Thylakoid Membrane Energization Research Articles

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes — As revealed by small-angle neutron scattering

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H (pD) and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity — similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.

Thiol Modulation of the Chloroplast ATP Synthase is Dependent on the Energization of Thylakoid Membranes

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.

The galactolipid monogalactosyldiacylglycerol (MGDG) contributes to photosynthesis-related processes in Arabidopsis thaliana

Processes putatively dependent on the galactolipid monogalactosyldiacylglycerol (MGDG) were recently studied using the knockdown monogalactosyldiacylglycerol synthase 1 (mgd1-1) mutant (~40% reduction in MGDG). Surprisingly, targeting of chloroplast proteins was not affected in mgd1 mutants, suggesting they retain sufficient MGDG to maintain efficient targeting. However, in dark-grown mgd1 plants the photoactive to photoinactive protochlorophyllide (Pchlide) ratio was increased, suggesting that photoprotective responses are induced in them. Nevertheless, mgd1 could not withstand high light intensities, apparently due to impairment of another photoprotective mechanism, the xanthophyll cycle (and hence thermal dissipation). This was mediated by increased conductivity of the thylakoid membrane leading to a higher pH in the thylakoid interior, which impaired the pH-dependent activation of violaxanthin de-epoxidase (VDE) and PsbS. These findings suggest that MGDG contribute directly to the regulation of photosynthesis-related processes.Addendum to: Aronsson H, Schüttler MA, Kelly AA, Sundqvist C, Dörmann P, Karim S, Jarvis P. Monogalactosyldiacylglycerol deficiency in Arabidopsis thaliana affects pigment composition in the prolamellar body and impairs thylakoid membrane energization and photoprotection in leaves. Plant Physiol 2008; 148:580-92.

Monogalactosyldiacylglycerol deficiency in Arabidopsis affects pigment composition in the prolamellar body and impairs thylakoid membrane energization and photoprotection in leaves.

Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of chloroplast membranes and has been proposed to act directly in several important plastidic processes, particularly during photosynthesis. In this study, the effect of MGDG deficiency, as observed in the monogalactosyldiacylglycerol synthase1-1 (mgd1-1) mutant, on chloroplast protein targeting, phototransformation of pigments, and photosynthetic light reactions was analyzed. The targeting of plastid proteins into or across the envelope, or into the thylakoid membrane, was not different from wild-type in the mgd1 mutant, suggesting that the residual amount of MGDG in mgd1 was sufficient to maintain functional targeting mechanisms. In dark-grown plants, the ratio of bound protochlorophyllide (Pchlide, F656) to free Pchlide (F631) was increased in mgd1 compared to the wild type. Increased levels of the photoconvertible pigment-protein complex (F656), which is photoprotective and suppresses photooxidative damage caused by an excess of free Pchlide, may be an adaptive response to the mgd1 mutation. Leaves of mgd1 suffered from a massively impaired capacity for thermal dissipation of excess light due to an inefficient operation of the xanthophyll cycle; the mutant contained less zeaxanthin and more violaxanthin than wild type after 60 min of high-light exposure and suffered from increased photosystem II photoinhibition. This is attributable to an increased conductivity of the thylakoid membrane at high light intensities, so that the proton motive force is reduced and the thylakoid lumen is less acidic than in wild type. Thus, the pH-dependent activation of the violaxanthin de-epoxidase and of the PsbS protein is impaired.

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF1.

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.

Simultaneous analysis of prompt and delayed chlorophyll a fluorescence in leaves during the induction period of dark to light adaptation

An attempt is made to reveal the relation between the induction curves of delayed fluorescence (DF) registered at 0.35–5.5 ms and the prompt chlorophyll fluorescence (PF). A simple formulation was proposed to link the ratio of the transient values of delayed and variable fluorescence with the redox state of the primary electron acceptor of Photosystem II—Q A, and the thylakoid membrane energization. The term luminescence potential ( U L ) was introduced, defined as the sum of the redox potential of Q A and the transmembrane proton gradient. It was shown that U L is proportional to the ratio of DF to the variable part of PF. The theoretical model was verified and demonstrated by analysing induction courses of PF and millisecond DF, simultaneously registered from leaves of barley—wild-type and the chlorophyll b-less mutant chlorina f2. A definitive correlation between PF and DF was established. If the luminescence changes are strictly due to U L , the courses of DF and PF are reciprocal and the millisecond DF curve resembles the first derivative of the PF( t) function.

Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells

Fluorescent ΔpH and Δ Ψ indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp. PCC 6803. Acridine yellow and Acridine orange proved to be the best ΔpH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically. Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane. In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies. Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus. Different from Synechocystis sp. PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis.

Heat sensitivity of chloroplasts and leaves: Leakage of protons from thylakoids and reversible activation of cyclic electron transport

In illuminated intact spinach chloroplasts, warming to and beyond 40 °C increased the proton permeability of thylakoids before linear electron transport through Photosystem II was inhibited. Simultaneously, antimycin A-sensitive cyclic electron transport around Photosystem II was activated with oxygen or CO2, but not with nitrite as electron acceptors. Between 40 to 42 °C, activation of cyclic electron transport balanced the loss of protons so that a sizeable transthylakoid proton gradient was maintained. When the temperature of darkened spinach leaves was slowly increased to 40°C, reduction of the quinone acceptor of Photosystem II, QA, increased particularly when respiratory CO2 production and autoxidation of plastoquinones was inhibited by decreasing the oxygen content of the atmosphere from 21 to 1%. Simultaneously, Photosystem II activity was partially lost. The enhanced dark QA reduction disappeared after the leaf temperature was decreased to 20 °C. No membrane energization was detected by light-scattering measurements during heating the leaf in the dark. In illuminated spinach leaves, light scattering and nonphotochemical quenching of chlorophyll fluorescence increased during warming to about 40 °C while Photosystem II activity was lost, suggesting extra energization of thylakoid membranes that is unrelated to Photosystem II functioning. After P700 was oxidized by far-red light, its reduction in the dark was biphasic. It was accelerated by factors of up to 10 (fast component) or even 25 (slow component) after short heat exposure of the leaves. Similar acceleration was observed at 20 °C when anaerobiosis or KCN were used to inhibit respiratory oxidation of reductants. Methyl viologen, which accepts electrons from reducing side of Photosystem II, completely abolished heat-induced acceleration of P700+ reduction after far-red light. The data show that increasing the temperature of isolated chloroplasts or intact spinach leaves to about 40 °C not only inhibits linear electron flow through Photosystem II but also activates Photosystem I-driven cyclic electron transport pathways capable of contributing to the transthylakoid proton gradient. Heterogeneity of the kinetics of P700+ reduction after far-red oxidation is discussed in terms of Photosystem I-dependent cyclic electron transport in stroma lamellae and grana margins.

Identification of surface exposed amino acids of isolated and membrane-bound CF1 by protease accessibility

In order to compare surface-exposed amino acids in isolated and membrane-bound CF1 the technique of limited proteolysis was employed. The cleavage sites of several proteases were identified by sequence analysis of the resulting peptides after isolation by SDS-PAGE. In isolated CF1 the N-terminal region of the α subunit was found to be highy sensitive to proteases; the accessible peptide bonds included αE17-G18, αR21-E22, αE22-V23, and αK24-V25. Additional protease-attacked bonds in α subunit were αS86-S87, xαE125-S126. and αR127-L128. In the β subunit of isolated CF1 the bonds βL14-E15 and βV76-A77 were identified as being accessible. All identified protease accessible amino acids are located at the protein surface according to a molecular model of CF1 computed after the crystal structure of mitochondrial F1 by S. Engelbrecht (1997). In membrane bound CF1 the primarily accessible peptide bond of the N-terminal domain of α is αR21-E22. After this bond is cleaved by trypsin, the αK24-V25 becomes accessible to further trypsin attack. Moreover, the peptide bonds αR14O-S141 and αG16O-R161 are cleaved. According to the Engelbrecht model αG16O is almost completely shielded and actually this amino acid was hardly accessible to protease in isolated CF1. The β subunit in general is much more sensitive to proteolysis in membrane-bound than in solubilized CF1. In the β subunit of membrane-bound CF1 a papain-sensitive bond βG102-G103 was identified. The results indicate major structural alterations when CF1 is extracted from the CF0CF1 complex. Thiol modulation, i.e. reduction of the regulatory disulfide bond between γC199 and γC205 of y subunit, enhances the accesibility of a number of peptide bonds, in particular ααG160-R161, to proteolytic attack by papain. In contrast, thylakoid membrane energization results in masking of this peptide bond.

