Simulation of chlorophyll fluorescence rise and decay kinetics, and P700-related absorbance changes by using a rule-based kinetic Monte-Carlo method.

A model of primary photosynthetic reactions in the thylakoid membrane was developed and its validity was tested by simulating three types of experimental kinetic curves: (1) the light-induced chlorophyll a fluorescence rise (OJIP transients) reflecting the stepwise transition of the photosynthetic electron transport chain from the oxidized to the fully reduced state; (2) the dark relaxation of the flash-induced fluorescence yield attributed to the QA- oxidation kinetics in PSII; and (3) the light-induced absorbance changes near 820 or 705 nm assigned to the redox transitions of P700 in PSI. A model was implemented by using a rule-based kinetic Monte-Carlo method and verified by simulating experimental curves under different treatments including photosynthetic inhibitors, heat stress, anaerobic conditions, and very high light intensity.

Study of the effect of reducing conditions on the initial chlorophyll fluorescence rise in the green microalgae Chlamydomonas reinhardtii.

Incubation of Chlamydomonas reinhardtii cells under nutrient deficiency results in the faster initial rise in the light-induced chlorophyll fluorescence kinetic curve. We showed that short-term anaerobic incubation of algal cells altered initial fluorescence in a way similar to nutrient starvation, suggesting an important role of the plastoquinones redox state in the observed effect. Bi-component analysis of highly resolved initial fluorescence rise kinetics in sulfur- or oxygen-depleted C. reinhardtii cells suggested that one of the mechanisms underlying the observed phenomenon involves primary closure (photochemical inactivation via Qa reduction) of ß-type PSII as compared to α-PSII. Moreover, results of modeling of the fluorescence curve brought us to the conclusion that accumulation of closed centers in α-PSII supercomplexes may also cause a faster initial fluorescence rise. The observed correlations between nutrient supply rate and initial fluorescence rise pattern in green algae can serve to characterize culture nutritional status in vivo.

[Performance index as an indicator of the physiological state of trees in urban forest ecosystems].

Based on the measurements of fluorescence of bark chloroplasts by means of PAM and PEA fluorometers, the information capacity of the methods for assessing the physiological state of Tilia cordata L. from the maximum quantum efficiency of PS II photochemistry (Fv/Fm) and the performance index (PI) has been compared. The measurements were performed on annual shoots of linden trees growing in different environment. It was shown that the chlorophyll content in the bark of shoots growing near the busy urban street was twice less compared with trees growing out of the city. On the trees from the unsafe environment, a small decrease in the relative fluorescence variable (Fv/Fm) was registered, and there was a significant statistical deviation of this value compared to control trees. It was found that the PI and its constituent parameters calculated on the basis of light fluorescence induction curve (PEA-method) are more informative and allow one to recognize changes in the primary energy transformation processes in PS II when they are comparatively small. The results of our work show that PI can be used as a sensitive and a rapid test to evaluate the physiological state of trees and other plant objects even under minor environmental changes.

Probing of photosynthetic reactions in four phytoplanktonic algae with a PEA fluorometer.

High-resolution light-induced kinetics of chlorophyll fluorescence (OJIP transients) were recorded and analyzed in cultures of diatoms (Thalassiosira weissflogii, Chaetoceros mulleri) and dinoflagellates (Amphidinium carterae, Prorocentrum minimum). Fluorescence transients showed the rapid exponential initial rise from the point O indicating low connectivity between PS II units and high absorption cross-section of PS II antenna. Dark-adapted dinoflagellates revealed capability to maintain the PS I-mediated re-oxidation of the PQ pool at the exposure to strong actinic light that may lead to the underestimation of F(M) value. In OJIP transients recorded in phytoplanktonic algae the fluorescence yield at the point O exceeded F(O) level because Q(A) has been already partly reduced at 50 micros after the illumination onset. PEA was also employed to study the recovery of photosynthetic reactions in T. weissflogii during incubation of nitrogen starved cells in N-replete medium. N limitation caused the impairment of electron transport between Q(A) and PQs, accumulation of closed PS II centers, and the reduced ability to generate transmembrane DeltapH upon illumination, almost fully restored during the recovery period. The recovered cells showed much higher values of NPQ than control ones suggesting maximization of photoprotection mechanisms in the population with a 'stress history.'

[Effect of dibromothymoquinone on chlorophyll a fluorescence in Chlamydomonas reinhardtii cells incubated in complete or sulfur-depleted medium].

