Depletion of stromal Pi induces high 'energy-dependent' antenna exciton quenching (qE) by decreasing proton conductivity at CFO-CF1 ATP synthase

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P_i), which results in down-regulation of light capture via the exciton quenching (q_E) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P_i depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF_O-CF₁ ATP synthase to protons ( g _H⁺) is decreased, retarding proton efflux from the lumen. Sequestering of P_i by mannose infiltration increased sensitivities of q_E and pmf to LEF. The effects were attributable to decreases in g _H⁺, but not to CEF1 and were largely reversed by subsequent P_i feeding. Rapid recovery of g _H⁺ in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson–Calvin cycle intermediates or lowering of stromal P_i below its K _Mat the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.     

Global spectroscopic analysis to study the regulation of the photosynthetic proton motive force: A critical reappraisal

In natural variable environments, plants rapidly adjust photosynthesis for optimum balance between photochemistry and photoprotection. These adjustments mainly occur via changes in their proton motive force (pmf). Recent studies based on time resolved analysis of the Electro Chromic Signal (ECS) bandshift of photosynthetic pigments in the model plant Arabidopsis thaliana have suggested an active role of ion fluxes across the thylakoid membranes in the regulation of the pmf. Among the different channels and transporters possibly involved in this phenomenon, we previously identified the TPK3 potassium channel. Plants silenced for TPK3 expression displayed light stress signatures, with reduced Non Photochemical Quenching (NPQ) capacity and sustained anthocyanin accumulation, even at moderate intensities. In this work we re-examined the role of this protein in pmf regulation, starting from the observation that both TPK3 knock-down (TPK3 KD) and WT plants display enhanced anthocyanin accumulation in the light under certain growth conditions, especially in old leaves. We thus compared the pmf features of young “green” (without anthocyanins) and old “red” (with anthocyanins) leaves in both genotypes using a global fit analysis of the ECS. We found that the differences in the ECS profile measured between the two genotypes reflect not only differences in TPK3 expression level, but also a modified photosynthetic activity of stressed red leaves, which are present in a larger amounts in the TPK3 KD plants.     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

温度升高对高光强环境下蛋白核小球藻(Chlolorella pyrenoidosa)光能利用和生长的阻抑效应

以蛋白核小球藻（Cholorella pyrenoidosa）为实验材料,研究了温度变化对不同光照水平下蛋白核小球藻的光能利用和生长的影响,以明确光照强度对微藻的光能利用和生长的影响是否因温度不同而发生变化。实验中共设置了3个光照强度水平（50,150,300μmol·m^-2s^-1）和2个温度水平（15℃,25℃）。实验结果表明,不同光照水平下小球藻叶绿素荧光的非光化学淬灭（NPQ）大小与温度有关,光照强度为150,300μmol·m^-2s^-1时,温度升高使小球藻叶绿素荧光NPQ提高,并且光照强度越高小球藻叶绿素荧光NPQ增大越多,50μmol·m^-2s^-1光照强度下温度升高对叶绿素荧光NPQ没有影响。实验发现,25℃培养温度下小球藻的光合电子传递速率（ETR）随光照强度增高而上升的速率要低于15℃时小球藻ETR上升的速率;随着光照强度增高,温度升高使小球藻ETR降低程度增大。实验结果还表明,15℃时小球藻培养液叶绿素a浓度随光照强度升高而增高,300μmol·m^-2s^-1培养光强下具有最高的叶绿素a浓度。但在25℃时,光照强度升高叶绿素a浓度并不一定增高,300μmol·m^-2s^-1光照强度下的叶绿素a浓度比150μmol·m^-2s^-1光照强度下要低。本研究表明,温度升高增大了高光照水平下蛋白核小球藻对光能的热耗散,使光照增强对小球藻生长的促进作用减弱。由于温度升高对小球藻光能利用和生长的阻抑作用,小球藻生长的适宜光照水平因温度升高而降低。     

Citrulline and DRIP-1 Protein (ArgE Homologue) in Drought Tolerance of Wild Watermelon

Drought-affected plants experience more than just desiccationof their organs due to water deficit. Plants transpire 1000times more molecules of water than of CO₂ fixed by photosynthesisin full sunlight. One effect of transpiration is to cool theleaves. Accordingly, drought brings about such multi-stressesas high temperatures, excess photoradiation and other factorsthat affect plant viability. Wild watermelon serves as a suitablemodel system to study drought responses of C₃ plants, sincethis plant survives drought by maintaining its water contentwithout any wilting of leaves or desiccation even under severedrought conditions. Under drought conditions in the presenceof strong light, wild watermelon accumulates high concentrationsof citrulline, glutamate and arginine in its leaves. The accumulationof citrulline and arginine may be related to the induction ofDRIP-1, a homologue of ArgE in Escherichia coli, where it functionsto incorporate the carbon skeleton of glutamate into the ureacycle. Immunogold electron microscopy reveals the enzyme tobe confined exclusively to the cytosol. DRIP-1 is also inducedby treating wild watermelon with 150 mM NaCl, but is not inducedfollowing treatment with 100 µM abscisic acid. The salttreatment causes the accumulation of -aminobutyrate, glutamineand alanine, in addition to a smaller amount of citrulline.Citrulline may function as a potent hydroxyl radical scavenger.     