Electron flow accompanying the ascorbate peroxidase cycle in maize mesophyll chloroplasts and its cooperation with linear electron flow to NADP + and cyclic electron flow in thylakoid membrane energization

Potential roles for cyclic and pseudocyclic electron flow in C4 plants are to provide ATP for the C4 cycle and, under excess light, to down-regulate PS II activity through membrane energization. Intact mesophyll chloroplasts of maize were used to evaluate forms of electron transport including the Mehler peroxidase reaction (linear electron flow to O2, formation of H2O2 which is reduced by ascorbate, and linear flow linked to reduction of oxidized ascorbate). Addition of H2O2 to isolated chloroplasts in the light in the presence of an uncoupler induced Photosystem (PS) II activity, as determined from increases in photochemical quenching of chlorophyll fluorescence (qp) and the quantum yield of PS II. H2O2 also induced dissipation of energy by thylakoid membrane energization and non-photochemical fluorescence quenching (qn), which was inhibited by addition of an uncoupler. These effects of H2O2 on qp and qn were inhibited by addition of KCN, an inhibitor of ascorbate peroxidase. The results suggest that H2O2 is reduced via ascorbate, and that the oxidized ascorbate is then reduced by linear electron flow contributing to photochemistry and thylakoid membrane energization. Evidence for function of pseudocyclic electron flow via the Mehler peroxidase reaction was obtained with only oxygen as an electron acceptor, as well as in the presence of oxaloacetate a natural electron acceptor in C4 photosynthesis. KCN decreased qp and PS II yield in the absence and presence of oxaloacetate and, in the former case, it severely reduced q_n. KCN also decreased ΔpH formation across the thylakoid membrane based on its decrease in the light-induced quenching of 9-aminoacridine fluorescence, particularly in the absence of oxaloacetate. Antimycin A, an inhibitor of cyclic electron flow, also diminished ΔpH formation. These results provide evidence for shared energization of thylakoid membranes by the Mehler peroxidase reaction, cyclic electron flow, and linear electron flow linked to the C4 pathway.

Relationships among violaxanthin deepoxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (ΔZ; peak at 506 nm) and by light scattering (ΔS; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (ΔSg) for absorbance changes around 540 nm that are due to a spectral overlap with ΔZ. This protocol isolated the component which is caused by light-scattering changes alone (ΔSn). In control leaves, the kinetics and temperature dependence of the initial rate of rise in ΔSn that takes place upon illumination, closely matched that of ΔZ. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both ΔSn and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on ΔAn and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of ΔSn and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of ΔZ upon darkening was much slower than the relaxation of ΔSn and NPQ. The relaxation of ΔSn and NPQ showed quantitatively similar kinetics and temperature dependencies (Q10=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause ΔSn or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by ΔSn. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained ΔSn and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O2 in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b 6-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b 6-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and 'poises' the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO2 can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.

Thylakoid membrane energization and swelling in photoinhibited Chlamydomonas cells is prevented in mutants unable to perform cyclic electron flow

Photoinhibition of Photosystem II in unicellular algae in vivo is accompanied by thylakoid membrane energization and generation of a relatively high ΔpH as demonstrated by (14)C-methylamine uptake in intact cells. Presence of ammonium ions in the medium causes extensive swelling of the thylakoid membranes in photoinhibited Chlamydomonas reinhardtii but not in Scenedesmus obliquus wild type and LF-1 mutant cells. The rise in ΔpH and the related thylakoid swelling do not occur at light intensities which do not induce photoinhibition. The rise in ΔpH and membrane energization are not induced by photoinhibitory light in C. reinhardtii mutant cells possessing an active Photosystem II but lacking cytochrome b6/f, plastocyanin or Photosystem I activity and thus being unable to perform cyclic electron flow around Photosystem I. In these mutants the light-induced turnover of the D1 protein of Reaction Center II is considerably reduced. The high light-dependent rise in ΔpH is induced in the LF-1 mutant of Scenedesmus which can not oxidize water but otherwise possesses an active Reaction Center II indicating that PS II-linear electron flow activity and reduction of plastoquinone are not required for this process. Based on these results we conclude that photoinhibition of Photosystem II activates cyclic electron flow around Photosystem I which is responsible for the high membrane energization and ΔpH rise in cells exposed to excessive light intensities.

