G. P. Kukarskikh

Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity.

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

Hydrogen photoproduction in green algae Chlamydomonas reinhardtii under magnesium deprivation

The effect of Mg-deprivation on green algae Chlamydomonas reinhardtii was studied. It resulted in the decrease of photosynthetic activity, increased respiration and accumulation of starch. After 35 hours anaerobic conditions were established and sustained H2 evolution (>7 days) was detected.

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700+ in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.

Performance index in assessing the physiological state of trees in urban ecosystems

Based on PAM and PEA measurements of fluorescence of bark chloroplasts, we have compared the information capacity of the methods for assessing the physiological state of Tilia cordata Mill. by the maximal quantum efficiency of PS II photochemistry (Fv/Fm) and by the performance index (PI). The measurements were made on annual shoots of linden trees growing in different environs. It was shown that the chlorophyll content in the bark of shoots growing near a busy urban street was twice less than in controls growing out of town. For the trees in the unfavorable environment, a small decrease in (Fv/Fm) was registered, and there was a significant statistical scatter in these values as compared with controls. The PI and its constituent parameters calculated from fluorescence induction curves (PEA method) are more informative and allow recognizing changes in the primary energy conversion processes in PS II when they are still small. Thus, PI can be used as a sensitive, robust, and rapid test to evaluate the physiological state of trees and other plant objects even under minor environmental changes.

Examination of chlorophyll fluorescence in sulfur-deprived cells of Chlamydomonas reinhardtii

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F0 and Fm) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F0 and Fm) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b559.

Comparative analyses of H2 photoproduction in magnesium‐ and sulfur‐starved Chlamydomonas reinhardtii cultures

Magnesium (Mg)-deprived Chlamydomonas reinhardtii cells are capable to sustain hydrogen (H2 ) photoproduction at relatively high photosystem II (PSII) activity levels for an extended time period as compared with sulfur (S)-deprived cells. Herein, we present a comparative study of H2 photoproduction induced by Mg and S shortage to unravel the specific rearrangements of the photosynthetic machinery and cell metabolism occurring under the two deprivation protocols. The exhaustive analysis of photosynthetic activity and regulatory pathways, respiration and starch metabolism revealed the specific rearrangements of the photosynthetic machinery and cellular metabolism, which occur under the two deprivation conditions. The obtained results allowed us to conclude that the expanded time period of H2 production upon Mg-deprivation is due to the less harmful effects that Mg-depletion has on viability and metabolic performance of the cells. Unlike S-deprivation, the photosynthetic light and dark reactions in Mg-deprived cells remained active over the whole H2 production period. However, the elevated PSII activity in Mg-deprived cells was counteracted by the operation of pathways for O2 consumption that maintain anaerobic conditions in the presence of active water splitting.

Effect of dibromothymoquinone on Chlorophyll a Fluorescence in Chlamydomonas reinhardtii cells incubated in complete or sulfur-depleted medium

The effect of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. Dibromothymoquinone was shown to affect differently control cells incubated in complete medium and S-starved cells. The fluorescence yield in the control suspension considerably increased in the presence of the inhibitor. Presumably, this can be due to inactivation of protein kinase, as a result of which part of light-harvesting complex II that could have diffused from the stacking zone of the membrane into the lamellar zone towards photosystem I remains close to photosystem II. In S-starved cells, whose photosynthetic apparatus is in state 2, the fluorescence level declines in the presence of dibromothymoquinone. The JIP testing of induction curves (O-J-I-P fluorescence transient) suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to the control, while in the starved cells it acts predominantly as an electron acceptor.

Leaf Morphology, Pigment Complex, and Productivity in Wild-Type and afila Pea Genotypes

The effects of genetically determined changes in leaf morphology on the characteristics of growth, pigment complex, and productivity were studied in pea plants (Pisum sativum L.). The homeotic afila(af ) mutation, which transformed leaflets into tendrils, decreased the leaf area and the chlorophyll (Chl) content per plant (CCP) in the af/af plants 1.5-fold as compared to the wild type (Af/Af). The loss of leaflets in the af/af plants was partly recompensed by expansion of the tendrils and stipules and by extra accumulation of Chl (a + b). The mutation did not affect Chl (a + b) that fell to the share of light-harvesting complexes (LHC) and the ratio of Chl a/b (representing the relative distribution of chlorophylls between LHC and the reaction centers); neither it affected the quantum efficiency of the primary charge separation (Fv/Fm). The diminished assimilating area (AA) in the af/af plants at the preflowering period did not reduce the final biomass and grain yield. The measurement of the area shaded by plants in the glasshouse experiments and the direct assessment of the vertical profile of solar radiation in the field stand canopies demonstrated that this phenomenon was in particular related to the fact that, in the af/af plants, the solar radiation was available to the apical and subapical leaves (as in the wild-type plants) and also to the lower metamers. As a result, the actively functioning AA expanded, and the photoassimilating potential of the af/af plants was enhanced. Our data presume the direct relationship between plant production and CCP.

A Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactiors

The development of high-performance photobioreactors equipped with automatic systems for non-invasive real-time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light-induced fluorescence rise and the dark relaxation of the flash-induced fluorescence yield (Qa- - re-oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas-tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long-term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress-sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real-time monitoring of algal photosynthesis in photobioreactors.

Ferredoxin-NADP reductase is involved in the ferredoxin-dependent cyclic electron transport in isolated thylakoids.

Mechanisms of the ferredoxin (Fd)-dependent cyclic electron transport (CET) mediated by Photosystem (PS) I in chloroplasts of higher plants are still a subject of considerable discussion [1–3]. The mechanism of electron flow distribution between noncyclic (from reduced Fd through a linear chain of carriers to NADP + ) and cyclic (from reduced Fd back to the donor side of PS I) is the key problem of electron transport regulation in thylakoid membranes. This problem cannot be solved unless the CET components are identified. Inhibitory analysis and studies on redox reactions of cytochromes showed that plastoquinone and the cytochrome b 6 / f complex are components of CET [2, 4, 5]. However, the mechanisms of plastoquinone reduction mediated by reduced Fd remain obscure. The Fd– plastoquinone reductase reaction (FQRR) is an Fddependent component of the CET that does not coincide with the linear electron flow through PS I. None of isolated and identified chloroplast proteins (e.g., protein responsible for specific sensitivity of CET to antimycin A) have been found to be related to FQRR so far [4, 6]. The possible contribution of a specific form of cytochrome b 559 to this process was suggested in [5]. The existence of an alternative antimycin-insensitive pathway of plastoquinone reduction by electrons from the acceptor side of PS I was also assumed [7, 8]. Although the problem of the possible role of ferredoxin–NADP reductase (FNR) in FQRR was addressed by many researchers [6, 7], the available experimental data are insufficient to solve it, and the data cannot be interpreted in terms of a consistent general scheme of this process. In this work, we studied the effects of some inhibitors of electron transport, FNR inhibitors, and pyridine nucleotides on the kinetics of dark reduction of photooxidized ê700 + . The results of the study supported the suggestion that plastoquinone and the cytochrome complex are components of CET. It was also clearly demonstrated that FNR is involved in CET.