Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis

In this study we focused on the effects of light irradiation and the addition of L-galactono-1,4-lactone (L-GalL) on the conversion of exogenous L-GalL to L-ascorbate (AsA) and the total AsA pool size in detached leaves of Arabidopsis plants and transgenic plants expressing the rat L-gulono-1,4-lactone oxidase gene. Increases in the total AsA level in L-GalL-treated leaves depended entirely on light irradiation. Treatment with an inhibitor of photosynthetic electron transport together with L-GalL reduced the increase in total AsA under light. Light, particularly the redox state of photosynthetic electron transport, appeared to play an important role in the regulation of the conversion of L-GalL to AsA in the mitochondria, reflecting the cellular level of AsA in plants.     

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     

Modeling of the photosynthetic electron transport regulation in cyanobacteria

In this work we have performed a computer analysis of electron and proton transport in cyanobacterial cells using a mathematical model of light-dependent stages of photosynthesis taking into account the key stages of pH-dependent regulation of electron transport on both acceptor and donor sides of photosystem 1 (PS1). Comparison of theoretical and experimental data shows that the model adequately describes the multiphase kinetics of photoinduced redox transformations of P₇₀₀ (the primary electron donor in PS1). Our computer simulation describes the effect of variations of atmospheric gases (CO₂ and O₂) on the induction events in cyanobacteria (P₇₀₀ photooxidation, generation of transmembrane ΔpH), which strongly depends on the preillumination conditions (aerobic or anaerobic atmosphere). It has been shown that the variations of CO₂ concentration in the cell interior may noticeably affect the kinetics of electron transport, acidification of lumen, and ATP synthesis. The contributions of alternative pathways of electron transport (cyclic electron transport around PS1 and electron outflow to O₂) to the function of cyanobacterial photosynthetic apparatus have been analyzed. At the initial stage of induction period, cyclic electron flows around PS1 (“short” and “long” pathways) substantially contribute to photosynthetic electron transport. These flows, however, attenuate with the light-induced activation of the Calvin-Benson cycle reactions. In the meantime, the outflow of electrons from PS1 to O₂ (or to other metabolic chains) increases with oxygen accumulation in the medium. The effects of ferredoxin oxidation by hydrogenase catalyzing the H₂ formation on the kinetics of P700 photooxidation and distribution of electron flows on the acceptor side of PS1 have been modeled.     

The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency

Copper is an essential micronutrient that functions as a redox cofactor in multiple plant processes, including photosynthesis. Arabidopsis thaliana possesses a conserved family of CTR-like high-affinity copper transport proteins denoted as COPT1-5. COPT1, the only family member that is functionally characterized, participates in plant copper acquisition. However, little is known about the function of the other Arabidopsis COPT proteins in the transport and distribution of copper. Here, we show that a functional fusion of COPT5 to the green fluorescent protein localizes in Arabidopsis cells to the prevacuolar compartment. Plants defective in COPT5 do not exhibit any significant phenotype under copper-sufficient conditions, but their growth is compromised under copper limitation. Under extreme copper deficiency, two independent copt5 knockout mutant lines exhibit severe defects in vegetative growth and root elongation, low chlorophyll content, and impairment in the photosynthetic electron transfer. All these phenotypes are rescued when the wild-type copy of the COPT5 gene is retransformed into a copt5 knockout line or when copper, but not other metals, are added to the medium. COPT5 is expressed in vascular tissues, with elevated levels in roots. Taken together, these results suggest that COPT5 plays an important role in the plant response to environmental copper scarcity, probably by remobilizing copper from prevacuolar vesicles, which could act as internal stores or recycling vesicles to provide the metal cofactor to key copper-dependent processes such as photosynthesis.     

Participation of ferredoxin in oxygen reduction by the photosynthetic electron transport chain

Oxygen reduction by isolated pea thylakoids was studied in the presence of ferredoxin (Fd), Fd + NADP, and cytochrome c. At Fd concentrations optimal for NADP reduction, it contributed 30–50% of the reducing equivalents (as deduced by comparing the rates of oxygen reduction and light oxidation of reduced Fd). The oxygen reduction rate in the presence of Fd + NADP was 3–4 times lower than with Fd alone, and comparable to that with cyt c. It is supposed that the process involves a photosystem I component whose reaction with oxygen depends on the rate of electron efflux from the PS I terminal acceptors, and that this component is phylloquinone.     

Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-gamma-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.     

