Altered photosynthetic electron channelling into cyclic electron flow and nitrite assimilation in a mutant of ferredoxin:NADP(H) reductase

The mechanism by which plants regulate channelling of photosynthetically derived electrons into different areas of chloroplast metabolism remains obscure. Possible fates of such electrons include use in carbon assimilation, nitrogen assimilation and redox signalling pathways, or return to the plastoquinone pool through cyclic electron flow. In higher plants, these electrons are made accessible to stromal enzymes, or for cyclic electron flow, as reduced ferredoxin (Fd), or NADPH. We investigated how knockout of an Arabidopsis ( Arabidopsis thaliana ) ferredoxin:NADPH reductase (FNR) isoprotein and the loss of strong thylakoid binding by the remaining FNR in this mutant affected the channelling of photosynthetic electrons into NADPH- and Fd-dependent metabolism. Chlorophyll fluorescence data show that these mutants have complex variation in cyclic electron flow, dependent on light conditions. Measurements of electron transport in isolated thylakoid and chloroplast systems demonstrated perturbed channelling to NADPH-dependent carbon and Fd-dependent nitrogen assimilating metabolism, with greater competition in the mutant. Moreover, mutants accumulate greater biomass than the wild type under low nitrate growth conditions, indicating that such altered chloroplast electron channelling has profound physiological effects. Taken together, our results demonstrate the integral role played by FNR isoform and location in the partitioning of photosynthetic reducing power.     

Participation of ferredoxin in oxygen reduction in a photosynthetic electron-transport chain

Oxygen reduction in a photosynthetic electron-transport chain (PETC) was studied in isolated pea thylakoids in the presence of either ferredoxin, or ferredoxin + NADP+, or cytochrome c. The contribution of the electron flow through ferredoxin to the total oxygen reduction was evaluated by comparing the rate of oxygen reduction and the rate of oxidation of reduced ferredoxin in the light. It was found that at ferredoxin concentrations optimal for NADP+ reduction, 30-50% of electrons transferred to oxygen went through ferredoxin both in the absence and presence of NADP+. However, the absolute rate of oxygen reduction by membrane components of PETC in the presence of NADP+ was 3-4 times less than that in the presence of ferredoxin alone and close to the rate of oxygen reduction in the presence of cytochrome c. It was assumed that a Photosystem I component, whose role in this process depends on the rate of electron outflow from terminal acceptors of this photosystem, participates in oxygen reduction, and this component is phylloquinone.     

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

Effect of copper deficiency on photosynthetic electron transport in wheat plants

Copper deficiency in wheat ( Triticum aestivum L. cv. Nazareno Stramppeli) markedly affects photosynthetic activity. Flag leaves of copper-deficient plants showed a 50% reduction of the photosynthetic rate expressed as mg CO₂ dm⁻²h⁻¹. The activities of PSI and PSII, determined for isolated chloroplasts, as well as fluorescence measurements on intact leaves of copper-deficient plants, indicated a low activity of photosynthetic electron transport. Ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity was not affected by copper deficiency but copper deficiency affected the chloroplast ultrastructure, especially at the level of grana, where a disorganization of thylakoids is evident.     

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP⁺. The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 μM in the absence of NADP⁺ increased the proportion of oxygen reduction by Fd from 25–35 to 40–60% in different experiments. This proportion decreased with increasing light intensity. When NADP⁺ was added in the presence of 15 μM Fd, which was optimal for the NADP⁺ reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP⁺ and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.     

Feedback regulation of photosynthetic electron transport by NADP(H) redox poise

When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process — feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines — one having reduced levels of ferredoxin NADP⁺-reductase (FNR), the enzyme responsible for reducing NADP⁺, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.     

