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.     

Effect of cadmium on photosystem II activity in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: alteration of O–J–I–P fluorescence transients indicating the change of apparent activation energies within photosystem II

In this study, we evaluated how cadmium inhibitory effect on photosystem II and I electron transport may affect light energy conversion into electron transport by photosystem II. To induce cadmium effect on the photosynthetic apparatus, we exposed Chlamydomonas reinhardtii 24 h to 0–4.62 μM Cd²⁺. By evaluating the half time of fluorescence transients O–J–I–P at different temperatures (20–30°C), we were able to determine the photosystem II apparent activation energies for different reduction steps of photosystem II, indicated by the O–J–I–P fluorescence transients. The decrease of the apparent activation energies for PSII electron transport was found to be strongly related to the cadmium-induced inhibition of photosynthetic electron transport. We found a strong correlation between the photosystem II apparent activation energies and photosystem II oxygen evolution rate and photosystem I activity. Different levels of cadmium inhibition at photosystem II water-splitting system and photosystem I activity showed that photosystem II apparent activation energies are strongly dependent to photosystem II donor and acceptor sides. Therefore, the oxido-reduction state of whole photosystem II and I electron transport chain affects the conversion of light energy from antenna complex to photosystem II electron transport.     

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⁺.     

Lack of the small plastid-encoded PsbJ polypeptide results in a defective water-splitting apparatus of photosystem II, reduced photosystem I levels, and hypersensitivity to light

Photosystem II is a large pigment-protein complex catalyzing water oxidation and initiating electron transfer processes across the thylakoid membrane. In addition to large protein subunits, many of which bind redox cofactors, photosystem II particles contain a number of low molecular weight polypeptides whose function is only poorly defined. Here we have investigated the function of one of the smallest polypeptides in photosystem II, PsbJ. Using a reverse genetics approach, we have inactivated the psbJ gene in the tobacco chloroplast genome. We show that, although the PsbJ polypeptide is not principally required for functional photosynthetic electron transport, plants lacking PsbJ are unable to grow photoautotrophically. We provide evidence that this is due to the accumulation of incompletely assembled water-splitting complexes, which in turn causes drastically reduced photosynthetic performance and extreme hypersensitivity to light. Our results suggest a role of PsbJ for the stable assembly of the water-splitting complex of photosystem II and, in addition, support a control of photosystem I accumulation through photosystem II activity.     

Proposals for the naming of chloroplast genes. II. Update to the nomenclature of genes for thylakoid membrane polypeptides

The nomenclature for genes for components of the photosynthetic membranes has been reviewed and updated. Newly discovered genes have been added to the existing convention for gene nomenclature. Genes designatedpetA throughpetI are described for components of the photosynthetic electron transport systems,psaA throughpsaK for photosystem I components, andpsbA throughpsbR for photosystem II, including the extrinsic polypeptides of the oxygen-evolving complex. References for representative examples of each gene are given.     

Properties of Photosynthetic Mutants Isolated from Euglena gracilis

Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation.     

Electron donors and acceptors in photosynthetic reaction centers

Summary A review is given of primary and associated electron transport reactions in various division of photosynthetic bacteria and in the two photosystems of plant photosynthesis. Two types of electron acceptor chains are distinguished: type Q, found in purple bacteria, Chloroflexus and system II of oxygenic photosynthesis and type F, found in green sulfur bacteria, Heliobacterium and photosystem I. Secondary donor reactions are discussed in relation to plant photosystem II.Dedicated to the memory of Warren L. Butler     

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b ₆/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.Abbreviations ENDOR electron nuclear double resonance - ESEEM electron spin echo envelope modulation - LHC light harvesting complex - PSI Photosystem I - PS II Photosystem II - P₆₈₀ primary electron donor in PS II - P₇₀₀ primary electron donor in PS I     

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.     

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.     

Influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation, regulation and organisation of photosynthesis in Solanum nigrum

The influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation of the photosynthetic apparatus was examined on 4-weeks-old climate chamber-grown Solanum nigrum plant. To have an indication on the relative performance of the photosynthetic apparatus of ALS-treated plants, the level of carbon dioxide (CO₂) fixation, the relative quantum efficiency of photosystem I (Φ_PSI) or photosystem II (Φ_PSII) electron transport and leaf chlorophyll content were assessed for both control and treated plants at 2, 4 and 7 days after application of the herbicide. Results indicated a progressive inhibition of the level of CO₂ fixation, the relative quantum efficiency of photosystem I (Ф_PSI) and II (Ф_PSII) electron transport and the leaf chlorophyll content already 2 days after application of the herbicide. The linear relationship between the photosystem I and II was unaltered by herbicidal treatment and was sustained under conditions where large changes in pigment composition of the leaves occurred. It appears that the stress-induced loss of leaf chlorophyll is not a catastrophic process but rather is the consequence of a well-organised breakdown of components. Under photorespiratory and non-photorespiratory conditions, the relationship between the index of electron transport flow through photosystem I and II and the rate of CO₂ fixation is altered so that electron transport becomes less efficient at driving CO₂ fixation.     