The influence of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. The reagent affected differently control cells incubated in complete medium and S-starved cells. Thus, the fluorescence yield in the control essentially increased in the presence of dibromothymoquinone, which can be due to the inactivation of light-harvesting complex II protein kinase, followed by the suppression of membrane transition from high-fluorescence state 1 to low-fluorescence state 2. On the contrary, S-starved cells with membranes in state 2 showed a lower fluorescence yield in the presence of dibromothymoquinone than without it. The JIP test of OJIP fluorescence transients suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to control, while in starved cells, it acts predominantly as an electron acceptor.

Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii.

Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark ('Fo conditions') or treated to reduce Qa ('Fm conditions'). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under 'Fo' and 'Fm conditions', respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed.

[Examination of chlorophyll fluorescence in sulfur-deprived cells of Chlamydomonas reinhardtii].

Modulated fluorometry (PAM) was applied for probing the photosynthesis in cells of C. reinhardtii during sulfur deprivation. A significant (up to a fourfold) increase in chlorophyll fluorescence yield (parameters F(o) and F(m)) normalized to chlorophyll concentration was shown for deprived cells. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in PS II of sulfur-deprived cells. Thus, starved cells exhibited a lower deltapH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in reaction centers of PS II, as well as the transition of the photosynthetic apparatus primarily to state 2. However, these changes cannot cause the elevation of chlorophyll fluorescence in the cells under sulfur limitation. The phenomenon observed may be due to a partial dissociation of light-harvesting complexes from reaction centers of PS II and/or dysfunction of the dissipative cycle in PS II with cytochrome b559 as an intermediate.

[Photochemical activity of photosystem II and hydrogen photoproduction in sulfur-deprived Chlamydomonas reinhardtii mutants D1-R323D and D1-R323L].

The role of photosystem II in hydrogen photoproduction by Chlamydomonas reinhardtii cells was studied in mutants with modified D1-protein. In D1-R323D and D1-R323L mutants, the replacement of arginine by aspartate or leucine, respectively, resulted in the disruption of electron transport at the donor side of photosystem II. The rate of oxygen evolution in D1-R323D decreased twice as compared to the pseudo-wild type (pWT), and in D1-R323L no oxygen evolution was detected. The latter mutant was not capable of photoautotrophical growth. The dynamics of changes in oxygen content, the reduction of photosystem II active reaction centers (deltaF/F(1)m), and hydrogen production rate in pWT were found to be similar to the wild type if cultivated under sulfur deprivation in a closed bioreactor. The observed gradual decrease in the deltaF/F(1)m value turned to a sharp drop almost to zero followed by a partial recovery during which the production of hydrogen set in. The transition to the anaerobic phase in D1-R323D cultured in a sulfur-deprived medium occurred earlier than it happened in pWt under the same conditions. However, the partial recovery of photosystem II activity and hydrogen production started at a later time, and the rate of hydrogen production was low. The D1-R323L mutant incapable of oxygen evolution entered the rapidly anaerobiosis but produced no hydrogen. The kinetics of photoinduced redox transitions in P700 was similar in all investigated strains and was not affected by diuron addition. This implies that the mutants had a pool of reducers, which could donate electrons through the quinone pool or cytochrome to photosystem I. However, in D1-R323L mutant lacking the active photosystem II, this condition was not sufficient to support hydrogenase activity.

[Effect of methylmercury chloride on the primary photosynthetic activity of higher plants]. / Vliianie khlorida metilrtuti na pervichnye protsessy fotosinteza u vysshikh rastenii.

The effect of methylmercury chloride (MeHg) on the fluorescence characteristics of pea seedling leaves and thylakoids isolated from these leaves was studied by the pulse-amplitude-modulation (PAM) fluorometric method. In 3-4 days after the addition of MeHg (20 microM) to the nutritious solution, the maximal (Fv/Fm) and real (under steady state actinic light illumination) (deltaF/F'm) quantum photochemical yield of PS II decreased. The nonphotochemical fluorescence quenching coefficient in control (qN) decreased after its maximum value has been reached. In MeHg-treated samples, this decrease was not observed, possibly due to the disturbance of delta pH energy transducing processes in ATP synthase. This was confirmed by the results of experiments on isolated thylakoids. After MeHg (5 microM) treatment of thylakoids, the photophosphorylation rate and light-triggered Mg2+-dependent H+-ATPase activity were suppressed by 20-40%, depending on the duration of MeHg exposure. However, in experiments with isolated thylakoids, no decrease either in the electron transport rate or in the Fv/Fm ratio was observed. In total, the results obtained allow one to assume that MeHg at concentrations and time duration used directly damages the coupling complex. The PS II inactivation in leaves and algae cells may be a result of the oxidative stress processes.