Functional diversity of photosynthesis during drought in a model tropical rainforest – the contributions of leaf area, photosynthetic electron transport and stomatal conductance to reduction in net ecosystem carbon exchange

The tropical rainforest mesocosm within the Biosphere 2 Laboratory, a model system of some 110 species developed over 12 years under controlled environmental conditions, has been subjected to a series of comparable drought experiments during 2000–2002. In each study, the mesocosm was subjected to a 4–6 week drought, with well‐defined rainfall events before and after the treatment. Ecosystem CO₂ uptake rate (A_eco) declined 32% in response to the drought, with changes occurring within days and being reversible within weeks, even though the deeper soil layers did not become significantly drier and leaf‐level water status of most large trees was not greatly affected. The reduced A_eco during the drought reflected both morphological and physiological responses. It is estimated that the drought‐induced 32% reduction of A_eco has three principal components: (1) leaf fall increased two‐fold whereas leaf expansion growth of some canopy dominants declined to 60%, leading to a 10% decrease in foliage coverage of the canopy. This might be the main reason for the persistent reduction of A_eco after rewatering. (2) The maximum photosynthetic electron transport rate at high light intensities in remaining leaves was reduced to 71% for three of the four species measured, even though no chronic photo‐inhibition occurred. (3) Stomata closed, leading to a reduced ecosystem water conductance to water vapour (33% of pre‐drought values), which not only reduced ecosystem carbon uptake rate, but may also have implications for water and energy budgets of tropical ecosystems. Additionally, individual rainforest trees responded differently, expressing different levels of stress and stress avoiding mechanisms. This functional diversity renders the individual response heterogeneous and has fundamental implications to scale leaf level responses to ecosystem dynamics.     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

Effects of long-term exposure to elevated CO2 and N fertilization on the development of photosynthetic capacity and biomass accumulation in Quercus suber L.

The effects of long‐term (4 year) CO₂ enrichment (70 Pa versus 35 Pa) and nitrogen nutrition (8 mm versus 1 mm NO₃^–) on biomass accumulation and the development of photosynthetic capacity in leaves of cork oak (Quercus suber L., a Mediterranean evergreen tree) were studied. The evolution of photosynthetic parameters with leaf development was estimated by fitting the biochemical model of Farquhar et al. (Planta 149, 78–90, 1980) with modifications by Sharkey (Botanical Review 78, 71–75, 1985) to A–C_i response curves. CO₂ enrichment had a small reduction effect on the development of the maximum CO₂ fixation capacity by Rubisco (V_Cmax), and no effect over maximum electron transport capacity (J_max), day‐time respiration (R_d) and Triose‐P utilization (TPU). However, there was a statistically significant effect of N fertilization and the interaction CO₂ × N over the evolution of V_Cmax, J_max and TPU. Relative stomatal limitation (estimated from A–C_i curves) was higher (+20%) for plants grown under ambient CO₂ than for plants grown under elevated CO₂. There was a significant effect of CO₂ and N fertilization over total biomass accumulation as well as leaf area. Plants grown at elevated CO₂ had 27% more biomass than plants grown at ambient CO₂ when given high N. However, for plants grown under low N there was no significant effect of CO₂ enrichment on biomass accumulation. Plants grown under low N also had significantly higher root : shoot ratios whereas there were no differences between CO₂ treatments. The larger biomass accumulation of Q. suber under elevated CO₂ is attributable to a higher availability of CO₂ coupled to a larger leaf area, with no significant decrease in photosynthetic capacity under CO₂ enrichment and elevated N fertilization. For low N fertilization, the effects of CO₂ enrichment over leaf area and biomass accumulation are lost, suggesting that in native ecosystems with low N availability, the effects of CO₂ enrichment may be insignificant.     

