Influence of State-2 transition on the proton motive force across the thylakoid membrane in spinach chloroplasts

The proton motive force (pmf) across the thylakoid membrane is composed of the proton gradient and the membrane potential, which promotes millisecond-delayed light emission (ms-DLE). In this study, the time courses of LHC II phosphorylation and ms-DLE were investigated in spinach chloroplast during State-2 transition. Red light illumination resulted in an exponential rise in LHC II phosphorylation and a biphasic time course of ms-DLE. The phospho-LHC II appeared upon ∼ 1 min illumination. The phosphorylation level increased exponentially when illumination was elongated to 20 min. The t_&frac; of saturated LHC II phosphorylation was estimated 4–5 min under present illumination. During this process, the amplitudes of ms-DLE increased transiently to a maximal amplitude within 0.5 min illumination, and the reached maximum of the fast phase of ms-DLE was ∼ 140% of the dark control. Then, ms-DLE decreased from the maximum. After ≥3 min illumination, ms-DLE decreased to a lower level than the dark control. In the presence of uncouplers and inhibitors, the transient increase in the biphasic time course of ms-DLE was removed by nigericin and DCMU, and the sequential decrease was delayed by DCCD. The time course was not affected significantly by valinomycin and DBMIB. Moreover, the level of LHC II phosphorylation was enhanced by nigericin, valinomycin and DCCD, and was inhibited completely by DCMU and partially by DBMIB. Taken together, we proposed that the PS II photochemical activity remained unaffected even with a higher level of LHC II phosphorylation, which was reflected by the effect of DCCD on the time course of ms-DLE. Probably, the evidence of LHC II phosphorylation is the rearrangement of LHC II–PS II complex and the thylakoid, a feedback to light-exposure, rather than the redistribution of excitation energy from PS II to PS I.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Photosynthetic free energy transduction related to the electric potential changes across the thylakoid membrane

A model based on our present knowledge of photosynthetic energy transduction is presented. Calculated electric potential profiles are compared with microelectrode recordings of the thylakoid electric potential during and after actinic illumination periods of intermediate duration. The information content of the measured electric response is disclosed by a comparison of experimental results with calculations. The proton flux through the ATP synthase complex is seen to markedly influence the electric response. Also the imbalance in maximum turnover rate between the two photosystems, common to obligate shade plants like Peperomia metallica used in the microelectrode experiments, is clearly reflected in the electric potential profile.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Synthesis of soluble, thylakoid, and envelope membrane proteins by spinach chloroplasts purified from gradients.

Intact spinach chloroplasts that had been purified on gradients of silica sol incorporated [³⁵S]methionine into soluble and membrane-bound products, using light as the sole energy source. The labeled chloroplasts were lysed osmotically and fractionated on a discontinuous gradient of sucrose into the soluble fraction and the thylakoid and envelope membranes. About 29% of the radioactivity in the chloroplast was recovered in the soluble fraction, 59% in the thylakoid membranes, and 0.1% in the envelope membranes. The products of protein synthesis in the different fractions, as well as in the whole chloroplast, were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. There were two zones of radioactivity in the gels of the soluble fraction, the major zone coincident with the large subunit of ribulose diphosphate carboxylase at a molecular weight of about 50,000. The thylakoid membranes contained five labeled polypeptides, the most active having a molecular weight of about 31,000. The envelope membranes contained a major radioactive component of a molecular weight of about 50,000 and two other minor components.     

Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes.

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.     

On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells.

The potential defference across the thyladoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illunimation. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3-3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.     

Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (ΔΨ) and pH difference (ΔpH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K⁺ diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K⁺ permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with ΔΨ formed by valinomycin-K⁺ did not significantly alter 9-AA fluorescence, but unexpectedly dissipated ΔΨ. The ΔΨ preformed by valinomycin-K⁺ decreased gradually following the addition of increasing concentrations of anacardic acid. The ΔΨ dissipation rate was dependent on the pre-existing magnitude of ΔΨ, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of ΔpH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by ΔΨ, and a ‘proton carrier’ that dissipates the transmembrane proton gradient formed.     

Synthesis of thylakoid membrane proteins by chloroplasts isolated from spinach. Cytochrome b559 and P700-chlorophyll a-protein

