No abstract

In nature, plants are challenged by constantly changing light conditions. To reveal the molecular mechanisms behind acclimation to sometimes drastic and frequent changes in light intensity, we grew Arabidopsis thaliana under fluctuating light conditions, in which the low light periods were repeatedly interrupted with high light peaks. Such conditions had only marginal effect on photosystem II but induced damage to photosystem I (PSI), the damage being most severe during the early developmental stages. We showed that PROTON GRADIENT REGULATION5 (PGR5)-dependent regulation of electron transfer and proton motive force is crucial for protection of PSI against photodamage, which occurred particularly during the high light phases of fluctuating light cycles. Contrary to PGR5, the NAD(P)H dehydrogenase complex, which mediates cyclic electron flow around PSI, did not contribute to acclimation of the photosynthetic apparatus, particularly PSI, to rapidly changing light intensities. Likewise, the Arabidopsis pgr5 mutant exhibited a significantly higher mortality rate compared with the wild type under outdoor field conditions. This shows not only that regulation of PSI under natural growth conditions is crucial but also the importance of PGR5 in PSI protection.

Photoinhibition ofphotosynthesis was studied in isolated photosystem II membranes by using chlorophyll fluorescence and electron paramagnetic resonance (EPR) spectroscopy combined with protein analysis. Under anaerobic conditions four sequentially intermediate steps in the photoinhibitory process were identified and characterized. These intermediates show high dark chlorophyll fluorescence (Fo) with typical decay kinetics (fast, semistable, stable, and nondecaying). The fast-decaying state has no bound Q. but possesses a single reduced QA species with a 30-s decay half-time in the dark (QB, second quinone acceptor; QA, first quinone acceptor). In the semistable state, Q-is stabilized for 2-3 min most likely by protonation, and gives rise to the Q-Fe2' EPR signal in the dark. In the stable state, QA has become double reduced and is stabilized for 0.5-2 hr by protonation and a protein conformational change. The final, nondecaying state is likely to represent centers where QA H2 has left its binding site. The first three photoinhibitory states are reversible in the dark through reestablishment of QA to QB electron transfer. Significantiy, illumination at 4 K of anaerobically photoinhibited centers trapped in all but the fast state gives rise to a spinpolarized triplet EPR signal from chlorophyll Pawl (primary electron donor). When oxygen is introduced during anaerobic illumination, the light-inducible chlorophyll triplet is lost concomitant with induction of D1 protein degradation. The results are integrated into a model for the photoinhibitory process involving initial loss of bound QuB followed by stable reduction and subsequent loss of QA facilitating chlorophyll P6w, triplet formation. This in turn mediates lght-induced formation of highly reactive and damaging singlet oxygen.Oxidative damage to proteins is a normal consequence of a variety ofbiochemical reactions (1). The repair often requires degradation and resynthesis of the damaged proteins. Such protein turnover is known to occur for one protein in plant chloroplasts under conditions of high light stress (2-4). This protein, designated D1, is one of the reaction center subunits of photosystem II (PSII) that catalyzes the important watersplitting reaction (4). The turnover of the D1 protein is an event subsequent to the photoinactivation of PSII electron transport that occurs at high light intensities. There is at present no consensus with respect to the molecular mechanism behind these photoinactivation and protein-damaging events in plants normally referred to as photoinhibition.The first inactivation of PSII electron transport has been proposed to be targeted to the primary (QA) (4,5) or the secondary (QB) quinone acceptor (2,3,6) or the primary charge separation (7). The photoinactivation is thought to result in damage to the D1 protein, which thereby becomes triggered for degradation. This damage has been suggested to include formation of toxic oxygen species (8, 9). Under certain conditions, D1 protein degradation may be triggered by lo...

Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. Native-PAGE preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion owing to a great variety of native-PAGE and thylakoid-solubilization systems. In the present paper, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly: (i) the use of detergent; (ii) the use of solubilization buffer; and (iii) the gel electrophoresis method. Special attention is paid to separation of high-molecular-mass thylakoid membrane super- and mega-complexes from Arabidopsis thaliana leaves. Several novel super- and mega-complexes including PS (photosystem) I, PSII and LHCs (light-harvesting complexes) in various combinations are reported.

