Structural and Functional Aspects of Electron Transport Thermoregulation and ATP Synthesis in Chloroplasts.

The review is focused on analysis of the mechanisms of temperature-dependent regulation of electron transport and ATP synthesis in chloroplasts of higher plants. Importance of photosynthesis thermoregulation is determined by the fact that plants are ectothermic organisms, whose own temperature depends on the ambient temperature. The review discusses the effects of temperature on the following processes in thylakoid membranes: (i) photosystem 2 activity and plastoquinone reduction; (ii) electron transfer from plastoquinol (via the cytochrome b6f complex and plastocyanin) to photosystem 1; (iii) transmembrane proton transfer; and (iv) ATP synthesis. The data on the relationship between the functional properties of chloroplasts (photosynthetic transfer of electrons and protons, functioning of ATP synthase) and structural characteristics of membrane lipids (fluidity) obtained by electron paramagnetic resonance studies are presented.

Temperature-dependent regulation of electron transport and ATP synthesis in chloroplasts in vitro and in silico.

The significance of temperature-dependent regulation of photosynthetic apparatus (PSA) is determined by the fact that plant temperature changes with environmental temperature. In this work, we present a brief overview of temperature-dependent regulation of photosynthetic processes in class B chloroplasts (thylakoids) and analyze these processes using a computer model that takes into account the key stages of electron and proton transport coupled to ATP synthesis. The rate constants of partial reactions were parametrized on the basis of experimental temperature dependences of partial photosynthetic processes: (1) photosystem II (PSII) turnover and plastoquinone (PQ) reduction, (2) the plastoquinol (PQH2) oxidation by the cytochrome (Cyt) b6f complex, (3) the ATP synthase activity, and (4) the proton leak from the thylakoid lumen. We consider that PQH2 oxidation is the rate-limiting step in the intersystem electron transport. The parametrization of the rate constants of these processes is based on earlier experimental data demonstrating strong correlations between the functional and structural properties of thylakoid membranes that were probed with the lipid-soluble spin labels embedded into the membranes. Within the framework of our model, we could adequately describe a number of experimental temperature dependences of photosynthetic reactions in thylakoids. Computer modeling of electron and proton transport coupled to ATP synthesis supports the notion that PQH2 oxidation by the Cyt b6f complex and proton pumping into the lumen are the basic temperature-dependent processes that determine the overall electron flux from PSII to molecular oxygen and the net ATP synthesis upon variations of temperature. The model describes two branches of the temperature dependence of the post-illumination reduction of [Formula: see text] characterized by different activation energies (about 60 and ≤ 3.5 kJ mol-1). The model predicts the bell-like temperature dependence of ATP formation, which arises from the balance of several factors: (1) the thermo-induced acceleration of electron transport through the Cyt b6f complex, (2) deactivation of PSII photochemistry at sufficiently high temperatures, and (3) acceleration of the passive proton outflow from the thylakoid lumen bypassing the ATP synthase complex. The model describes the temperature dependence of experimentally measured parameter P/2e, determined as the ratio between the rates of ATP synthesis and pseudocyclic electron transport (H2O → PSII → PSI → O2).

Photosynthetic Electron and Proton Transport in Chloroplasts: EPR Study of ΔpH Generation, an Overview.

This is a brief overview focused on the electron paramagnetic resonance applications to the study of the proton transport processes in chloroplasts. After brief description of structural and functional organization of the chloroplast electron transport chain, our attention is focused on the measurements of trans-thylakoid pH difference (ΔpH) with pH-sensitive spin-probes. The use of spin-probes is based either (i) on measuring the ΔpH-partitioning of spin-probes between the thylakoid lumen and external volume, or (ii) on monitoring changes in the electron paramagnetic resonance spectra of pH-sensitive nitroxide radicals located in the lumen. Along with the use of spin-probes, the intra-thylakoid pH (pHin) can be determined by the "kinetic" method, which relies on the fact that the rate-limiting step in the chain of photosynthetic electron transfer (plastoquinol oxidation by the cytochrome b 6 f complex) is controlled by pHin. The results of ΔpH determinations in chloroplasts based on the use of pH-sensitive spin-probes and measurements of post-illumination reduction of photoreaction centers of Photosystem I are discussed in the context of the problem of energy coupling in laterally heterogeneous lamellar system of chloroplasts.

Acclimation of shade-tolerant and light-resistant Tradescantia species to growth light: chlorophyll a fluorescence, electron transport, and xanthophyll content.