Local Anesthetic Binding to Thylakoid Membranes. Relation to Inhibition of Light-Induced Membrane Energization and Photophosphorylation

Abstract The association of the lipophilic tertiary amine and local anesthetic dibucaine with osmotically shocked chloroplasts of Spinacia oleracea L. cv. Monatol was investigated. Dibucaine, known as an effective inhibitor of thylakoid membrane energization and ATP synthesis, ex­hibited three distinct binding classes with chloroplasts: partitioning in the lipid phase of the membranes, electrostatic screening of negative electrical charges on the thylakoid surface and light-induced association of an as yet unknown nature. Evidence is presented that the mecha­nism of inhibition of the transthylakoid pH gradient, ∆pH, by dibucaine is distinct from ‘classical’ amine-type uncoupling: The inhibitory effect of dibucaine on ∆pH was independent of the initial strength of ∆pH. Light-induced dibucaine binding was independent of the volume of the intrathylakoid space and of the strength of ∆pH as varied by medium pH. Judged from a comparison of the data on dibucaine binding and on inhibition of ∆pH and photophosphorylation, dibucaine bound via partitioning in the membrane lipid phase is responsible for the uncoupler-like effects of the local anesthetic. A mechanism for the inhibition of thylakoid energization by local anesthetic amines is discussed.

Kinetic Relationship between Energy-Dependent Fluorescence Quenching, Light Scattering, Chlorophyll Luminescence and Proton Pumping in Intact Leaves

Abstract A measuring system was designed for simultaneous recording of modulated chlorophyll fluorescence and light scattering changes. The kinetic relationship was investigated between lightinduced changes in non-photochemical fluorescence quenching, as determined by the saturation pulse method, and in light scattering, as measured via the apparent absorbance change at 543 nm. Very similar, but not identical kinetics were observed, reflecting a close non-linear relationship between these two indicators of thylakoid membrane energization. Fluorescence was found more sensitive at low levels of energization, while scattering continued indicating further increases in energization when quenching already was saturated. A general relationship between quenching and scattering is demonstrated which holds irrespective of whether energization is varied during induction or via changes in light intensity or CO2 concentration. In the light-off responses, only part of fluorescence quenching was found to relax with the same kinetics as scattering. It is suggested that at high levels of energization slowly reversible membrane changes may be induced which have the potential of non-photochemical quenching at a low level of energization, and which are not accompanied by scattering changes. Neither quenching nor scattering changes displayed kinetics sufficiently fast to be taken as a direct expression of internal thylakoid acidificati on in intact leaves. This conclusion is drawn from comparative measurements of proton-uptake, as reflected by CO2-solubilization upon light-induced stroma alkalization, and of chlorophyll luminescence. Both, the initial CO2 - gulp and the pH-dependent luminescence rise were found to clearly precede the development of energy-dependent quenching.

Fluorescence Quenching and Gas Exchange in a Water Stressed C3 Plant, Digitalis lanata

A leaf cuvette has been adapted for use with a pulse-modulation fluorometer and an open gas exchange system. Leaf water potential (psi) was decreased by withholding watering from Digitalis lanata EHRH. plants. At different stages of water deficiency the photochemical (q(Q)) and nonphotochemical (q(E)) fluorescence quenching was determined during the transition between darkness and light-induced steady state photosynthesis of the attached leaves. In addition, the steady state CO(2) and H(2)O gas exchange was recorded. Following a decrease of leaf water potential with increasing water deficiency, the transition of photochemical quenching was almost unaffected, whereas nonphotochemical quenching increased. This is indicative of an enhanced thylakoid membrane energization during the transition and is interpreted as a partial inhibition of either the ATP generating or the ATP consuming reaction sequences. Complete reversion of the stress induced changes was achieved within 6 hours after rewatering. In contrast to the variations during transition, the final steady state values of q(Q) and q(E) remained unchanged over the entire stress range from -0.7 to -2.5 megapascals. From these results we conclude that, once established, electron transport via photosystem II and the transmembrane proton gradient remain unaffected by water stress. These data are indicative of a protective mechanism against photoinhibition during stress, when net CO(2) uptake is limited.

