Elucidating the site of action of oxalate in photosynthetic electron transport chain in spinach thylakoid membranes

The effects of oxalate on PS II and PS I photochemistry were studied. The results suggested that in chloride-deficient thylakoid membranes, oxalate inhibited activity of PS II as well as PS I. To our knowledge, this is the only anion so far known which inhibits both the photosystems. Measurements of fluorescence induction kinetics, YZ* decay, and S2 state multiline EPR signal suggested that oxalate inhibited PS II at the donor side most likely on the oxygen evolving complex. Measurements of re-reduction of P700+ signal in isolated PS I particles in oxalate-treated samples suggested a binding site of oxalate on the donor, as well as the acceptor side of PS I.     

Effect of high magnesium ion concentration on the electron transport rate and proton exchange in thylakoid membranes in higher plants]

The effects of magnesium ion concentration on the rate of electron transport in isolated pea thylakoids were investigated in the pH range from 4.0 up to 8.0. In the absence of magnesium ions in the medium and in the presence of 5 mM MgCl2 in the experiments not only without added artificial acceptors but also with ferricyanide or methylviologen as an acceptor, this rate had a well-expressed maximum at pH 5.0. It was shown that, after depression to minimal values at pH 5.5-6.5, it gradually rose with increasing pH. An increase in magnesium ion concentration up to 20 mM essentially affected the electron transfer rate: it decreased somewhat at pH 4.0-5.0 but increased at higher pH values. At this magnesium ion concentration, the maximum rate was at pH 6.0-6.5 and the minimum, at pH 7.0. Subsequent rise upon increasing pH to 8.0 was expressed more sharply. The influence of high magnesium ion concentration on the rate of electron transport was not observed in the presence of gramicidin D. It was found that without uncoupler, the changes in the electron transfer rate under the influence of magnesium ions correlated to the changes in the first-order rate constant of the proton efflux from thylakoids. It is supposed that the change in the ability of thylakoids to keep protons by the action of magnesium ions is the result of electrostatic interactions of these ions with the charges on the external surface of membranes. A possible role of regulation of the electron transport rate by magnesium ions in vivo is discussed.     

Sugar transport across the plasma membranes of higher plants

The fluxes of carbohydrates across the plasma membranes of higher-plant cells are catalysed mainly by monosaccharide and disaccharide-H⁺ symporters. cDNAs encoding these different transporters have been cloned recently and the functions and properties of the encoded proteins have been studied extensively in heterologous expression systems. Several of the proteins have been identified biochemically in these expression systems and their location in plants has been shown immunohistochemically or with transgenic plants which were transformed with reporter genes, expressed under the control of the promoters of individual transporter genes. In this paper we summarize the current knowledge on the molecular biology and biochemistry of higher-plant sugar transport proteins.     

Physiology of PSI cyclic electron transport in higher plants

Having long been debated, it is only in the last few years that a concensus has emerged that the cyclic flow of electrons around Photosystem I plays an important and general role in the photosynthesis of higher plants. Two major pathways of cyclic flow have been identified, involving either a complex termed NDH or mediated via a pathway involving a protein PGR5 and two functions have been described-to generate ATP and to provide a pH gradient inducing non-photochemical quenching. The best evidence for the occurrence of the two pathways comes from measurements under stress conditions-high light, drought and extreme temperatures. In this review, the possible relative functions and importance of the two pathways is discussed as well as evidence as to how the flow through these pathways is regulated. Our growing knowledge of the proteins involved in cyclic electron flow will, in the future, enable us to understand better the occurrence and diversity of cyclic electron transport pathways. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

The importance of grana stacking for xanthophyll cycle-dependent NPQ in the thylakoid membranes of higher plants

In the present study we have examined the effects of grana stacking on the rate of violaxanthin (Vx) de-epoxidation and the extent of non-photochemical quenching of chlorophyll a fluorescence (NPQ) in isolated thylakoid membranes of spinach. Our results show that partial and complete unstacking of thylakoids in reaction media devoid of sorbitol and MgCl₂ did not significantly affect the efficiency of Vx de-epoxidation. Under high light (HL) illumination we found slightly higher values of Vx conversion in stacked membranes, whereas in thylakoids incubated at pH 5.2 in the dark, representing the pH-optimum of Vx de-epoxidase, de-epoxidation was slightly increased in the unstacked membranes. Partial and complete unstacking of grana membranes, however, had a dramatic effect on the HL-induced NPQ. High NPQ values could only be achieved in stacked thylakoid membranes in the presence of MgCl₂ and sorbitol. In unstacked membranes NPQ was drastically decreased. The effects of grana stacking on the xanthophyll cycle-dependent component of NPQ were even more pronounced, and complete unstacking of thylakoid membranes led to a total loss of this quenching component. Our data imply that grana stacking in the thylakoid membranes of higher plants is of high importance for the process of overall NPQ. For the xanthophyll cycle-dependent component of NPQ it may even be essential. Possible effects of grana stacking on the mechanism of zeaxanthin-dependent quenching are discussed.     