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.Abbreviations Chl Chlorophyll - EDTA Ethylenediamine Tetraacetic Acid - FeCN Potassium Ferricyanide - Hepes N-2-Hydroxyethylpiperazine-N¹-2-ethanesulfonic acid - pBQ Para-benzoquinone - PSII photosystem II     

Alternative photosynthetic electron flow to oxygen in marine Synechococcus

Cyanobacteria dominate the world's oceans where iron is often barely detectable. One manifestation of low iron adaptation in the oligotrophic marine environment is a decrease in levels of iron-rich photosynthetic components, including the reaction center of photosystem I and the cytochrome b₆f complex [R.F. Strzepek and P.J. Harrison, Photosynthetic architecture differs in coastal and oceanic diatoms, Nature 431 (2004) 689-692.]. These thylakoid membrane components have well characterised roles in linear and cyclic photosynthetic electron transport and their low abundance creates potential impediments to photosynthetic function. Here we show that the marine cyanobacterium Synechococcus WH8102 exhibits significant alternative electron flow to O₂, a potential adaptation to the low iron environment in oligotrophic oceans. This alternative electron flow appears to extract electrons from the intersystem electron transport chain, prior to photosystem I. Inhibitor studies demonstrate that a propyl gallate-sensitive oxidase mediates this flow of electrons to oxygen, which in turn alleviates excessive photosystem II excitation pressure that can often occur even at relatively low irradiance. These findings are also discussed in the context of satisfying the energetic requirements of the cell when photosystem I abundance is low.     

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.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO₂ concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

高温伤害光合机构原初位点的研究进展

本文介绍了高温伤害光合机构原初位点的研究进展,分析了不同观点产生的可能原因,为进一步研究高温对植物光合作用的影响提供思路。     

Measurements of mesophyll conductance,photosynthetic electron transport and alternative electron sinks of field grown wheat leaves

Photosynthetic electron transport drives the carbon reduction cycle, the carbon oxidation cycle, and any alternative electron sinks such as nitrogen reduction. A chlorophyll fluorescence— based method allows estimation of the total electron transport rate while a gas-exchange-based method can provide estimates of the electron transport needed for the carbon reduction cycle and, if the CO₂ partial pressure inside the chloroplast is accurately known, for the carbon oxidation cycle. The gas-exchange method cannot provide estimates of alternative electron sinks. Photosynthetic electron transport in flag leaves of wheat was estimated by the fluorescence method and gasexchange method to determine the possible magnitude of alternative electron sinks. Under non-photorespiratory conditions the two measures of electron transport were the same, ruling out substantial alternative electron sinks. Under photorespiratory conditions the fluorescence-based electron transport rate could be accounted for by the carbon reduction and carbon oxidation cycle only if we assumed the CO₂ partial pressure inside the chloroplasts to be lower than that in the intercellular spaces of the leaves. To further test for the presence of alternative electron sinks, carbon metabolism was inhibited by feeding glyceraldehyde. As carbon metabolism was inhibited, the electron transport was inhibited to the same degree. A small residual rate of electron transport was measured when carbon metabolism was completely inhibited which we take to be the maximum capacity of alternative electron sinks. Since the alternative sinks were small enough to ignore, the comparison of fluorescence and gas-exchange based methods for measuring the rate of electron transport could be used to estimate the mesophyll conductance to CO₂ diffusion. The mesophyll conductance estimated this way fell as wheat flag leaves senesced. The age-related decline in photosynthesis may be attributed in part to the reduction of mesophyll conductance to CO₂ diffusion and in part to the estimated decline of ribulose 1,5-bisphosphate carboxylase amount.     

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co‐factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF‐deficient mutant plants, and we show that re‐initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.     

Control of misses in oxygen evolution by the oxido-reduction state of plastoquinone in Dunaliella tertiolecta

The way misses happen in oxygen evolution is subject to debate (Govindjee et al. 1985). We recently observed a linear lowering of the miss probability with the flash number (Meunier and Popovic 1989). Therefore, we investigated in Dunaliella tertiolecta the link between the average miss probability and the redox state of plastoquinone after n flashes. The effect of flashes was to oxidize the plastoquinone pool; we found that the oxidation of plastoquinone highly correlated (linear regression: R ²=0.996) with the lowering of the miss probability. The flash frequency was found to affect both the miss probability and the redox state of plastoquinone. When pre-flashes were given using a high flash frequency (10 Hz), the plastoquinone pool was oxidized and misses were low; however, if long dark intervals between flashes were used, the oxidizing effect of flashes was lost and the misses were high. We could not explain our results by assuming equal misses over all S-states; but unequal misses, caused by deactivations, were coherent with our results. We deduced that chlororespiration was responsible for the reduction of plastoquinone in the dark interval between flashes. We compared oxygen evolution with and without benzoquinone, using a low flash frequency (0.5 Hz) for maximum misses. Benzoquinone lowered the misses from 34% to 3%, and raised the amplitude of oxygen evolution by more than a factor of two (2). From this we deduced that the charge carrier C postulated to explain misses (Lavorel and Maison-Peteri 1983) did not account for more than 3% of miss probability in Dunaliella tertiolecta. These results indicate that the misses in oxygen evolution are controlled by the redox state of plastoquinone, through deactivations.     