All cyanobacteria, algae, and plants use a similar water-oxidizing catalyst for water oxidation. This catalyst is housed in Photosystem II, a membrane-protein complex that functions as a light-driven water oxidase in oxygenic photosynthesis. Water oxidation is also an important reaction in artificial photosynthesis because it has the potential to provide cheap electrons from water for hydrogen production or for the reduction of carbon dioxide on an industrial scale. The water-oxidizing complex of Photosystem II is a Mn-Ca cluster that oxidizes water with a low overpotential and high turnover frequency number of up to 25-90 molecules of O2 released per second. In this Review, we discuss the atomic structure of the Mn-Ca cluster of the Photosystem II water-oxidizing complex from the viewpoint that the underlying mechanism can be informative when designing artificial water-oxidizing catalysts. This is followed by consideration of functional Mn-based model complexes for water oxidation and the issue of Mn complexes decomposing to Mn oxide. We then provide a detailed assessment of the chemistry of Mn oxides by considering how their bulk and nanoscale properties contribute to their effectiveness as water-oxidizing catalysts.

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the β-lactamase–like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O 2 without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δ flv1(A) and/or Δ flv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis.

Light induces phosphorylation of photosystem II (PSII) proteins in chloroplasts by activating the protein kinase(s) via reduction of plastoquinone and the cytochrome b6f complex. The recent finding of high-light-induced inactivation of the phosphorylation of chlorophyll a͞b-binding proteins (LHCII) of the PSII antenna in floated leaf discs, but not in vitro, disclosed a second regulatory mechanism for LHCII phosphorylation. Here we show that this regulation of LHCII phosphorylation is likely to be mediated by the chloroplast ferredoxin-thioredoxin system. We present a cooperative model for the function of the two regulation mechanisms that determine the phosphorylation level of the LHCII proteins in vivo, based on the following results: (i) Chloroplast thioredoxins f and m efficiently inhibit LHCII phosphorylation. (ii) A disulfide bond in the LHCII kinase, rather than in its substrate, may be a target component regulated by thioredoxin. (iii) The target disulfide bond in inactive LHCII kinase from dark-adapted leaves is exposed and easily reduced by external thiol mediators, whereas in the activated LHCII kinase the regulatory disulfide bond is hidden. This finding suggests that the activation of the kinase induces a conformational change in the enzyme. The active state of LHCII kinase prevails in chloroplasts under low-light conditions, inducing maximal phosphorylation of LHCII proteins in vivo. (iv) Upon high-light illumination of leaves, the target disulfide bond becomes exposed and thus is made available for reduction by thioredoxin, resulting in a stable inactivation of LHCII kinase. R eversible protein phosphorylation is a unique regulatory mechanism for modification of the structure and function of proteins in both prokaryotic and eukaryotic organisms. It plays a fundamental role in signal transduction pathways that relay information from outside of the cell to the gene level. Bennett (1, 2) discovered the reversible, light-dependent phosphorylation of proteins in thylakoid membranes of plant chloroplasts. All thylakoid phosphoproteins identified so far are closely associated with photosystem II (PSII), a light-driven waterplastoquinone-oxidoreductase enzyme. Four core proteins of PSII, including the D1 and D2 reaction center proteins, the 43-kDa chlorophyll a-binding protein (CP43 protein), and the psbH gene product (PsbH protein) are prone to redox-regulated reversible phosphorylation. Three of six chlorophyll a͞b-binding proteins of the PSII antenna, Lhcb1 and Lhcb2 (designated LHCII) as well as Lhcb4 proteins, also undergo light-dependent phosphorylation. The protein kinase(s) involved in phosphorylation of the PSII proteins is associated with thylakoid membranes (2) and is activated by light via reduction of electron transfer components, plastoquinone and cytochrome b 6 ͞f complex (2-6).A large majority of the experimental data support the existence of two different kinases for PSII phosphoproteins, one for LHCII and another for PSII core proteins with distinct redox regulation systems (3, 6-10). Th...