Characterization of thylakoid lipid membranes from cyanobacteria and higher plants by molecular dynamics simulations

The thylakoid membrane is mainly composed of non-common lipids, so called galactolipids. Despite the importance of these lipids for the function of the photosynthetic reaction centers, the molecular organization of these membranes is largely unexplored. Here we use multiscale molecular dynamics simulations to characterize the thylakoid membrane of both cyanobacteria and higher plants. We consider mixtures of up to five different galactolipids plus phosphatidylglycerol to represent these complex membranes. We find that the different lipids generally mix well, although nanoscale heterogeneities are observed especially in case of the plant membrane. The fluidity of the cyanobacterial membrane is markedly reduced compared to the plant membrane, even considering elevated temperatures at which thermophilic cyanobacteria are found. We also find that the plant membrane more readily undergoes a phase transformation to an inverted hexagonal phase. We furthermore characterized the conformation and dynamics of the cofactors plastoquinone and plastoquinol, revealing of the fast flip-flop rates for the non-reduced form. Together, our results provide a molecular view on the dynamical organization of the thylakoid membrane.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

Mechanism of bicarbonate action on photosynthetic electron transport in broken chloroplasts

In CO2-depleted chloroplasts electron transport between the Photosystem II electron acceptor Q and plastoquinone is largely suppressed. In the presence of a high concentration of sodium formate (greater than 10 mM), which probably binds to the bicarbonate site, addition of bicarbonate restores the ferricyanide Hill reaction only after incubation in the dark. With lower formate concentrations bicarbonate is able to restore electron transport in the light. The Hill reaction rate in CO2-depleted chloroplasts after bicarbonate addition, divided by the rate in CO2-depleted chloroplasts before bicarbonate addition, shows a sharp optimum at pH 6.5. Furthermore, the rate-limiting step in bicarbonate action is probably diffusion. The results are explained in terms of a hypothetical model: the bicarbonate-binding site is located at the outer side of the thylakoid membrane, but not directly accessible from the "bulk". To reach the site from the bulk, the molecule has to pass a channel with negatively charge groups on its side walls. In the light these groups are more negatively charged than in the dark. Therefore, the formate ion cannot exchange for bicarbonate in the light, and a dark period is necessary to enable exchange of formate for bicarbonate.     

Studies on thylakoid phosphorylation and noncyclic electron transport

The effect of thylakoid phosphorylation on noncyclic electron transport in spinach chloroplasts was investigated by measuring both the reduction of nicotinamide adenine dinucleotide phosphate (NADP) and the steady-state redox level of the primary electron acceptor quinone of photosystem II (Q) during electron flow to NADP. These data are compared with the theoretical predictions for an electron transport model which relates both the redox levels of Q and the photosystem II optical cross section to the overall velocity of noncyclic electron flow. It is demonstrated that transfer of 15-20% of the photosystem II antenna to photosystem I may stimulate electron flow to NADP only if Q is less than 60-70% oxidized (this condition exists with our thylakoids, even at extremely low absorption fluxes, when the illumination is not specifically enriched in photosystem I absorbed wavelengths); in phosphorylated thylakoids the steady-state redox level Q is substantially shifted to a more oxidized one (measurements of this parameter using light of different wavelengths quantitatively support the idea that thylakoid phosphorylation leads to increased photosystem I and decreased photosystem II cross sections); thylakoid phosphorylation leads to stimulated noncyclic electron flow to NADP only when the increased photosystem I antenna is able to bring about large increases in the steady-state level of oxidized Q.     

The action of proteolytic enzymes on chloroplast thylakoid membranes

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analyzed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans.

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Sidedness studies of thylakoid phosphatidylglycerol in higher plants.

The transmembrane distribution of phosphatidylglycerol was determined in thylakoids from barley (Hordeum vulgare), lettuce (Lactuca sativa) and pea (Pisum sativum) chloroplasts. Phospholipase A2 and phospholipase D digestion and chemical-labelling methods were used. Phosphatidylglycerol was preferentially localized in the outer (stromal) leaflet. The proportion of the phospholipid in this leaflet ranged from about 66% in pea to about 75% for barley and lettuce thylakoids. One of the main fatty acids, trans-delta 3-hexadecenoic acid, was exclusively located in the outer leaflet in all three plant types. The data are discussed in relation to suggested roles for phosphatidylglycerol in thylakoid function.     

