Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O₂ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP⁺ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.     

Thylakoid protein phosphorylation and the thiol redox state

Illumination of thylakoid membranes leads to the phosphorylation of a number of photosystem II-related proteins, including the reaction center proteins D1 and D2 as well as the light-harvesting complex (LHCII). Regulation of light-activated thylakoid protein phosphorylation has mainly been ascribed to the redox state of the electron carrier plastoquinone. In this work, we show that this phosphorylation in vitro is also strongly influenced by the thiol disulfide redox state. Phosphorylation of the light-harvesting complex of photosystem II was found to be favored by thiol-oxidizing conditions and strongly downregulated at moderately thiol-reducing conditions. In contrast, phosphorylation of the photosystem II reaction center proteins D1 and D2 as well as that of other photosystem II subunits was found to be stimulated up to 2-fold by moderately thiol-reducing conditions and kept at a high level also at highly reducing conditions. These responses of the level of thylakoid protein phosphorylation to changes in the thiol disulfide redox state are reminiscent of those observed in vivo in response to changes in the light intensity and point to the possibility of a second loop of redox regulation of thylakoid protein phosphorylation via the ferredoxin-thioredoxin system.     

Influence of clinorotation on some parameters of photosynthetic apparatus

This study aimed to examine the electron transport rates in the thylakoids, isolated from leaves of pea plants grown under clinorotation and in vertical control, to measure the chlorophyll a/b (Chl a/b) ratio in such thylakoids and in photosystem I (PSI) particles isolated from them, to elucidate if there are any differences in changes of PS II activity in thylakoids and Chl a/b ratio in PS I particles under phosphorylation of polypeptides of thylakoid pigment-protein complexes.     

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Electron and proton transport in chloroplasts taking into account lateral heterogeneity of thylakoids. Mathematical model]

A mathematical model of a chloroplast was constructed, which takes into account the inhomogeneous distribution of complexes of photosystems I and II between granal and intergranal thylakoids. The structural and functional complexes of photosystems I and II, which are localized in intergranal and granal thylakoids, respectively, and the b/f complex, which is uniformly distributed in thylakoid membranes, are assumed to be immobile. The interactions between spatially distant electron transport complexes are provided by plastoquinone and plastocyanine, which diffuse in the thylakoid membrane and intrathylakoid space, respectively. The main stages of proton transport associated with the functioning of photosystem II and oxidation-reduction transformations of plastoquinone are considered. The model takes into account the interactions of protons with membrane-bound buffer groups, the lateral diffusion of hydrogen ions in the intrathylakoid space and in the lumen between adjacent granal thylakoids, and the transmembrane proton transport associated with the function of ATP synthase and passive leakage of protons from thylakoids outside. The numerical integration of two systems of differential equations describing the behavior of some variables in two different regions: granal and intergranal thylakoids was performed. The model describes adequately the kinetics of processes being studied and predicts the occurrence of inhomogeneous lateral profiles of proton potentials and redox state of electron carriers. Modeling the electron and proton transport with allowance for the topological features of chloroplasts (lateral heterogeneity of thylakoids) is important for correct interpretation of "power-flux" interactions and the experimentally measured kinetic parameters averaged over the entire spatially inhomogeneous thylakoid system.     

Electron paramagnetic resonance of electron transport in photosynthetic systems. XI. Effects of photosynthetic control: dependence of the rate of electron transport on the energization of bean chloroplast thylakoid membrane

The kinetics of light-induced P700 redox transients in bean chloroplast was studied. It has been shown that the rate of electron transport decreased during few seconds of illumination of coupled chloroplasts without addition of ADP and inorganic phosphate. The evidence were obtained that there is a feedback inhibition of electron transport governed by the internal pH of thylakoid. This results in the overshoot in the kinetics of P700 redox transients induced by continuous actinic light. Under the phosphorylation condition (addition of Mg-ADP and inorganic phosphate) the effect of decreasing of the rate of electron transport between two photosystems was not observed. Addition of uncouplers (FCCP or gramicidine) also increased the steady-state rate of noncyclic electron transport. After adding only Mg-ADP (without phosphate) or Mg-ATP to coupled chloroplasts the effect of the light-driven inhibition of electron transport was observed as in the case of chloroplasts without any additions. We showed that the regulation for the electron transport rate was realized at the step of the plastoquinol oxidation by photosystem 1. Light-driven energization of the thylakoid membrane also leads to the the slowing of the reduction of spin label TEMPO. Evidences were obtained that TEMPO interacts with the semiquinone localized in the acceptor side of photosystem 2. From the comparative study of P700+ and TEMPO reduction by photosystem 2 we have concluded that there are two points of inhibitory action of DCMU localized at the acceptor and donor sides of photosystem 2. The mechanisms of photosynthetic control and the role of transmembrane proton gradient for energy transmission in chloroplasts are discussed.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP(+), and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700(+).     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Computer simulation study of pH-dependent regulation of electron transport in chloroplasts

