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In nature, sessile plants have to adapt to their environment and to the never ending changes they are exposed to. They do so mainly by proteomic and metabolomic changes. In all cells, there are complex networks of auxiliary proteins that are responsible for quality control of all the cell's proteins. The auxiliary proteins are divided into chaperones and proteases, and these are further separated into different groups. Chaperones help other proteins in terms of stability and folding. In order for a protein to achieve its function, the three-dimensional structure has to be precise. A protease is a helper protein that is able to break peptide bonds in a process termed proteolysis. Chaperones and proteases can work independently, but sometimes the chaperone unfolds the substrate of the protease to ensure full degradation of the protein. In some cases, the chaperone and the protease functions are combined in one protein.

All proteins studied within this thesis are localized in the chloroplast, the organelle that originated from cyanobacteria, in which plants and algae convert the energy from sunlight into carbohydrates in the process called photosynthesis. Molecular oxygen is released as a by-product, and carbon dioxide is consumed. Photosystem II (PSII), one of the major protein complexes involved in photosynthesis, consists of more than 30 protein subunits, where around half of them are termed low molecular weight (LMW) proteins with a molecular size less than 10 kDa. In this thesis, data identifying one PSII LMW protein, PsbY, as a chaperone for the PSII subcomplex Cytochrome b₅₅₉ are presented. In the absence of PsbY, Arabidopsis plants were more sensitive to photoinhibition, and the protective circular electron transport around PSII is completely blocked.

Data on members of the Filamentation temperature sensitive protein H (FtsH) protease family are also discussed, with a focus on FtsH11 and FtsHi1-i5. Members of the FtsH protease family carry a protease domain and a chaperone domain. Our data show that FtsH11 has an influence on the structure and function of chloroplasts of Arabidopsis plants grown under continuous light along with protein import into the same. FtsHi1-5 are five members with mutations within the proteolytic motif, most probably rendering them proteolytically inactive, hence they are referred to as ''inactive FtsH proteases''. Knock-out plants of the inactive members are embryo lethal, and knock-down plants grow slower than wild type, probably because of an affected level of plastid proteins at the translational level.

The membrane-integrated metallo-protease FtsH11 of Arabidopsis thaliana is proposed to be dual targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast FtsH11 was found to be located exclusively in the envelope. Two chloroplast-located proteins of unknown function (Tic22-like protein and YGGT-A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsH11, and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6 weeks old ftsH11 compared to wild type and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light the amount of PSI decreased relative to PSII, accompanied by a drastic change of the chloroplast morphology and a drop of NPQ. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.

Assembly of Photosystem (PS) II in plants has turned out to be a highly complex process which, at least in part, occurs in a sequential order and requires many more auxiliary proteins than subunits present in the complex. Owing to the high evolutionary conservation of the subunit composition and the three-dimensional structure of the PSII complex, most plant factors involved in the biogenesis of PSII originated from cyanobacteria and only rarely evolved de novo. Furthermore, in chloroplasts the initial assembly steps occur in the non-appressed stroma lamellae, whereas the final assembly including the attachment of the major LHCII antenna proteins takes place in the grana regions. The stroma lamellae are also the place where part of PSII repair occurs, which very likely also involves assembly factors. In cyanobacteria initial PSII assembly also occurs in the thylakoid membrane, in so-called thylakoid centers, which are in contact with the plasma membrane. Here, we provide an update on the structures, localisations, topologies, functions, expression and interactions of the low molecular mass PSII subunits PsbY, PsbW and the auxiliary factors HCF136, PsbN, TerC and ALB3, assisting in PSII complex assembly and protein insertion into the thylakoid membrane.

Abstract Photosystem II is a protein complex embedded in the thylakoid membrane of photosynthetic organisms and performs the light driven water oxidation into electrons and molecular oxygen that initiate the photosynthetic process. This important complex is composed of more than two dozen of intrinsic and peripheral subunits, of those half are low molecular mass proteins. PsbY is one of those low molecular mass proteins; this 4.7–4.9 kDa intrinsic protein seems not to bind any cofactors. Based on structural data from cyanobacterial and red algal Photosystem II PsbY is located closely or in direct contact with cytochrome b559. Cytb559 consists of two protein subunits (PsbE and PsbF) ligating a heme-group in-between them. While the exact function of this component in Photosystem II has not yet been clarified, a crucial role for assembly and photo-protection in prokaryotic complexes has been suggested. One unique feature of Cytb559 is its redox-heterogeneity, forming high, medium and low potential, however, neither origin nor mechanism are known. To reveal the function of PsbY within Photosystem II of Arabidopsis we have analysed PsbY knock-out plants and compared them to wild type and to complemented mutant lines. We show that in the absence of PsbY protein Cytb559 is only present in its oxidized, low potential form and plants depleted of PsbY were found to be more susceptible to photoinhibition.

In the genome of Arabidopsis thaliana five genes encode members of the FtsH (Filamentation temperature sensitive protein H) protease family with mutations or deletions in their proteolytic site. Despite of not being active proteases these so called FtsHi (i for inactive) enzymes still seem to be highly important for plant development. All five enzymes have been localized within the plant chloroplast, most of them seem to be inserted in the chloroplast envelope. As homozygous ftshi mutants are seed lethal, here we compared different heterozygous ftshi mutants with respect to their growth at various light periods and at semi-natural growth conditions in the field. Their photosynthetic efficiency was evaluated by pulse-amplitude modulated fluorescence and the composition of their photosynthetic complexes was analysed by native polyacrylamide gel electrophoresis. Co-expression analyses were performed to find clues about the function of the five FtsHi inactive proteases.

In the thylakoid lumen of Arabidopsis thaliana, three pentapeptide repeat family proteins of unknown function are localized. Pentapeptide repeat proteins (PRP) are comprised of at least eight tandem repeats of five amino acids of the consensus sequence A(D/N)LXX, which fold into a quadrilateral beta helix structure. Here we have solved the crystal structure of the mature form of the lumenal PRP protein TL15 to 1.3 Å resolution. TL15 is comprised of a main pentapeptide domain, consisting of a total of 19 pentapeptide repeats which form five turns of a beta helix, and a C-terminal alpha helix domain consisting of two alpha helices. The alpha helices form a ‘cap’ at the C-terminal end of the beta helix and are connected by a disulphide bond between the conserved cysteine residues C122 and C142. Furthermore we show that the lumenal PRPs TL15 and TL20.3 can assist in refolding of a chemically denatured substrate in vitro, indicating foldase chaperone activity. The three lumenal PRPs have been previously identified as targets of thioredoxin, and interestingly we observed a greatly increased chaperone activity of TL15 and TL20.3 after reduction of their disulphide bonds. Our results provide the high resolution crystal structure of the TL15 protein and our analysis of chaperone activity suggests that TL15 and TL20.3 may constitute a novel type of redox-regulated molecular chaperones in the chloroplast lumen of higher plants.