Reprint of: physiology of PSI cyclic electron transport in higher plants

Having long been debated, it is only in the last few years that a concensus has emerged that the cyclic flow of electrons around Photosystem I plays an important and general role in the photosynthesis of higher plants. Two major pathways of cyclic flow have been identified, involving either a complex termed NDH or mediated via a pathway involving a protein PGR5 and two functions have been described-to generate ATP and to provide a pH gradient inducing non-photochemical quenching. The best evidence for the occurrence of the two pathways comes from measurements under stress conditions-high light, drought and extreme temperatures. In this review, the possible relative functions and importance of the two pathways is discussed as well as evidence as to how the flow through these pathways is regulated. Our growing knowledge of the proteins involved in cyclic electron flow will, in the future, enable us to understand better the occurrence and diversity of cyclic electron transport pathways. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

The action of the sesquiterpenic benzoquinone, perezone, on electron transport in biological membranes

Perezone (2-(1,5-dimethyl-4-hexenyl)-3-hydroxymethyl-p-benzoquinone) is a sesquiterpenic benzoquinone isolated from roots of plants of the genus Perezia. It exhibits oxido-reduction characteristics which suggest that the compound can be used for studies of the electron transfer chain of rat liver mitochondria. Perezone at 50 microM inhibits mitochondrial electron transport through a process which differs from that of rotenone, amytal, and Antimycin A. The inhibition is temperature dependent; at 35 degrees C it fails to inhibit valinomycin-induced mitochondrial respiration, but at 20 degrees C it inhibits respiration by 80-90%. Perezone is an electron-donor and electron-acceptor compound that behaves similarly to naphtoquinone. It mediates electron transport from a reaction center preparation isolated from Rhodopseudomonas sphaeroides and added cytochrome c. The low respiration of rat liver mitochondria depleted of coenzyme Q10 (CoQ) is increased by perezone. The electron transport activity of perezone was also demonstrated with CoQ-deficient yeast mutant E3-24.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Effects of severe CO2 starvation on the photosynthetic electron transport chain in tobacco plants

Tobacco plants (Nicotiana tabacum) were kept in CO₂ free air for several days to investigate the effect of lack of electron acceptors on the photosynthetic electron transport chain. CO₂ starvation resulted in a dramatic decrease in photosynthetic activity. Measurements of the electron transport activity in thylakoid membranes showed that a loss of Photosystem II activity was mainly responsible for the observed decrease in photosynthetic activity. In the absence of CO₂ the plastoquinone pool and the acceptor side of Photosystem I were highly reduced in the dark as shown by far-red light effects on chlorophyll fluorescence and P700 absorption measurements. Reduction of the oxygen content of the CO₂ free air retarded photoinhibitory loss of photosynthetic activity and pigment degradation. Electron flow to oxygen seemed not to be able to counteract the stress induced by severe CO₂ starvation. The data are discussed in terms of a donation of reducing equivalents from mitochondria to chloroplasts and a reduction of the plastoquinone pool via the NAD(P)H-plastoquinone oxidoreductase during CO₂ starvation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxygen as an alternative electron acceptor in the photosynthetic electron transport chain of C3 plants

This study deals with effects of oxygen on the kinetics of P(700) photoinduced redox transitions and on induction transients of chlorophyll fluorescence in leaves of C(3) plants Hibiscus rosa-sinensis and Vicia faba. It is shown that the removal of oxygen from the leaf environment has a conspicuous effect on photosynthetic electron transport. Under anaerobic conditions, the concentration of oxidized P700 centers in continuous white light was substantially lower than under aerobic conditions. The deficiency of oxygen released non-photochemical quenching of chlorophyll fluorescence, thus indicating a decrease in the trans-thylakoid pH gradient (DeltapH). Quantitative analysis of experimental data within the framework of an original mathematical model has shown that the steady-state electron flux toward oxygen in Chinese hibiscus leaves makes up to approximately 40% of the total electron flow passing through photosystem 1 (PS1). The decrease in P700+ content under anaerobic conditions can be due to two causes: i) the retardation of electron outflow from PS1, and ii) the release of photosynthetic control (acceleration of electron flow from PS2 to P700+) owing to lower acidification of the intra-thylakoid space. At the same time, cyclic electron transport around PS1 was not stimulated in the oxygen-free medium, although such stimulation seemed likely in view of possible rearrangement of electron flows on the acceptor side of PS1. This conclusion stems from observations that the rates of P700+ reduction in DCMU-poisoned samples, both under aerobic and anaerobic conditions, were negligibly small compared to rates of electron flow from PS2 toward P700+ in untreated samples.