Intact chloroplasts, purified from spinach leaves by sedimentation in density gradients of colloidal silica, incorporate labeled amino acids into at least 16 different polypeptides of the thylakoid membranes, using light as the only source of energy. The thylakoid products of chloroplast translation were visualized by subjecting membranes purified from chloroplasts labeled with [35S]methionine to electrophoresis in high-resolution, SDS-containing acrylamide gradient slab gels and autoradiography. The apparent mol wt of the labeled products ranged from less than 10,000 to greater than 70,000. One of the labeled products is the apoprotein of the P700-chlorophyll a- protein (CPI). The CPI apoprotein is assembled into a pigment-protein complex which is electrophoretically indistinguishable from the native CPI complex. Isolated spinach chloroplasts also incorporate [3H]leucine and [35S]methionine into cytochrome b559. The radioactive label remains with the cytochrome through all stages of purification: extraction of the thylakoid membranes with Triton X-100 and urea, adsorption of impurities on DEAE cellulose, two cycles of electrophoresis in Triton- containing polyacrylamide gels and electrophoresis in SDS-containing gradient gels. Cytochrome b559 becomes labeled with both [3H]leucine and [35S]methionine and accounts for somewhat less than 1% of the total isotopic incorporation into thylakoid protein. The lipoprotein appears to be fully assembled during the time-course of our labeling experiments.     

Effect of methanol on spinach thylakoid ATPase

Methanol at 35% ($\text{v}\text{v}$) overcomes the latency of spinach thylakoid ATPase. Activation is immediate and reversible involving changes in the V_max, not the K_m of the enzyme, MgATP is a much better substrate than CaATP; free Mg²⁺ noncompetitively inhibits activity. This inhibition can be overcome by the addition of Na₂SO₃. While both MgATP and MgGTP act as substrates, free ATP and GTP both inhibit activity. ADP and MgADP are also inhibitory. Insensitivity to certain inhibitors indicates that methanol neither induces the same conformational changes in CF₁ as illumination does, nor does it lead to coupling between H⁺ movement through CF₀ and ATP hydrolysis. Methanol activation provides a much improved method for assaying thylakoid ATPase.     

Influence of adenine nucleotides on the inhibition of photophosphorylation in spinach chloroplasts by N-ethylmaleimide.

The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites.     

On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells

The potential difference across the thylakoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illumination. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3–3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.     

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [¹⁴C]5,5-dimethyl-2,4-oxazolidinedione and [¹⁴C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO₂ fixation by superoptimal medium Pi or by adding glyceraldehyde—an inhibitor of the Calvin cycle—leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific `low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP<sup>+</sup> and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N′-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N′-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific `low phosphate' proton leak in the thylakoids, and its origin is discussed.     

Redox potential of plastoquinone A in spinach chloroplasts.

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is -30 mV per pH between pH 4.0 and 5.7, and -60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Temporary suppression of the flash-induced electrical potential across the thylakoid membrane upon energization

Energization of the chloroplast thylakoid membrane causes a temporary decrease in the amplitude of the flash-induced transmembrane electrical potential as monitored by the micro-electrode technique and by the electrochromic absorbance band shift at 518 nm in chloroplasts of Peperomia metallica. This energization-dependent decrease of the flash-induced potential has a relaxation time of recovery in the dark of about 23±4 s. The phenomenon can neither be explained by a decrease of the intrinsic efficiency of photosystem I and II (PSI and PSII) nor by a partial closure of reaction centers of PSI and PSII. This leads us to propose that the energization-dependent decrease of the amplitude of the flash-induced electrical potential is caused by either the formation of a fraction of PSI and/or PSII reaction centers with fast charge recombination or by an increase of the membrane capacitance. The dark recovery after energization of the amplitude of the transmembrane electrical potential and that of non-photochemical fluorescence quenching were found to be comparable, which suggests a common cause for both phenomena.     

Structural changes of the thylakoid membrane network induced by high light stress in plant chloroplasts

Land plants live in a challenging environment dominated by unpredictable changes. A particular problem is fluctuation in sunlight intensity that can cause irreversible damage of components of the photosynthetic apparatus in thylakoid membranes under high light conditions. Although a battery of photoprotective mechanisms minimize damage, photoinhibition of the photosystem II (PSII) complex occurs. Plants have evolved a multi-step PSII repair cycle that allows efficient recovery from photooxidative PSII damage. An important feature of the repair cycle is its subcompartmentalization to stacked grana thylakoids and unstacked thylakoid regions. Thus, understanding the crosstalk between stacked and unstacked thylakoid membranes is essential to understand the PSII repair cycle. This review summarizes recent progress in our understanding of high-light-induced structural changes of the thylakoid membrane system and correlates these changes to the efficiency of the PSII repair cycle. The role of reversible protein phosphorylation for structural alterations is discussed. It turns out that dynamic changes in thylakoid membrane architecture triggered by high light exposure are central for efficient repair of PSII.     

Electrophoretic analysis of the membrane proteins of spinach chloroplasts

The polypeptide heterogeneity of the chloroplast envelope has been compared with these of other fractions of Spinach chloroplasts: thylakoïds and stroma by means of SDS-polyacrylamide gel electrophoresis. The envelope electrophoretic pattern shows many polypeptides whose mobility differ from those of the thylakoïds. The possible identity of the small subunit of RudPcarboxylase with a polypeptide of the chloroplast external membrane is discussed.