[The photochemical activity of photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells depends on the redox state of the quinone pool during the transition to anaerobiosis]. / Fotokhimicheskaia aktivnost' fotosistemy II pri perekhode golodaiushchikh po sere kletok Chlamydomonas reinhardtii v anaérobnye usloviia zavisit ot redoks-sostoianiia pula khinonov.

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

[Regulation of the primary photosynthesis processes]. / Reguliatsiia pervichnykh protsessov fotosinteza.

The mechanisms of primary processes of photosynthesis and macromolecular conformational changes that control the efficiency of primary energy transformation in photosynthesis are discussed. Special attention is focused on the analysis of chlorophyll fluorescence as an integrated parameter indicative of the efficiency and dynamics of primary steps of photosynthesis. Sharp changes in environmental conditions and other unfavorable factors may lead to the distortions of the coupling between consecutive electron transfer steps. As a result, an excess of electrons and/or electronic excitation energy may form at some sites of the electron transport chain. This may lead to the generation of reactive oxygen species responsible for the subsequent oxidative stress. The results of the application of these data in the areas of biotechnology and ecology are demonstrated.

[Effect of mercuric chloride and methylmercury on photosynthetic activity of the diatom Thalassiosira weissflogii by a fluorescent method]. / Izuchenie deistviia khlorida rtuti i metilrtuti na fotosinteticheskuiu aktivnost' diatomovoi vodorosli Thalassiosira weissflogii fluorestsentnymi metodami.

It was shown by the pulse-amplitude modulation fluorescent method that, at a weak illumination (6 microE m-2.s-1), methylmercury at a concentration of 10(-6)-10(-7) M decreases the photochemical activity of the reaction centers of photosystem II in cells of microalgae Thalassiosira weissflogii after a prolonged lag phase. Cells resistant to methylmercury at these low concentrations were detected by the microfluorimetric method. Chloride mercury decreased the activity of photosystem II of the algae only when at higher concentrations. Both toxicants at a concentration of 10(-6) M decreased the rate of recovery of photoinduced damage of centers of photosystem II and led to an increase in the energization component of nonphotochemical fluorescence quenching. These results indicate that the complex of fluorescent methods can be used to monitor early changes in the photosynthetic apparatus of algae in response to the toxic action of heavy metals.

The dependence of algal H2 production on Photosystem II and O2 consumption activities in sulfur-deprived Chlamydomonas reinhardtii cells.

Chlamydomonas reinhardtii cultures, deprived of inorganic sulfur, undergo dramatic changes during adaptation to the nutrient stress [Biotechnol. Bioeng. 78 (2002) 731]. When the capacity for Photosystem II (PSII) O(2) evolution decreases below that of respiration, the culture becomes anaerobic [Plant Physiol. 122 (2000) 127]. We demonstrate that (a) the photochemical activity of PSII, monitored by in situ fluorescence, also decreases slowly during the aerobic period; (b) at the exact time of anaerobiosis, the remaining PSII activity is rapidly down regulated; and (c) electron transfer from PSII to PSI abruptly decreases at that point. Shortly thereafter, the PSII photochemical activity is partially restored, and H(2) production starts. Hydrogen production, which lasts for 3-4 days, is catalyzed by an anaerobically induced, reversible hydrogenase. While most of the reductants used directly for H(2) gas photoproduction come from water, the remaining electrons must come from endogenous substrate degradation through the NAD(P)H plastoquinone (PQ) oxido-reductase pathway. We propose that the induced hydrogenase activity provides a sink for electrons in the absence of other alternative pathways, and its operation allows the partial oxidation of intermediate photosynthetic carriers, including the PQ pool, between PSII and PSI. We conclude that the reduced state of this pool, which controls PSII photochemical activity, is one of the main factors regulating H(2) production under sulfur-deprived conditions. Residual O(2) evolved under these conditions is probably consumed mostly by the aerobic oxidation of storage products linked to mitochondrial respiratory processes involving both the cytochrome oxidase and the alternative oxidase. These functions maintain the intracellular anaerobic conditions required to keep the hydrogenase enzyme in the active, induced form.

[Effect of methylmercury on primary photosynthesis processes in green microalgae Chlamydomonas reinhardtii]. / Vliianie metilrtuti na pervichnye protsessy fotosinteza u zelenoi mikrovodorosli Chlamydomonas reinhardtii.