Stimulation and inhibition of photosystem II electron transport in cyanobacteria by ions interacting with the cytoplasmic face of thylakoids

The mechanism by which suspension medium ions regulate the rate of photoinduced electron transport across photosystem II was investigated with ion permeabilized cells of the cyanobacterium Anacystis nidulans. Electron transport was measured as the reduction of the electroneutral acceptor dichlorophenol indophenol, whose surface concentration is independent of electrostatic membrane potential. Potassium salts stimulate photoinduced electron transport at low concentrations and inhibit it at higher concentrations. No inhibition is observed when an antichaotropic anion is associated with potassium, while the inhibition is more severe the stronger the chaotropic character of the anion. Neutralization of the surface charge by potassium ions ligated to negatively charged membrane sites at the cytoplasmic side is a prerequisite for the expression of the chaotropic inhibition of photosystem II electron transport.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPC 1,5-diphenyl carbazide - FeCN ferricyanide anion - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - PS photosystem - TEC³⁺ tris ethylene diamine cobalt cation     

Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase

The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO₂ (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously.     

Acclimation to high-light conditions in cyanobacteria: from gene expression to physiological responses

Photosynthetic organisms have evolved various acclimatory responses to high-light (HL) conditions to maintain a balance between energy supply (light harvesting and electron transport) and consumption (cellular metabolism) and to protect the photosynthetic apparatus from photodamage. The molecular mechanism of HL acclimation has been extensively studied in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Whole genome DNA microarray analyses have revealed that the change in gene expression profile under HL is closely correlated with subsequent acclimatory responses such as (1) acceleration in the rate of photosystem II turnover, (2) downregulation of light harvesting capacity, (3) development of a protection mechanism for the photosystems against excess light energy, (4) upregulation of general protection mechanism components, and (5) regulation of carbon and nitrogen assimilation. In this review article, we survey recent progress in the understanding of the molecular mechanisms of these acclimatory responses in Synechocystis sp. PCC 6803. We also briefly describe attempts to understand HL acclimation in various cyanobacterial species in their natural environments.     

Effects of moderate heat stress on photosynthesis: importance of thylakoid reactions, rubisco deactivation, reactive oxygen species, and thermotolerance provided by isoprene

Photosynthesis is particularly sensitive to heat stress and recent results provide important new insights into the mechanisms by which moderate heat stress reduces photosynthetic capacity. Perhaps most surprising is that there is little or no damage to photosystem II as a result of moderate heat stress even though moderate heat stress can reduce the photosynthetic rate to near zero. Moderate heat stress can stimulate dark reduction of plastoquinone and cyclic electron flow in the light. In addition, moderate heat stress may increase thylakoid leakiness. At the same time, rubisco deactivates at moderately high temperature. Relationships between effects of moderate heat on rubisco activation and thylakoid reactions are not yet clear. Reactive oxygen species such as H₂O₂ may also be important during moderate heat stress. Rubisco can make hydrogen peroxide as a result of oxygenase side reactions and H₂O₂ production by rubisco was recently shown to increase substantially with temperature. The ability to withstand moderately high temperature can be improved by altering thylakoid lipid composition or by supplying isoprene. In my opinion this indicates that thylakoid reactions are important during moderate heat stress. The deactivation of rubisco at moderately high temperature could be a parallel deleterious effect or a regulatory response to limit damage to thylakoid reactions.     

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.     

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes

The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion.     

Chloroplast Reactions of Photosynthetic Mutants in Zea mays

Three seedling lethal mutants of Zea mays with impaired photosynthesis are described. These recessive mutants were selected on the basis of high chlorophyll fluorescence. They have normal chlorophyll pigmentation but are unable to fix CO₂ fully. Evidence is presented from fluorescence characteristics of isolated chloroplasts that both photosystem I and II mutants were isolated. Using conventional measures of photosynthetic electron transport, we suggest that the photosystem I mutant has limited ability to reduce NADP. The other two mutants are clearly blocked in photosystem II, one possibly lacking the primary electron acceptor.     

Inhibition of chlororespiration by myxothiazol and antimycin A in Chlamydomonas reinhardtii

Myxothiazol and antimycin A are shown to suppress the oxygen transient previously attributed to the flash-induced inhibition of chlororespiration in Chlamydomonas reinhardtii (Peltier et al. 1987, Biochim Biophys Acta 893: 83–90). However, these two compounds do not affect the photosynthetic electron transport chain as inferred by the insensitivity of the CO₂-dependent photosynthetic O₂ evolution and of the flash-induced electrochromic effect. Chlorophyll fluorescence induction measurements carried out in dark-adapted cells of a mutant of Chlamydomonas lacking photosystem 1, show that myxothiazol and antimycin A significantly increase the redox state of the photosystem 2 acceptors. We conclude from these results that chlororespiration is inhibited by myxothiazol and antimycin A and that the site of inhibition is located on the dark oxidation pathway of the plastoquinone pool. This inhibition is interpreted through the involvement of a myxothiazol and antimycin A sensitive cytochrome in the chlororespiratory chain.Abbreviations cyt cytochrome - PQ plastoquinone - PS photosystem