Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. II. Photosynthetic electron transport

Photoinhibition of photosynthesis in Lemna gibba L. was induced by exposing intact plants to a high photosynthetic photon flux density of 1 750 μmol m⁻² s⁻¹ at a low temperature of 3°C. Subsequently isolated chloroplasts showed pronounced reductions in the capacity of whole chain electron transport, measured as Hill activity, and in the efficiency of electron transport to the primary electron acceptor Q of photosystem II, measured as variable chlorophyll fluorescence at 20°C. These changes proceeded with similar kinetics (probably of the first-order reaction), suggesting that the site of photoinhibition is in the electron transfer to Q. A partial uncoupling of the whole chain electron transport also occured. The capacity of electron transport mediated by photosystem I was unaffected. The extent of photoinhibition of photosynthetic electron transport, as produced by a 2 h exposure of L. gibba to three different combinations of photon flux density and temperature was studied. It was shown that intrinsically similar states of photoinhibition, on the evidence of their time courses of recovery, were induced by either a high photon flux density and 25°C or by a moderate photon flux density and 3°C.     

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.     

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(+).     

Metabolic flux analysis in Synechocystis using isotope distribution from 13C-labeled glucose

Using the carbon isotope labeling technique, the response of cyanobacterial central carbon metabolism to the change in environmental conditions was investigated. Synechocystis was grown in the heterotrophic and mixotrophic cultures fed with 13C-labeled glucose. The labeling patterns of the amino acids in biomass hydrolysates for both cultures were detected by the two-dimensional 1H-13C correlation nuclear magnetic resonance (2D 1H-13C COSY NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) technique. The in vivo intracellular flux distributions were then quantitated from the labeling measurements and metabolite balances using a parameters fitting approach. From the estimated flux distributions, it was found that the pentose phosphate pathway was the major pathway of glucose catabolism in the heterotrophic culture, while in the mixotrophic culture, the flux of CO2 fixation through the Calvin cycle was about two-fold of the glucose input flux. The relative flux through the phosphoenolpyruvate carboxylase was very high in both cultures, and this reaction represented about 25% of the assimilated CO2 in the mixotrophic culture. More importantly, we found a substantial outflow from the tricarboxylic acid cycle to glycolysis pathway carried by the malic enzyme, demonstrating the operation of a C4 pathway in cyanobacterial cells through the PEP carboxylase and malic enzyme. The estimated flux distributions also revealed that the NADPH synthesis was in excess relative to its requirement, and the excess NADPH might be reoxidized in cyanobacterial respiration to provide the energy for cellular requirement. Moreover, the analyzed result also suggested that the activity of the respiratory electron transport chain in cyanobacterial cells was not inhibited by light.     

Psb29, a conserved 22-kD protein, functions in the biogenesis of Photosystem II complexes in Synechocystis and Arabidopsis

Photosystem II (PSII), the enzyme responsible for photosynthetic oxygen evolution, is a rapidly turned over membrane protein complex. However, the factors that regulate biogenesis of PSII are poorly defined. Previous proteomic analysis of the PSII preparations from the cyanobacterium Synechocystis sp PCC 6803 detected a novel protein, Psb29 (Sll1414), homologs of which are found in all cyanobacteria and vascular plants with sequenced genomes. Deletion of psb29 in Synechocystis 6803 results in slower growth rates under high light intensities, increased light sensitivity, and lower PSII efficiency, without affecting the PSII core electron transfer activities. A T-DNA insertion line in the PSB29 gene in Arabidopsis thaliana displays a phenotype similar to that of the Synechocystis mutant. This plant mutant grows slowly and exhibits variegated leaves, and its PSII activity is light sensitive. Low temperature fluorescence emission spectroscopy of both cyanobacterial and plant mutants shows an increase in the proportion of uncoupled proximal antennae in PSII as a function of increasing growth light intensities. The similar phenotypes observed in both plant and cyanobacterial mutants demonstrate that the function of Psb29 has been conserved throughout the evolution of oxygenic photosynthetic organisms and suggest a role for the Psb29 protein in the biogenesis of PSII.     

Function, structure and regulation of cyanobacterial and chloroplast ATP synthase

The proton-translocating ATP synthase from chloroplasts and cyanobacteria forms ATP upon photosynthetic electron transport by using the proton gradient across the thylakoid membrane. Both enzymes contain nine different subunits and from the similarity in gene organisation and the high degree of amino acid sequence homology of the subunits it appears that these ATP synthases might have a common ancestor. Both enzymes need to be activated by membrane energisation in order to perform catalytic activity but, in contrast to the chloroplast ATP synthase, that from the studied cyanobacteria (with the exception of Spirulina platensis ) shows no effect of the redox state on activation. Functionally, the cyanobacterial enzyme corresponds to the reduced form of the chloroplast ATP synthase. In the chloroplast enzyme a stretch of 9 amino acids, including two cysteines in the γ-subunit, is involved in this redox effect and this stretch is absent in cyanobacteria. With γ-mutants from the cyanobacterium Synechocystis 6803 the role of this stretch is studied. When active, both the cyanobacterial and the reduced chloroplast ATP synthase transport 4 protons per ATP synthesised and hydrolysed. This ratio may depend on the environment of the enzyme such as protein and lipid composition and pH.