In this study, we have compared the photosynthetic characteristics of two contrasting species of Tradescantia plants, T. fluminensis (shade-tolerant species), and T. sillamontana (light-resistant species), grown under the low light (LL, 50-125 µmol photons m-2 s-1) or high light (HL, 875-1000 µmol photons m-2 s-1) conditions during their entire growth period. For monitoring the functional state of photosynthetic apparatus (PSA), we measured chlorophyll (Chl) a emission fluorescence spectra and kinetics of light-induced changes in the heights of fluorescence peaks at 685 and 740 nm (F 685 and F 740). We also compared the light-induced oxidation of P700 and assayed the composition of carotenoids in Tradescantia leaves grown under the LL and HL conditions. The analyses of slow induction of Chl a fluorescence (SIF) uncovered different traits in the LL- and HL-grown plants of ecologically contrasting Tradescantia species, which may have potential ecophysiological significance with respect to their tolerance to HL stress. The fluorometry and EPR studies of induction events in chloroplasts in situ demonstrated that acclimation of both Tradescantia species to HL conditions promoted faster responses of their PSA as compared to LL-grown plants. Acclimation of both species to HL also caused marked changes in the leaf anatomy and carotenoid composition (an increase in Violaxanthin + Antheraxantin + Zeaxanthin and Lutein pools), suggesting enhanced photoprotective capacity of the carotenoids in the plants grown in nature under high irradiance. Collectively, the results of the present work suggest that the mechanisms of long-term PSA photoprotection in Tradescantia are based predominantly on the light-induced remodeling of pigment-protein complexes in chloroplasts.

Computer modeling of electron and proton transport in chloroplasts.

Photosynthesis is one of the most important biological processes in biosphere, which provides production of organic substances from atmospheric CO2 and water at expense of solar energy. In this review, we contemplate computer models of oxygenic photosynthesis in the context of feedback regulation of photosynthetic electron transport in chloroplasts, the energy-transducing organelles of the plant cell. We start with a brief overview of electron and proton transport processes in chloroplasts coupled to ATP synthesis and consider basic regulatory mechanisms of oxygenic photosynthesis. General approaches to computer simulation of photosynthetic processes are considered, including the random walk models of plastoquinone diffusion in thylakoid membranes and deterministic approach to modeling electron transport in chloroplasts based on the mass action law. Then we focus on a kinetic model of oxygenic photosynthesis that includes key stages of the linear electron transport, alternative pathways of electron transfer around photosystem I (PSI), transmembrane proton transport and ATP synthesis in chloroplasts. This model includes different regulatory processes: pH-dependent control of the intersystem electron transport, down-regulation of photosystem II (PSII) activity (non-photochemical quenching), the light-induced activation of the Bassham-Benson-Calvin (BBC) cycle. The model correctly describes pH-dependent feedback control of electron transport in chloroplasts and adequately reproduces a variety of experimental data on induction events observed under different experimental conditions in intact chloroplasts (variations of CO2 and O2 concentrations in atmosphere), including a complex kinetics of P700 (primary electron donor in PSI) photooxidation, CO2 consumption in the BBC cycle, and photorespiration. Finally, we describe diffusion-controlled photosynthetic processes in chloroplasts within the framework of the model that takes into account complex architecture of chloroplasts and lateral heterogeneity of lamellar system of thylakoids. The lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids have been calculated under different metabolic conditions. Analyzing topological aspects of diffusion-controlled stages of electron and proton transport in chloroplasts, we conclude that along with the NPQ mechanism of attenuation of PSII activity and deceleration of PQH2 oxidation by the cytochrome b6f complex caused by the lumen acidification, the intersystem electron transport may be down-regulated due to the light-induced alkalization of the narrow partition between adjacent thylakoids of grana. The computer models of electron and proton transport described in this article may be integrated as appropriate modules into a comprehensive model of oxygenic photosynthesis.

pH-dependent regulation of electron transport and ATP synthesis in chloroplasts.

This review is focused on pH-dependent mechanisms of regulation of photosynthetic electron transport and ATP synthesis in chloroplasts. The light-induced acidification of the thylakoid lumen is known to decelerate the plastoquinol oxidation by the cytochrome b 6 f complex, thus impeding the electron flow between photosystem II and photosystem I. Acidification of the lumen also triggers the dissipation of excess energy in the light-harvesting antenna of photosystem II, thereby protecting the photosynthetic apparatus against a solar stress. After brief description of structural and functional organization of the chloroplast electron transport chain, our attention is focused on the nature of the rate-limiting step of electron transfer between photosystem II and photosystem I. In the context of pH-dependent mechanism of photosynthetic control in chloroplasts, the mechanisms of plastoquinol oxidation by the cytochrome b 6 f complex have been considered. The light-induced alkalization of stroma is another factor of pH-dependent regulation of electron transport in chloroplasts. Alkalization of stroma induces activation of the Bassham-Benson-Calvin cycle reactions, thereby promoting efflux of electrons from photosystem I to NADP(+). The mechanisms of the light-induced activation of ATP synthase are briefly considered.