A mathematical model is presented that describes the key steps of photosynthetic electron transport and transmembrane proton transfer in chloroplasts. Numerical modeling has been performed with due regard for regulatory processes at the donor and acceptor parts of photosystem (PS) I. The influence of pH-dependent activation of the Calvin cycle enzymes and energy dissipation in PS II (nonphotochemical quenching of chlorophyll fluorescence) on the light-induced redox transients of P₇₀₀, plastoquinone, and NADP as well as on the changes in intrathylakoid pH and ATP level is examined. It is demonstrated that pH-dependent regulatory processes alter the distribution of electron fluxes on the acceptor side of PS I and the total rate of electron flow between PS II and PS I. The light-induced activation of the Calvin cycle leads to significant enhancement of the electron flow from PS I to NADP⁺ and attenuation of the electron flow to molecular oxygen.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled "Regulation of Electron Transport in Chloroplasts".     

Reprint of: Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Ferredoxin-Dependent Photosynthetic Electron Transport and Coupled Processes of Energy Storage in Chloroplasts of Wheat Plants Grown under Different Levels of Nitrogen Supply

The rates of electron transfer in the presence of natural cofactors, ferredoxin and NADP, which were added in the amounts catalyzing noncyclic or cyclic electron transfer, were studied in thylakoids isolated from 17-day-old wheat seedlings. Upon excitation of both photosystems (PS) of photosynthesis, the potential rate of NADP reduction in thylakoids isolated from plants grown on nitrogen-free nutrient solution did not differ from that in thylakoids from the control plants. However, the P/2e ratio was significantly lower in thylakoids isolated from nitrogen-deficient plants. On the contrary, in the presence of DCMU, the rate of PSI-driven electron transfer from an artificial donor to NADP was considerably higher in these than in the control thylakoids. In the presence of ferredoxin under anaerobic conditions, the rate of phosphorylation coupled to cyclic electron transport was also significantly higher in thylakoids isolated from nitrogen-deficient plants, than in thylakoids isolated from control plants. Our data show that PSI-driven electron transport and cyclic photophosphorylation are activated in nitrogen-starved wheat plants, at least at the initial stages of starvation.     

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.     

Inhibition of chloroplast electron transport reactions by trifluralin and diallate

The herbicides trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N, N-dipropyl-p-toluidine) and diallate (S-[2,3-dichloroallyl] diisopropylthiocarbamate) inhibit electron transport, ATP synthesis, and cytochrome f reduction by isolated spinach (Spinacia oleracea L.) chloroplasts. Both compounds inhibit noncyclic electron transport from H(2)O to ferricyanide more than 90% in coupled chloroplasts at concentrations less than 50 mum. Neither herbicide inhibits electron transport in assays utilizing only photosystem I activity, and the photosystem II reaction elicited by addition of oxidized p-phenylenediamine or 2,5-dimethylquinone is only partially inhibited by herbicide concentrations which block electron flow from H(2)O to ferricyanide. Inhibition of ATP synthesis parallels inhibition of electron flow in all noncyclic assay systems, and cyclic ATP synthesis catalyzed by either diaminodurene or phenazine metho-sulfate is susceptible to inhibition by both herbicides. These results indicate that trifluralin and diallate both inhibit electron flow in isolated chloroplasts at a point in the electron transport chain between the two photosystems.     

Deletion of the tobacco plastid psbA gene triggers an upregulation of the thylakoid-associated NAD(P)H dehydrogenase complex and the plastid terminal oxidase (PTOX)

We have constructed a tobacco psbA gene deletion mutant that is devoid of photosystem II (PSII) complex. Analysis of thylakoid membranes revealed comparable amounts, on a chlorophyll basis, of photosystem I (PSI), the cytochrome b6f complex and the PSII light-harvesting complex (LHCII) antenna proteins in wild-type (WT) and Δ psbA leaves. Lack of PSII in the mutant, however, resulted in over 10-fold higher relative amounts of the thylakoid-associated plastid terminal oxidase (PTOX) and the NAD(P)H dehydrogenase (NDH) complex. Increased amounts of Ndh polypeptides were accompanied with a more than fourfold enhancement of NDH activity in the mutant thylakoids, as revealed by in-gel NADH dehydrogenase measurements. NADH also had a specific stimulating effect on P700⁺ re-reduction in the Δ psbA thylakoids. Altogether, our results suggest that enhancement of electron flow via the NDH complex and possibly other alternative electron transport routes partly compensates for the loss of PSII function in the Δ psbA mutant. As mRNA levels were comparable in WT and Δ psbA plants, upregulation of the alternative electron transport pathways (NDH complex and PTOX) occurs apparently by translational or post-translational mechanisms.