Oxygenic photosynthesis is the major producer of both oxygen and organic compounds on earth and takes place in plants, green algae and cyanobacteria. The thylakoid membranes are the site of the photosynthetic light reactions that involve the concerted action of four major protein complexes known as photosystem II (PSII), cytochrome b6f complex, ATP synthase and photosystem I (PSI). The function of PSII is of particular interest as it performs the light–driven water splitting reaction driving the photosynthetic electron transport. My thesis addressed different aspects of PSII assembly and the functions of its low molecular weight PSII subunits PsbX and PsbW. Photosynthesis in green algae and higher plants is controlled by the nucleus. Many proteins of nuclear origin participate in the regulation of the efficient assembly of the photosynthetic protein complexes. In this investigation we have identified one of these nuclear encoded auxiliary proteins of photosystem II, REP27, which participates in the assembly of the D1 reaction center protein and repair of photodamaged PSII in the green algae Chlamydomonas reinhardtii. Interestingly, PSII is specially enriched in Low Molecular Weight (LMW) subunits that have masses less than 10kDa. These proteins account for more than the half of the PSII subunits. Several questions remains poorly understood regarding the LMW: Which is their evolutionary origin? What function do they perform in the protein complex? Where are they located in the protein structure? In this investigation the functions of two of these LMW subunits (PsbX and PsbW) have been studied using antisense inhibition and T-DNA knockout mutant plants in Arabidopsis thaliana. Deficiency of the PsbX protein leads to impaired accumulation and functionality of PSII. Characterization of PsbW knock-out plants show that PsbW participates in stabilization of the macro-organization of PSII and the peripheral antenna (Light Harvesting Complex, LHCII) in the grana stacks of the chloroplast, also known as PSII-LHCII supercomplexes.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.