The sensitivity of green microalgae Chlamydomonas reinhardtii to methylmercury chloride (MeHg) and chloride mercury (HgCl2) was evaluated by measuring chlorophyll fluorescence parameters by the pulse-amplitude-modulation (PAM) fluorometry. It was shown that MeHg at concentrations above 1 microM decreased the Fv/Fm ratio, which characterizes the maximal efficiency of energy utilization in photosystem II. The degree of inhibition depended on the time of treatment and was always higher under illumination conditions (50 microE.m-2.s-1) than under dark conditions. A similar regularity was observed for the delta F/Fm' ratio, which characterizes the real efficiency of energy storage at the given intensity of the photosynthesis-exciting light. Incubation with 5 microM HgCl2 for 5 h did not affect both ratios. The decrease in Fm at constant F0 as well as changes in the fast fluorescence kinetics after MeHg treatment of algae cells indicated the damage on the donor side of photosystem II and the damage of the electron transfer from QA to QB. The reduction of photochemical fluorescence quenching (qN) under MeHg treatment is also evidence of the increase in the fraction of closed reaction centers (QA-). At the same time, increase in the steady-state level of P700 photooxidation indicated a disturbance of electron transfer between photosystems. The present study demonstrates that methylmercury treatment damaged the photosynthetic electron transfer chain at several sites. The inhibitory effect of methylmercury is much stronger than the effect of mercury chloride on photosynthetic processes.

[Experimental and theoretical study of processes of cyclical electron transport around photosystem I]. / Ekspeirmental'noe i teoreticheskoe issledovanie protsessov tsiklicheskgo élektronnogo transporta vokrug fotosistemy I.

The kinetics of photoinduced EPR I signals at different concentrations of ferredoxin was studied on isolated pea chloroplasts. A kinetic model of ferredoxin-dependent electron transport around photosystem I was suggested. A multiparticle model was constructed, which makes it possible to "directly" model the processes of electron transfer in multiprotein complexes and limited diffusion in different compartments of the system (stroma, lumen, and intermembrane space). A comparison of the kinetic and "direct" models revealed an important role of spatial organization of the system in the kinetics of redox turnover of P700.

[Characteristics of the leaf photosynthetic machinery in broad bean grown in zinc chloride aqueous solutions]. / Osobennosti fotosinteticheskogo apparata list'ev bobov, vyrashchennykh na vodnykh rastvorakh khlorida tsinka.

The structural and functional characteristics of bean leaves (the content of chlorophyll, the rate of oxygen production, the slow fluorescence induction, and light-induced changes in the EPR signal I from oxidized reaction centers P700+) were investigated to obtain insight into the mechanism of influence of zinc chloride on the photosynthetic apparatus. Seedlings were grown on hydroponic medium containing ZnCl2 at concentrations from 10(-7) to 10(-3) M. At low concentrations of ZnCl2, a decrease in the content of chlorophyll per one unit of leaf mass was observed, while the rate of oxygen production per chlorophyll was increased. High concentrations of ZnCl2 in the hydroponic medium caused the slowed down the plant development and inhibited the light-induced production of oxygen. The changes in biophysical characteristics of leaves the parameter FM/FT of the slow fluorescence induction, and kinetics of redox transients of P700 induced by ZnCl2 were of similar character and correlated with the changes in photosynthetic activity. The data obtained demonstrate that structural and functional changes in the photosynthetic apparatus induced by the variations of growth conditions have adaptive character.

Effect of nitrite on primary photosynthetic processes in isolated chloroplasts of wheat grown under various conditions of nitrogen supply.

In wheat chloroplasts, NO2- (5 mM) inhibited non-cyclic phosphorylation coupled to the linear electron flow through both photosystems but stimulated cyclic phosphorylation with phenazine methosulfate (plus diuron) and electron flux from an artificial electron donor through PSI and methylviologen to oxygen. During light energization of chloroplasts, NO2- increased the F740/F695 ratio in the low-temperature fluorescence spectra, thus suggesting that the energy of absorbed quanta is redistributed in favor of PSI. Nitrite also stimulated the activity of Mg2+-dependent H+-ATPase. Changes in the slow component of the induction curve of delayed fluorescence also suggest that NO2- affects energy transformation processes that are coupled to electron transport in the chloroplasts. Nitrite had no effect on these functional characteristics of thylakoids and chloroplasts isolated from plants grown under nitrogen deficiency in the medium.