Plastocyanin is indispensable for photosynthetic electron flow in Arabidopsis thaliana

Plastocyanin is a soluble copper-containing protein present in the thylakoid lumen, which transfers electrons to photosystem I. In the chloroplast of the flowering plant Arabidopsis thaliana, a cytochrome c6-like protein is present, which was recently suggested to function as an alternative electron carrier to plastocyanin. We show that Arabidopsis plants mutated in both of the two plastocyanin-coding genes and with a functional cytochrome c6 cannot grow photoautotrophically because of a complete block in light-driven electron transport. Even increased dosage of the gene encoding the cytochrome c6-like protein cannot complement the double mutant phenotype. This demonstrates that in Arabidopsis only plastocyanin can donate electrons to photosystem I in vivo.     

On the site of electron donation to the photosynthetic electron transport chain by 1,5-diphenylcarbazide

A solubilized base-exchange enzyme activity was dependent upon the addition of phospholipids, added Ca⁺⁺ ion, and had optimum pH of 7.2. Phosphatidyl-ethanolamine was found to be the best stimulator of both[¹⁴C]-ethanolamine and [¹⁴C]-serine incorporation. Preliminary evidence suggests the presence of phospholipase D type activity in this solubilized preparation.     

Metal-induced inhibition of photosynthetic electron transport chain of the cyanobacterium Nostoc muscorum

Abstract: This study presents information on the mechanism of inhibition of the photosynthetic electron transport of Nostoc muscorum by chromium (Cr) and lead (Pb). Photosystem II (PS II) was found to be more sensitive both to low and high concentrations of test metals used. A considerable inhibition of photosystem I (PS I) was, however, observed at high concentrations only. Although Cr-induced inhibition of DCPIP photoreduction and lowering of chlorophyll a (Chl a ) fluorescence intensity ( F ₆₈₅) could not be reversed by artificial electron donors (diphenyl-carbazide (DPC), NH₂OH, MnCl₂ and benzidine) of PS II, these electron donors did substantially reverse the Pb-induced inhibition of DCPIP photoreduction as well as the lowering of Chl a fluorescence. Nevertheless, an increase in Chl a fluorescence at high concentrations of Pb suggested that this metal also arrests electron flow on the reducing side of the PS II reaction centre. Besides this, the suppression of fluorescence intensity of phycocyanin at low concentrations of both metals points to the involvement of phycobilisomes in the inhibition of PS II activity. The present study demonstrates that the modes of action of Cr and Pb on PS II are quite different.     

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.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

Divergent regulation of the HEMA gene family encoding glutamyl-tRNA reductase in Arabidopsis thaliana: expression of HEMA2 is regulated by sugars,but is independent of light and plastid signalling

The synthesis of 5-aminolevulinic acid (ALA) is a key regulatory step for the production of hemes and chlorophyll via the tetrapyrrole synthesis pathway. The first enzyme committed to ALA synthesis is glutamyl-tRNA reductase encoded in Arabidopsis by a small family of nuclear-encoded HEMA genes. To better understand the regulation of the tetrapyrrole synthesis pathway we have made a detailed study of HEMA2 expression with transgenic Arabidopsis thaliana L. Col. plants carrying chimeric HEMA2 promoter:gusA fusion constructs. Our results show that the HEMA2 promoter directs expression predominantly to roots and flowers, but that HEMA2 is also expressed at low levels in photosynthetic tissues. Deletion analysis of the HEMA2 promoter indicates that a ca. 850 bp fragment immediately upstream of the HEMA2 coding region is sufficient to drive regulated gusA expression. In contrast to HEMA1, HEMA2 is not up-regulated by red, far-red, blue, UV or white light. In addition, elimination of a promotive plastid signal by Norflurazon-induced photobleaching of plastids had no effect on HEMA2 expression while being required for normal white-light induction of HEMA1. HEMA2 expression in the cotyledons is inhibited by the presence of sucrose or glucose, but not fructose, and this response is light-independent. HEMA1 expression in cotyledons is also inhibited by sugars, but in a strictly light-dependent manner. The roles of HEMA1 and HEMA2 in meeting cellular tetrapyrrole requirements are discussed.     

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.     

Functional size of photosynthetic electron transport chain determined by radiation inactivation

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H₂O to methylviologen) was 623 ± 37 kilodaltons; for photosystem II (H₂O to dimethylquinone/ferricyanide), 174 ± 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 ± 11 kilodaltons. The difference between 364 ± 22 (the sum of 174 ± 11 and 190 ± 11) kilodaltons and 623 ± 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b₆/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.     

