ATP Synthesis In Chloroplasts Research Articles

Real-time response counterattack strategy of tolerant microalgae Chlorella vulgaris MBFJNU-1 in original swine wastewater and free ammonia

This work was the first time to systematically clarify the potential tolerance mechanism of an indigenous Chlorella vulgaris MBFJNU-1 towards the free ammonia (FA) during the original swine wastewater (OSW) treatment by transcriptome analysis using C. vulgaris UETX395 as the control group. The obtained results showed that C. vulgaris MBFJNU-1 was found to be more resistant to the high levels of FA (115 mg/L) and OSW in comparison to C. vulgaris UETX395 (38 mg/L). Moreover, the transcriptomic results stated that some key pathways from arginine biosynthesis, electron generation and transmission, ATP synthesis in chloroplasts, and glutathione synthesis of C. vulgaris MBFJNU-1 were greatly related with the OSW and FA. Additionally, C. vulgaris MBFJNU-1 in OSW and FA performed similar results in the common differentially expressed genes from these mentioned pathways. Overall, these obtained results deliver essential details in microalgal biotechnology to treat swine wastewater and high free ammonia wastewater.

The noninvasive monitoring of the redox status of photosynthetic electron transport chains in Hibiscus rosa-sinensis and Tradescantia leaves

We present an approach to the noninvasive determination of the electron capacity of the intersystem pool of electron carriers in chloroplasts in situ. As apt experimental models, we used the leaves of Hibiscus rosa-sinensis and Tradescantia species. Electron paramagnetic resonance and optical response of P700 (the primary electron donor in Photosystem I) were applied to measuring electron transport in chloroplasts. Electron capacities of the intersystem electron transport chain (ETC) were determined from redox transients of P700 upon chromatic transitions (white light → far-red light). During the induction period, we observed the nonmonotonic changes in the number of electron equivalents in the intersystem ETC per P700 (parameter Q). In Hibiscus rosa-sinensis, the light-induced rise of Q from ≈2.5 (in the dark) to Q ≈ 12 was followed by its decrease to Q ≈ 6. The data obtained are discussed in the context of pH-dependent regulation of electron transport in chloroplasts, which provides the well-balanced operation of the intersystem ETC. The decay of Q is explained by the attenuation of Photosystem II activity due to the lumen acidification and the acceleration of plastoquinol re-oxidation as a result of the Calvin-Benson cycle activation. Our computer model of electron and proton transport coupled to ATP synthesis in chloroplasts was used to analyze the up and down feedbacks responsible for pH-dependent regulation of electron transport in chloroplasts. The procedures introduced here may be important for subsequent works aimed at defining the plastoquinone participation in regulation of photosynthetic processes in chloroplasts in situ.

Flavodiiron proteins enhance the rate of CO2 assimilation in Arabidopsis under fluctuating light intensity.

The proton concentration gradient (ΔpH) and membrane potential (Δψ) formed across the thylakoid membrane contribute to ATP synthesis in chloroplasts. Additionally, ΔpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen. K+ exchange antiporter 3 (KEA3) relaxes this downregulation by substituting ΔpH with Δψ in response to fluctuation of light intensity. In the Arabidopsis (Arabidopsis thaliana) line overexpressing KEA3 (KEA3ox), the rate of electron transport is elevated by accelerating the relaxation of ΔpH after a shift from high light (HL) to low light. However, the plant cannot control electron transport toward photosystem I (PSI), resulting in PSI photodamage. In this study, we crossed the KEA3ox line with the line (Flavodiiron [Flv]) expressing the Flv proteins of Physcomitrium patens. In the double transgenic line (Flv-KEA3ox), electrons overloading toward PSI were pumped out by Flv proteins. Consequently, photodamage of PSI was alleviated to the wild-type level. The rate of CO2 fixation was enhanced in Flv and Flv-KEA3ox lines during HL periods of fluctuating light, although CO2 fixation was unaffected in any transgenic lines in constant HL. Upregulation of CO2 fixation was accompanied by elevated stomatal conductance in fluctuating light. Consistent with the results of gas exchange experiments, the growth of Flv and Flv-KEA3ox plants was better than that of WT and KEA3ox plants under fluctuating light.

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 ≤ 3.5kJmol-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).

The Diffusion Controlled Mode of the Photophosphorylation Process in Chloroplasts

The effect of the rate of ADP supply as a feedback signal on the rate of ATP synthesis in chloroplasts has been analyzed on the basis of literature data and our research results. Experimental data are discussed in the context of the diffusion controlled mode of photophosphorylation.