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.

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts.

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.

Determination of a transmembrane pH difference in chloroplasts with a spin label tempamine.

We present a method for measuring the transmembrane pH difference (deltapH=pHin-pHout) in chloroplasts with a spin label TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl) accumulating inside the thylakoids in response to generation of deltapH. Experiments with chloroplasts suspended in the media of different osmolarity demonstrated that most of TEMPAMINE (TA) molecules taken up by chloroplasts were localized in the bulk of the thylakoid lumen. The DeltapH value was determined from the relationship deltapH=lg([H+]in/[H+]out) approximately equal to lg(Cin/Cout), where Cin and Cout are the concentrations of TA inside and outside the thylakoids, respectively. To quantify the internal concentration Cin, we used the threshold nature of the concentration-dependent broadening of the EPR signal from TA. It was demonstrated that spin-exchange interactions between TA molecules caused an observable broadening of the signal only when the concentration of TA exceeded the threshold level, [TA]theta approximately 2.0-2.2mM. The concentration dependencies of the signal parameters (the peak-to-peak amplitude, App, and the linewidth, deltaHpp) were described within a model of the non-homogeneous broadening of an unresolved hyperfine multiplet from the protons of TA molecule. If the concentration of TA inside the thylakoids went beyond the threshold level, the spin-exchange broadening of the EPR signal was accompanied by a reversible decrease in the signal height (parameter deltaA). By measuring the signal behavior at different levels of microwave power, we were able to discriminate between the line broadening effects caused by concentrating TA molecules inside the thylakoids or the light-induced changes in the concentration of oxygen. We developed a general algorithm for determination of the deltapH value and the internal volume of thylakoids, Vin, from the non-linear dependence of parameter deltaA on the concentration C0 of TA in a chloroplast suspension. Advantages of this method are: (i) it avoids the use of a broadening agent; (ii) it allows the internal volume of thylakoids to be evaluated; and (iii) the concentrations of TA used to measure the deltapH are below the range of concentrations that could cause the uncoupling electron transport to ATP synthesis in chloroplasts. Results of our measurements are consistent with the literature data on deltapH determinations by other methods.

Water content and its intracellular distribution in intact and saline perfused rat hearts revisited.

Precise estimation of cellular water content is a necessary basis for quantitative studies of metabolic control in the heart; however, marked discrepancies in water spaces of heart tissue are found in the literature. Reasons for this wide diversity are analyzed, and the conclusion is that the most probable value of total intracellular water content is 615 ml H(2)O/kg of wet mass (wm) and intracellular content of dry substance is 189 g/kg wm in intact in vivo rat heart. An extracellular water of 174 ml per kg wm and 22 g of dry mass per kg wm in vascular and interstitium spaces account for the rest of the tissue mass. These values can be directly related to normoosmotic saline perfused hydrated hearts, characterized by water accumulation in the extracellular spaces. Due to essentially intact heart cells, the experimentally determined dry mass, water and metabolite contents of these hydrated hearts can be extrapolated to the original morphological configuration of an intact heart muscle before the onset of edema. Such an 'extrapolated' heart is defined as a standardized perfused heart (SPH). SPH is the heart in its original morphological configuration, characterized by cell density and cellular water contents of the intact heart, but with perfusate in the extracellular spaces. The total cellular water is distributed in the cell compartments of SPH and intact hearts according to volumes of particular compartments and density of their dry mass. The volumes of bulk water phases in different organelles, accessible to diffusion of low molecular metabolites, were obtained after corrections for the fraction of 'bound' water of 0.3 g per g of compartmental dry mass content. The diffusible water spaces are proposed to be 321, 55, 153, 21 and 8 ml/kg wm for myofibrils, sarcoplasm, mitochondria, sarcoplasmic reticulum and nuclei, respectively. The SPH model allows direct comparison of metabolic data for intact and perfused hearts. We used this model to analyze the penetration of extracellular marker into cells of intact and hydrated perfused rat hearts.