Divergent regulation of the HEMA gene family encoding glutamyl-tRNA reductase in Arabidopsis thaliana: expression of HEMA2 is regulated by sugars,but is independent of light and plastid signalling

The synthesis of 5-aminolevulinic acid (ALA) is a key regulatory step for the production of hemes and chlorophyll via the tetrapyrrole synthesis pathway. The first enzyme committed to ALA synthesis is glutamyl-tRNA reductase encoded in Arabidopsis by a small family of nuclear-encoded HEMA genes. To better understand the regulation of the tetrapyrrole synthesis pathway we have made a detailed study of HEMA2 expression with transgenic Arabidopsis thaliana L. Col. plants carrying chimeric HEMA2 promoter:gusA fusion constructs. Our results show that the HEMA2 promoter directs expression predominantly to roots and flowers, but that HEMA2 is also expressed at low levels in photosynthetic tissues. Deletion analysis of the HEMA2 promoter indicates that a ca. 850 bp fragment immediately upstream of the HEMA2 coding region is sufficient to drive regulated gusA expression. In contrast to HEMA1, HEMA2 is not up-regulated by red, far-red, blue, UV or white light. In addition, elimination of a promotive plastid signal by Norflurazon-induced photobleaching of plastids had no effect on HEMA2 expression while being required for normal white-light induction of HEMA1. HEMA2 expression in the cotyledons is inhibited by the presence of sucrose or glucose, but not fructose, and this response is light-independent. HEMA1 expression in cotyledons is also inhibited by sugars, but in a strictly light-dependent manner. The roles of HEMA1 and HEMA2 in meeting cellular tetrapyrrole requirements are discussed.     

Effect of light quality on the organization of photosynthetic electron transport chain of pea seedlings

The activity of NADP and O₂ photoreduction by water is essentially higher in chloroplasts isolated from pea seedlings (Pisum sativum L.) grown under blue light as compared with that from plants grown under red light. In contrast, the photoreduction of NADP and O₂ with photosystem I only is practically the same or even lower in chloroplasts isolated from plants grown under blue light. The addition of plastocyanin does not affect the rate or the extent of NADP photoreduction by water in the chloroplasts isolated from plants grown under blue light, whereas it sharply activates NADP reduction in the chloroplasts isolated from plants grown under red light. The extent of the light-induced oxidation of cytochrome f is appreciably higher in chloroplasts isolated from plants grown under blue light. Cytochrome b₅₅₉ plays the predominant role in the oxidoreductive reactions of these chloroplasts. Furthermore, the fluorescence measurements indicate more effective transfer of excitation energy from chlorophyll to the photosystem II reaction center in chloroplasts isolated from plants grown under blue light.     

Phylogenetic viewpoints on regulation of light harvesting and electron transport in eukaryotic photosynthetic organisms

The comparative study of photosynthetic regulation in the thylakoid membrane of different phylogenetic groups can yield valuable insights into mechanisms, genetic requirements and redundancy of regulatory processes. This review offers a brief summary on the current understanding of light harvesting and photosynthetic electron transport regulation in different photosynthetic eukaryotes, with a special focus on the comparison between higher plants and unicellular algae of secondary endosymbiotic origin. The foundations of thylakoid structure, light harvesting, reversible protein phosphorylation and PSI-mediated cyclic electron transport are traced not only from green algae to vascular plants but also at the branching point between the “green” and the “red” lineage of photosynthetic organisms. This approach was particularly valuable in revealing processes that (1) are highly conserved between phylogenetic groups, (2) serve a common physiological role but nevertheless originate in divergent genetic backgrounds or (3) are missing in one phylogenetic branch despite their unequivocal importance in another, necessitating a search for alternative regulatory mechanisms and interactions.     

Effect of phloridzin on photosynthetic electron transport

Phloridzin (2',4',6',4-tetraoxyhydrochalcon-2'-glucoside) was used to study the localization of synthesis of ATP in the electrontransporting chain of photosynthesis. It was shown that phloridzin inhibits the rate of photoreduction of NADP+ by isolated pea chloroplasts by 40%, electron transport via cytochrome f by 100% and via plastocyanin--by 50%. The "crossover" experiments demonstrated that phloridzin inhibits ADP-induced photoreduction of cytochrome f, having no effect on plastocyanin under identical conditions. It is assumed that the site of ATP synthesis is localized on the reduced site of cytochrome f, while the carrier itself is located in the electron transporting chain coupled to phosphorylation. It is possible that only part of the plastocyanin molecules are located in the phosphorylating pathway of electron transport.