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...

evaaro@utu.®). Abbreviations: PSII, photosystem II, PSI, photosystem I, LHCII, light-harvesting chlorophyll a/b complex of PSII, F v /F max , variable/maximal¯uorescence ratio, Q p , photochemical quenching of PSII, CF1, chloroplast coupling factor, PFD, photon¯ux density, P-LHCII, P-CP29 and P-D1, phosphorylated forms of LHCII, CP29 and D1, respectively. SummaryWinter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, re¯ecting the plastoquinone redox state, and the phosphorylation level of thylakoid light-harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short-term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.

Antibiotics are widely used to monitor signalling cascades within a plant cell, for example between the nucleus and chloroplasts, and to study the function of the photosynthetic machinery. In the present study, we attempted to test various antibiotics with respect to their expected modes of function and also to monitor their possible side effects on metabolic processes in mature leaves of pea (Pisum sativum L.). Streptomycin, despite its reported prokaryotic nature, prevented translation not only in the chloroplast, but also in the cytosol. Application of puromycin, an inhibitor of protein synthesis in both the pro- and eukaryotes, resulted in severe photoinhibition of photosystem II upon illumination, yet had no effect on plastid translation, thus implying a severe side effect on plastid metabolism. Prokaryotic-type translation inhibitors lincomycin, spectinomycin and erythromycin blocked translation in the chloroplast without any direct effects on cytoplasmic protein synthesis. More detailed studies with lincomycin, however, revealed a strong modulation of the expression of nuclear-encoded genes by slowing down the transcription rate of photosynthesis-related Lhcb and RbcS genes, and furthermore, lincomycin clearly decreased the phosphorylation level of the LHCII proteins.

Phosphothreonine antibody was used to explore reversible thylakoid protein phosphorylation in vivo in evolutionally divergent organisms with oxygenic photosynthesis. Three distinct groups of organisms were found. Cyanobacteria and red algae, both with phycobilisome antenna system, did not show phosphorylation of any of the photosystem II (PSII) proteins and belong to group 1. Group 2 species, consisting of a moss, a liverwort and a fern, phosphorylated both the light-harvesting chlorophyll a/b proteins (LHCII) and the PSII core proteins D2 and CP43, but not the D1 protein. Reversible phosphorylation of the D1 protein seems to be the latest event in the evolution of PSII protein phosphorylation and was found only in seed plants, in group 3 species. Light-intensity-dependent regulation of LHCII protein phosphorylation was similar in group 2 and 3 species, with maximal phosphorylation of LHCII at low light and nearly complete dephosphorylation at high light.z 1998 Federation of European Biochemical Societies.

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b 6 f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.Light induces the phosphorylation of a number of photosystem II (PSII)-related proteins in the thylakoid membranes of plant chloroplasts (Bennett, 1977(Bennett, , 1991, including two light-harvesting chlorophyll a/b complex II (LHCII) proteins, Lhcb1 and Lhcb2, of the PSII outer antenna (Larsson et al., 1987). Phosphorylation of LHCII proteins has been proposed to balance the excitation energy between PSII and photosystem I (PSI) in plant thylakoid membranes (Bennett, 1991;Allen, 1992). The phosphorylation of LHCII proteins is regulated in response to light via activation of the LHCII kinase with reduced plastoquinone pool and cytochrome b 6 f complex (Cyt b 6 f; Vener et al., 1995Vener et al., , 1997Gal et al., 1997;Zito et al., 1999). Moreover, LHCII protein phosphorylation is down-regulated under high light conditions in vivo, revealing the existence of an inhibitory control mechanism in chloroplasts (Rintamäki et al., 1997). We have recently shown that the chloroplast thioredoxins f and m are effective inhibitors of LHCII protein phosphorylation in vitro (Rintamäki et al., 2000). It has become apparent that the correct thiol redox state is critical for in vitro thylakoid protein phosphorylation (Carlberg et al., 1999;Rinta...