Abstract: In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na(+)-dependent inorganic phosphate (P(i)) transporters. The function of ANTR1 as a Na(+)-dependent P(i) transporter was demonstrated by heterologous expression and uptake of radioactive P(i) into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated P(i) uptake in a pH- and Na(+)-dependent manner as compared with the control cells. Among various tested effectors, P(i) was the preferred substrate. Although it competed with the uptake of P(i), glutamate was not transported by ANTR1 into E. coli. In relation to its function as a P(i) transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of P(i) produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H(+) electrochemical gradient storage.

Abstract: The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.

Abstract: In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.

Abstract: Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the beta-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn(2+) and Ca(2+) ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.

Abstract: The apparatus of photosynthetic energy conversion in chloroplasts is quite well characterized with respect to structure and function. Light-driven electron transport in the thylakoid membrane is coupled to synthesis of ATP, used to drive energy-dependent metabolic processes in the stroma and the outer surface of the thylakoid membrane. The role of the inner (luminal) compartment of the thylakoids has, however, remained largely unknown although recent proteomic analyses have revealed the presence of up to 80 different proteins. Further, there are no reports concerning the presence of nucleotides in the thylakoid lumen. Here, we bring three lines of experimental evidence for nucleotide-dependent processes in this chloroplast compartment. (i) The thylakoid lumen contains a protein of 17.2 kDa, catalyzing the transfer of the gamma-phosphate group from ATP to GDP, proposed to correspond to the nucleoside diphosphate kinase III. (ii) The 33-kDa subunit of photosystem II, bound to the luminal side of the thylakoid membrane and associated with the water-splitting process, can bind GTP. (iii) The thylakoid membrane contains a nucleotide transport system that is suggested to be associated with a 36.5-kDa nucleotide-binding protein. Our results imply, against current dogmas, that the thylakoid lumen contains nucleotides, thereby providing unexpected aspects on this chloroplast compartment from a metabolic and regulatory perspective and expanding its functional significance beyond a pure bioenergetic function.

Abstract: A chromatography fraction, prepared from isolated thylakoids of a fatty acid desaturation mutant (Fad6/desA Colon, two colons Km(r)) of the cyanobacterium Synechocystis 6803, could induce an initial cleavage of the D1 protein in Photosystem II (PSII) particles of Synechocystis 6803 mutant and Synechococcus 7002 wild type as well as in supercomplexes of PSII-light harvesting complex II of spinach. Proteolysis was demonstrated both in darkness and in light as a reduction in the amount of full-length D1 protein or as a production of C-terminal initial degradation fragments. In the Synechocystis mutant, the main degradation fragment was a 10-kDa C-terminal one, indicating an initial cleavage occurring in the cytoplasmic DE-loop of the D1 protein. A protein component of 70-90 kDa isolated from the chromatographic fraction was found to be involved in the production of this 10-kDa fragment. In spinach, only traces of the corresponding fragment were detected, whereas a 24-kDa C-terminal fragment accumulated, indicating an initial cleavage in the lumenal AB-loop of the D1 protein. Also in Synechocystis the 24-kDa fragment was detected as a faint band. An antibody raised against the Arabidopsis DegP2 protease recognized a 35-kDa band in the proteolytically active chromatographic fraction, suggesting the existence of a lumenal protease that may be the homologue DegP of Synechocystis. The identity of the other protease cleaving the D1 protein in the DE-loop exposed on the stromal (cytoplasmic) side of the membrane is discussed.

Abstract: The light exposure history and/or binding of different herbicides at the Q(B) site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea, C. , Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6547-6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38-44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photosytem II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosystem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.

Abstract: The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

Abstract: Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Abstract: Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction center.