Electrons and proton transfer in chloroplasts in silico: 1. The effect of topological factors on energy coupling in chloroplasts with a nonuniform distribution of protein complexes

This paper presents a theoretical study of the effects of topological factors (density of thylakoid packing in grana) on the efficiency of energy coupling in chloroplasts. The study is based on a mathematical model of electron and proton transport processes coupled to ATP synthesis in chloroplasts. The model was developed by the authors earlier, and the nonuniform distribution of electron transport and ATP synthase complexes in the membranes of granal and intergranal thylakoids was taken into account in the model. The results of numerical experiments enabled the analysis of the distribution of lateral profiles of the transmembrane pH difference and the concentrations of mobile plastoquinone and plastocyanin electron transporters in granal and intergranal thylakoids and the dependence of this distribution on the metabolic state of class B chloroplasts (photosynthetic control state or the conditions of intensive ATP synthesis). Moreover, the influence of topological factors (the density of thylakoid packing in grana and the degree of thylakoid swelling) that affect the rate of diffusion of protons and mobile electron carriers in the intrathylakoid space and in the interthylakoidal gap was investigated. The results of numerical experiments that involved the variation of geometric parameters of the system revealed the influence of thylakoid thickness and the distance between the granal thylakoids on the lateral pH profiles inside the thylakoids (pHi) and in the interthylakoidal gap (pHo). Acidification of the intrathylakoid space characterized by the pHi value increased concomitantly to the increase of the width of the interthylakoidal gap l o and decreased concomitantly to the increase of the width of the intrathylakoidal space l i.

André Tridon Jagendorf (1926-2017): a personal tribute.

We present here our tribute to an outstanding unique scientist, a pioneer of plant biochemistry, best known for his seminal ground-breaking discoveries related to electron and proton transport, and their intimate relation to ATP synthesis in chloroplasts. His dedication and in-depth understanding of science was matched by his humor. Here, we offer a modest précis of his life and research. We have also included Reminiscences from: Leonard Fish, Eric Larson, Donald Ort, Thomas Owens, and Robert Turgeon.

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.

Synergism of ammonium and palmitic acid in uncoupling of electron transfer and ATP synthesis in chloroplasts.

Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 microM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H+). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H+ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.

Three Sets of Translocation Intermediates Are Formed during the Early Stage of Protein Import into Chloroplasts

During the early stage of protein import into chloroplasts, precursor proteins synthesized in the cytosol irreversibly bind to chloroplasts to form the early translocation intermediate under stringent energy conditions. Many efforts have been made to identify the components involved in protein import by analyzing the early intermediate. However, the state of the precursor within the intermediate has not been well investigated so far. In this study, an attempt was made to evaluate the extent of translocation of the precursor by determining the state of the precursor in the early intermediate under various conditions and analyzing the fragments generated by limited proteolysis of the precursors docked to chloroplasts. Our results indicate that three different sets of early intermediate are formed based on temperature and the hydrolysis of GTP/ATP. These have been identified based on the size of proteolytic fragments of the precursor as "energy-dependent association," "insertion," and "penetration" states. These findings suggest two individual ATP-hydrolyzing steps during the early stage of protein import, one of which is temperature-sensitive. Our results also demonstrate that translocation through the outer envelope membrane is mainly dependent on internal ATP.

Effects of Diffusion and Topological Factors on the Efficiency of Energy Coupling in Chloroplasts with Heterogeneous Partitioning of Protein Complexes in Thylakoids of Grana and Stroma. A Mathematical Model

In this work, we studied theoretically the effects of diffusion restrictions and topological factors that could influence the efficiency of energy coupling in the heterogeneous lamellar system of higher plant chloroplasts. Our computations are based on a mathematical model for electron and proton transport in chloroplasts coupled to ATP synthesis in chloroplasts that takes into account the nonuniform distribution of electron transport and ATP synthase complexes in the thylakoids of grana and stroma. Numerical experiments allowed the lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids to be simulated under different metabolic conditions (in the state of photosynthetic control and under conditions of photophosphorylation). This model also provided an opportunity to simulate the effects of steric constraints (the extent of appression of thylakoids in grana) on the rates of non-cyclic electron transport and ATP synthesis. This model demonstrated that there might be two mechanisms of regulation of electron and proton transport in chloroplasts: 1) slowing down of non-cyclic electron transport due to a decrease in the intra-thylakoid pH, and 2) retardation of plastoquinone reduction due to slow diffusion of protons inside the narrow gap between the thylakoids of grana. Numerical experiments for model systems that differ with respect to the arrangement of thylakoids in grana allowed the effects of osmolarity on the photophosphorylation rate in chloroplasts to be explained.

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments.

Despite recent progress in sequencing the complete genome of rice ( Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60-98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.

