flavodiiron proteins whose operation circumvents the rate-limiting electron transport steps controlled by Calvin cycle reactions.