Light-driven reduction of oxygen as a method for studying electron transport in the green photosynthetic bacterium Chlorobium limicola

The use of O₂ uptake as a valid assay for non-cyclic photosynthetic electron flow in membranes from Chlorobium limicola is discussed. It is recommended that methyl viologen, catalase and superoxide dismutase should be added to the experimental medium. The addition of methyl viologen more than doubled the rate of O₂ uptake observed on illumination with 1 mM sulphide as donor. Superoxide dismutation was shown to be efficient under the experimental conditions by means of standard additions of potassium superoxide dissolved in dimethylsulphoxide. The highest rates of light stimulated O₂ uptake were obtained with sulphide as electron donor, and approached 50 mol O₂ · h^-1 · mg bacteriochlorophyll c ^-1 with 0.2 mM sulphide. The presence of 5 mM 2-mercaptoethanol or 3 mM sulphite as electron donor led to lower light stimulated rates of O₂ uptake, while 5 mM thiosulphate had little effect. The rates were insensitive to uncoupler. The light stimulated O₂ uptake with 0.2 mM sulphide as donor was 20–30% inhibited by 10 M antimycin A and 50 M cyanide.Abbreviations APS Adenosine 5-phosphosulphate - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MeV methyl viologen - P-840 the photoreactive bacteriochlorophyll     

Regulation of the photosynthetic electron transport chain

The regulation of electron transport between photosystems II and I was investigated in the plant Silene dioica L. by means of measurement of the kinetics of reduction of P₇₀₀ following a light-to-dark transition. It was found that, in this species, the rate constant for P₇₀₀ reduction is sensitive to light intensity and to the availability of CO₂. The results indicated that at 25 °C the rate of electron transport is down-regulated by approximately 40–50% relative to the maximum rate achievable in saturating CO₂ and that this down-regulation can be explained by regulation of the electron transport chain itself. Measurements of the temperature sensitivity of this rate constant indicated that there is a switch in the rate-limiting step that controls electron transport at around 20 °C: at higher temperatures, CO₂ availability is limiting; at lower temperatures some other process regulates electron transport, possibly a diffusion step within the electron transport chain itself. Regulation of electron transport also occurred in response to drought stress and sucrose feeding. Measurements of non-photochemical quenching of chlorophyll fluorescence did not support the idea that electron transport is regulated by the pH gradient across the thylakoid membrane, and the possibility is discussed that the redox potential of a stromal component may regulate electron transport. Received: 4 March 1999 / Accepted: 25 May 1999     

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.     

Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

Chloroplast-encoded small subunits of the three multiprotein complexes in photosynthetic electron transport

The photosystem I, photosystem II, and cytochromeb ₆ f complexes that are involved in electron transport of oxygenic photosynthesis consist of a number of subunits encoded by either the chloroplast or nuclear genomes. In addition to the major subunits that carry redox components or photosynthetic pigments, these complexes contain several to more than ten subunits with molecular masses of less than 10 kDa. Directed mutagenesis has served as a powerful tool for investigation of the roles of these small subunits in the organization or function of the complexes. Various chloroplast transformants of the green algaChlamydomonas reinhardtii and mutants of cyanobacteria in which a gene encoding a small subunit was deleted or altered have been constructed. Evidence has accumulated suggesting that these small subunits function in the assembly, stabilization, or protection from photoinhibition of the complexes or in the modulation or regulation of electron transport. This article presents an overview of the properties and functions of the chloroplast-encoded small subunits of the three multiprotein complexes of photosynthetic electron transport that have been mainly analyzed with chloroplast transformants ofC. reinhardtii and the corresponding cyanobacterial transformants. Recipient of the Botanical Society Award for Young Scientists, 1995.