PROTON GRADIENT REGULATION 5 contributes to ferredoxin-dependent cyclic phosphorylation in ruptured chloroplasts

Antimycin A-sensitive cyclic electron flow (CEF) was discovered as cyclic phosphorylation by Arnon et al. (1954). Because of its sensitivity to antimycin A, PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-like Photosynthetic Phenotype 1 (PGRL1)-dependent CEF has been considered identical to the CEF of Arnon et al. However, this conclusion still needs additional supportive evidence, mainly because of the absence of definitive methods of evaluating CEF activity. In this study, we revisited the classical method of monitoring cyclic phosphorylation in ruptured chloroplasts to characterize two Arabidopsis mutants: pgr5, which is defective in antimycin A-sensitive CEF, and chlororespiratory reduction 2-1 (crr2-1), which is defective in chloroplast NDH-dependent CEF. We observed a significant reduction in CEF-dependent pmf formation and consequently ATP synthesis in the pgr5 mutant, although LEF-dependent pmf formation and ATP synthesis were not impaired at photosynthetic photon flux densities below 130?μmol?m^?2?s^?1. In contrast, the contribution of chloroplast NDH complex to pmf formation and ATP synthesis was not significant. Antimycin A partially inhibited CEF-dependent pmf formation, although there may be further inhibition sites. Unlike in the observation in leaves, the proton conductivity of ATP synthase, monitored as g_H⁺, was not enhanced in ruptured chloroplasts of the pgr5 mutant.     

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO₂. It was found that at the first maximum, light-and CO₂-saturated photosynthesis can be much faster than steady-state photosynthesis. Both Q_A in the reaction center of PS II and P₇₀₀ in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P₇₀₀ oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force F_A, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(P_i), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO₂, the assimilatory force F_A declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P₇₀₀. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.Abbreviations PGA 3-phosphoglycerate - DHAP dihydroxyacetone phosphate - P₇₀₀ electron-donor pigment in the reaction enter of PS I - Q_A quinone acceptor in the reaction center of PS II This work received support from the Estonian Academy of Sciences, the Bavarian Ministry of Science and Art and the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful for criticism by D.A. Walker, Robert Hill Institute, University of Sheffield, U.K. and by Mark Stitt, Institute of Botany, University of Heidelberg, FRG.     

The regulatory subunit ε in Escherichia coli FOF1-ATP synthase

F-type ATP synthases are extraordinary multisubunit proteins that operate as nanomotors. The Escherichia coli (E. coli) enzyme uses the proton motive force (pmf) across the bacterial plasma membrane to drive rotation of the central rotor subunits within a stator subunit complex. Through this mechanical rotation, the rotor coordinates three nucleotide binding sites that sequentially catalyze the synthesis of ATP. Moreover, the enzyme can hydrolyze ATP to turn the rotor in the opposite direction and generate pmf. The direction of net catalysis, i.e. synthesis or hydrolysis of ATP, depends on the cell's bioenergetic conditions. Different control mechanisms have been found for ATP synthases in mitochondria, chloroplasts and bacteria. This review discusses the auto-inhibitory behavior of subunit ε found in F_OF₁-ATP synthases of many bacteria. We focus on E. coli F_OF₁-ATP synthase, with insights into the regulatory mechanism of subunit ε arising from structural and biochemical studies complemented by single-molecule microscopy experiments.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores flash-induced cytochrome b reduction

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an α-band maximum at 562 nm in the presence of antimycin.The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood.Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation.These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.     

Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol

Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820 nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5 min) mild (40 °C) or strong (44 °C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F_V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg²⁺. It is also demonstrated that the increase in F_V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases

Recent time-resolved optical and electrometric experiments have provided a sequence of events for the proton-translocating mechanism of cytochrome c oxidase. These data also set limits for the mechanistic, kinetic, and thermodynamic parameters of the proton pump, which are analysed here in some detail. The analysis yields limit values for the pK of the “pump site”, its modulation during the proton-pumping process, and suggests its identity in the structure. Special emphasis is made on side-reactions that may short-circuit the pump, and the means by which these may be avoided. We will also discuss the most prominent proton pumping mechanisms proposed to date in relation to these data.     

Functional and topological aspects of pH-dependent regulation of electron and proton transport in chloroplasts in silico

In this work, we summarize results of computer simulation of electron and proton transport processes coupled to ATP synthesis in chloroplasts performed within the frames of a mathematical model developed as a system of differential equations for concentrations of electron carriers and hydrogen ion inside and outside the granal and stromal thylakoids. The model takes into account topological peculiarities and lateral heterogeneity of the chloroplast lamellar system. This allowed us to analyze the influence of restricted diffusion of protons inside small compartments of a chloroplast (e.g., in the narrow inter-thylakoid gap) on electron transport processes. The model adequately describes two modes of pH-dependent feedback control of electron transport associated with: (i) the acidification of the thylakoid lumen, which causes the slowing down of plastoquinol oxidation and stimulates an increase in dissipation of excess energy in PS2, and (ii) the alkalization of stroma, inducing the activation of the BBC (Bassham-Benson-Calvin) cycle and intensified consumption of ATP and NADPH. The influence of ATP on electron transport is mediated by modulation of the thylakoid membrane conductivity to protons through the ATP synthase complexes. We also analyze the contribution of alternative electron transport pathways to the maintenance of optimal balance between the energy donating and energy consuming stages of the light-induced photosynthetic processes.     

Laurdan fluorescence spectroscopy in the thylakoid bilayer: The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach (Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 °C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.