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

We present a method for measuring the transmembrane pH difference (ΔpH=pH in−pH out) 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 ΔpH. 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 ΔpH value was determined from the relationship ΔpH=lg([H +] in/[H +] out)≅lg( C in/ C out), where C in and C out are the concentrations of TA inside and outside the thylakoids, respectively. To quantify the internal concentration C in, 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] ϑ≈2.0–2.2 mM . The concentration dependencies of the signal parameters (the peak-to-peak amplitude, A pp, and the linewidth, Δ H pp) 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 Δ A). 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 ΔpH value and the internal volume of thylakoids, V in, from the non-linear dependence of parameter Δ A on the concentration C 0 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 ΔpH 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 ΔpH determinations by other methods.

Regulation of proton flow and ATP synthesis in chloroplasts.

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 micromol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the gamma and epsilon subunits of CF(1) play key roles in both regulation of activity and proton translocation.

Long‐ and short‐term effects of decreased sink demand on carbohydrate levels and photosynthesis in wheat leaves

ABSTRACTWheat (Triticum aestivum L.) ears were removed to investigate long‐term regulation of photosynthesis by sink demand at ambient CO2 and 22 °C. The CO2 level was also increased to 660 μmol mol−1 and temperature was lowered to 5 °C to examine short‐term responses of photosynthesis to low sink demand. Sink removal inhibited photosynthesis and increased leaf levels of glucose, fructose and ribulose‐1, 5‐bisphosphate (RuBP), and the glucose‐6‐phosphate (G6P)/fructose‐6‐phosphate (F6P) and RuBP/3‐phosphoglycerate (PGA) ratios under growth conditions, but had no effect on the activity and activation state of ribulose‐1, 5‐bisphosphate carboxylase oxygenase (Rubisco) either under growth or short‐term conditions, suggesting an inhibition of photosynthesis by decreased in vivo catalysis of Rubisco. Photosynthesis increased similarly in eared and earless shoots after a rise in CO2 concentration, and the ratio of triose‐phosphates (glyceraldehyde 3‐phosphate and dihydroxyacetone phosphate, TP) to PGA was similar or higher for removed than intact ears, suggesting that feedback inhibition of photosynthesis was not caused by a limitation of ATP synthesis in chloroplasts. Under short‐term conditions (660 μmol mol−1 CO2, 5 °C), TP and RuBP levels and the TP/PGA and TP/RuBP ratios were increased by sink removal, indicating an additional limitation of photosynthesis by the rate of RuBP regeneration.

Cooperation among electron‐transfer complexes in ATP synthesis in chloroplasts

We have investigated the extent to which redox reactions of thylakoid membranes cooperate in ATP synthesis. This was done by measuring the onset of ATP synthesis following a series of single-turnover light-flashes at various levels of electron transport inhibition. In the presence of antibiotics that prevent the formation of a membrane potential, the onset of ATP synthesis seems to depend entirely on the formation of an adequately large delta pH. Under our conditions, the accumulation of about 60 mmol H+ X mol chlorophyll-1 is then necessary to form the requisite delta pH, which in turn requires about 15 saturating flashes in uninhibited thylakoid samples. Inhibition of some of the electron transfer centers by limiting the light intensity of the flashes, by dichlorophenyldimethylurea, by heat treatment, or by NH2OH-treatment caused an increase in the number of flashes required for the onset of ATP synthesis. The increase in the requisite number of flashes reflected the decreased number of electrons transferred in each flash, almost exactly the same number of electrons being transferred before ATP synthesis could begin. This effect of inhibitors was true when the two photosystems were operating in unison and when either of the two photosystems was acting alone. However, when either photosystem acted alone, there was an increase in the number of flashes required for the onset of ATP synthesis, an increase which was consistent with the observed lower flash-induced proton accumulation. A mathematical analysis of the onset of flash-induced ATP synthesis shows that at least several hundred proton-translocating electron transport complexes must be cooperating to form the threshold delta pH. In spite of this evidence for extensive cooperation among different electron transport complexes in ATP formation, the implied pooling of H+ ions does not seem to involve inner vesicle regions accessible to exogenous buffers. Thus, even when the number of H+ ions accumulated per flash was reduced by 70% through attenuation of the intensity of the flashes, exogenous hydrogen ion buffers present within the lumen of the thylakoid vesicle had no effect on the number of flashes required for the onset of ATP synthesis.

Flash-induced ATP synthesis in pea chloroplasts in relation to proton flux

Chloroplasts extracted from preilluminated pea seedling leaves have an active ATP synthase. Single turnover flashes induce ATP synthesis in these chloroplasts. This synthesis has been measured by the luminescence of the luciferin-luciferase assay system and the absorbance changes at 515 nm monitored to determine the concomitant proton flux through the ATP-synthase. Variations in the ratio H + ATP are observed and can be explained by the way ATP synthase is operating. The newly synthesized ATP molecules remain bound to the enzyme, and have variable probabilities to be hydrolysed or released from the enzyme, dependent upon the energized state of the membranes. The lowest value found for the ratio, H + ATP , is 1.90 ± 0.55, which should be very close to the true value.