Effect of Electron Transfer Inhibitors and Uncoupling Agents on the Chlorophyll Fluorescence Lifetime during Slow Fluorescence Decline in Bean Leaves and Intact Chloroplasts

Abstract Fluorescence measurements were performed with bean leaves and intact chloroplasts, which are characterized by a slow fluorescence decline. The fluorescence lifetime (ι) and the fluorescence relative quantum yield (φ) of intact chloroplasts, measured with the phase fluorometer, showed nearly proportional light-induced decrease with a halftime of ∼ 30 s, which was removed by 3-(3.4-dichlorophenyl)-1.1 -dimethylurea (DCMU), by 4.5,6,7-tetrachloro-2′-trifluoromethylbenzimidazole (TTFB). by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at a low concentration and by gramicidin D. Simultaneously the slow light-induced increase in absorbance at 518 nm (ΔA518), reflecting thylakoid membrane energization, was eliminated. The data on ι of intact chloroplasts are original, the other results agree with literature data. In leaves, the slow light-induced fluorescence decline, with ι dropping from ∼ 2 to ∼ 0.6 ns, was abolished by FCCP at a concentration of 5 μm. However, while ι was stabilized at a level close to the initial (maximum) one or somewhat higher, φ became close to the minimum value. Besides, the amplitude of ΔA518 was lowered about three times. These effects seemed to be due to multiple action of FCCP as a protonophoric uncoupler and an electron transfer inhibitor. In the presence of another uncoupling agent, TTFB, which, besides, is a diuron-like inhibitor of the electron transfer in chloroplasts, we observed the light-dependent, but hardly linked to changes in membrane potential, great increase in ι of a leaf from ∼ 1.9 to ∼ 4 .3 ns, with φ decreasing slightly. Addition of DCMU together with FCCP to the incubation medium or infiltration of a leaf with DCMU alone stimulated the rise in ι only to ∼ 3 ns. The increase in ι of a leaf observed in the presence of FCCP and DCMU, and especially with TTFB, may be associated with protein conformation changes which (i) alter the lifetime of nanosecond recombination luminescence of the photosystem II and/or (ii) disturb excitation energy transfer from the light-harvesting chlorophyll a/b complex to other pigment-protein complexes.

Hydrogen exchange into soluble spinach chloroplast coupling factor during heat activation of its ATPase

Exchange of 500–600 atoms of 3H per mol of solubilized spinach chloroplast coupling factor (CF 1) occurs when the enzyme is incubated for 4 min in 3H 2O at 63°C. These 3H atoms are bound in parts of the protein where exchange is hindered by the three-dimensional structure at 25°C. Back-exchange at 25°C shows complex kinetics, with at least two kinetic components having half-times of 1.4 and 40 h, respectively. Back-exchange from the denatured enzyme is extremely rapid with an apparent half-time of the order of 20–30 s. The time courses for exchange and ATPase activation are very similar at 63°C, and reasonably close at 25°C. Both reactions have an optimum temperature of 60°C when measured after 4 min. Activation of ATPase requires a strong reducing agent to be present, but this is not needed for hydrogen exchange. It is suggested that an open conformation of CF 1 induced by heat may be a required intermediate for the rapid activation of ATPase, being a sporadic and rare occurrence at 25°C but also a required step in ATPase activation. This open conformation could be related to that induced in bound CF 1 by thylakoid membrane energization.

On the role of membrane-bound ADP and ATP in photophosphorylation in chloroplast membranes

The role of CFr firmly-bound nucleotides in photophosphorylation is of current and widespread interest and importance; especially as regards the proposal that conformational changes of the membrane-bound protein result in altered binding affinities for the substrates, ADP, Pi and ATP [ 1,2]. In addition, a lightdependent and uncoupler-sensitive exchange of nucleotides into membrane-bound CFr was recently discovered by Harris and Slater [3]. The relationship between this type of exchange reaction and ATP formation in chloroplasts was further investigated by Strotmann et al. [4] and Magnusson and McCarty [S] . Energization of thylakoid membranes in the presence of labeled ADP or ATP resulted in the incorporation of ADP into CFr . The nucleotide bound to CF, in this manner appears for the most part to be associated with one of the two larger subunits of CFr . Recently, we reported on the light-dependent phosphorylation of CFr-bound ADP and attempted to correlate this activity with the phosphorylation of free ADP [6] . Using short term illumination (2 3 s), about one mole of bound ADP per mole of CFr was phosphorylated to yield an equivalent amount of [$“P] ATP. These studies have been further extended using short-term (2 30 ms), saturating illumination to attempt to compare the kinetics characteristic of phosphorylation of bound and free ADP. In this communication, it is shown that the rate of phosphorylation of bound nucleotide: (1) Is much lower than that observed with free ADP.