Plants, algae and photosynthetic bacteria are able to harvest the sunlight and use its energy to transform water and carbon dioxide to carbohydrate molecules and oxygen, both important to sustain life on Earth. This process is called photosynthesis and is the route by which almost all energy enters the biosphere. As most simple things in life, the process of photosynthesis is easily explained but unfortunately not that easy to reproduce. If we could, we would be living in a much different world with almost unlimited energy. Light energy is harvested by chlorophyll molecules, bound to proteins in the chloroplast thylakoid membrane and drives the oxygen-evolving complex, to extract electrons from water. Electrons are then transferred to NADPH through photosystem II (PSII) to cytochrome b6f and photosystem I, the major photosynthetic protein complexes. The cytochrome b6f complex also transfers protons into the lumenal space of the thylakoid. These protons together with those from water oxidation create an electrochemical gradient across the thylakoid membrane, which fuels the ATP synthase to produce ATP. ATP, NADPH and carbon dioxide are used during the dark reactions to produce sugars in the chloroplast stroma. The thylakoid lumenal space where the water oxidation occurs has until recently been viewed as a proton sink with very few proteins. With the publication of the genome of Arabidopsis thaliana it seems to be a much more complex compartment housing a wide variety of biochemical processes. ATP is a nucleotide and the major energy currency, but there are also other nucleotides such as AMP, ADP, GMP, GDP and GTP. Chloroplast metabolism has mostly been associated with ATP, but GTP has been shown to have a role in integration of light harversting complexes into the thylakoid. In this work, we have demonstrated the occurrence of nucleotide-dependent processes in the lumenal space of spinach by bringing evidence first for nucleotide (ATP) transport across the thylakoid membrane, second for nucleotide inter-conversion (ATP to GTP) by a nucleoside diphosphate kinase, and third the discovery that the PsbO extrinsic subunit of PSII complex can bind and hydrolyse GTP to GDP. The active PSII complex functions as a dimer but following light-induced damage, it is monomerised allowing for repair of its reaction center D1 protein. PsbO is ubiquitous in all oxygenic photosynthetic organisms and together with other extrinsic proteins stabilises the oxygen-evolving complex. We have modelled the GTP-binding site in the PsbO structure and showed that the GTPase activity of spinach PsbO induces changes in the protein structure, dissociation from the complex and stimulates the degradation of the D1 protein, possibly by inducing momerisation of damaged PSII complexes. As compared to spinach, Arabidopsis has two isoforms of PsbO, PsbO1 and PsbO2, expressed in a 4:1 ratio. A T-DNA insertion knockout mutant of PsbO1 showed a retarded growth rate, pale green leaves and a decrease in the oxygen evolution while a PsbO2 knockout mutant did not show any visual phenotype as compared to wild type. Unexpectedly, during growth under high light conditions the turnover rate of the D1 protein was impaired in the PsbO2 knockout, whereas it occurred faster in the PsbO1 knockout as compared to wild type. We concluded that the PsbO1 protein mainly functions in stabilizing the oxygen evolving complex, whereas the PsbO2 protein regulates the turnover of the D1 protein. The two PsbO proteins also differ in their GTPase-activity (PsbO2 >> PsbO1). Although their amino acid sequences are 90% identical, they differ in the GTP-binding region which could explain the difference in their GTPase activity. Based on these data, we propose that the GTPase activity of PsbO(2) leads to structural changes in interacting loops and plays a role in the initial steps of D1 turnover such as the PSII monomerisation step. The nucleotide-dependent processes we discovered in the thylakoid lumen raise questions of transporters to facilitate these processes. As stated earlier, we provided biochemical evidence of an ATP thylakoid transporter, and most recently have identified a transporter that may be important for the export of lumenal phosphate back to the stroma. More transporters for GDP, metal ions and others solutes have still to be identified.

Role of PsbO isoforms in Arabidopsis thaliana Abstract Photosystem II (PSII) uses sunlight to catalyze water oxidation and reduce plastoquinone. Water oxidation takes place in oxygen evolving complex (OEC). OEC is stabilized by extrinsic subunits of PSII. The largest and most important of them is PsbO, manganese-stabilizing protein which can be found in all known oxygenic photosynthetic organisms. Model plant Arabidopsis thaliana expresses two isoforms of psbO gene, namely PsbO1and PsbO2.Mutants psbo1 and psbo2 lacking PsbO1 and PsbO2, respectively, recently brought new findings on the particular roles of isoforms in maintaining photosynthesis. PsbO1 is commonly considered as the main isoform facilitating water splitting, whereas PsbO2 is believed to be involved in PSII repair process (replacement of photodamaged D1 subunit). This work focuses on particular roles of Arabidopsis PsbO isoforms in maintaining photosynthesis with special focus on response to light stress. Mutants psbo1, psbo2 and wild type plants Col-0 were used for extensive biochemical investigation. Our aim was to find out what is the impact on overall thylakoid structure and composition in mutants. Furthermore, to investigate response to light stress in wild type regarding to yields of particular subcompartments, changes in photosystem II...

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...