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CRISPR-Cas9 mutagenesis of psbO1 and psbO2 genes in Arabidopsis thaliana
Hlavsová, Kateřina ; Duchoslav, Miloš (advisor) ; Knoppová, Jana (referee)
PsbO protein is one of the essential extrinsic subunits of photosystem II, a protein complex that is embedded in thylakoid membranes of chloroplasts. PsbO is important in stabilizing manganese cluster, a structure that break down water molecules and evolves oxygen. Arabidopsis thaliana encodes for two isoforms which functions have not yet been clearly explained. Up-to- date information of PsbO1 and PsbO2 come from experiments using T-DNA insertion mutants psbo1 and psbo2. Finding viable double mutants psbo1 psbo2 had brought up questions about actual expression levels of mutated genes in psbo1 and psbo2 mutants. This is what led us to creating psbo1cr and psbo2cr knock-out mutant lines using the CRISPR-Cas9 mutagenesis and then comparing them to the frequently used T-DNA insertion mutants psbo1 and psbo2. We performed a comparison of basic phenotype characteristics, chlorophyll a fluorescence parameters and immunodetection of PsbO1 and PsbO2 proteins. Even though we might have observed slight expression of mutated genes in psbo1 and psbo2 lines, our results show that measured parameters of psbo1cr and psbo2cr are identical to those of psbo1 and psbo2. Key words: PsbO, photosystem II, CRISPR-Cas9, mutagenesis, Arabidopsis thaliana
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Role of PsbO isoforms in Arabidopsis thaliana
Svoboda, Václav ; Duchoslav, Miloš (advisor) ; Knoppová, Jana (referee)
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...
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Biogenesis of Photosystem II in the Model Cyanobacterium Synechocystis sp. PCC 6803 - The Role of Selected Auxiliary Protein Factors and Subcellular Localisation
KNOPPOVÁ, Jana
This thesis explores localisations and roles of three auxiliary protein factors involved in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 and contributes to subcellular localisation of the initial steps of PSII biogenesis and repair-related D1 synthesis. The main results consist in i) identification of a functional interaction of the protein factor Psb27 with a lumenal domain of the Photosystem II subunit CP43, ii) discovery of a novel pigment binding complex formed by the Ycf39 protein and high-light-inducible proteins implicated in photoprotection and delivery of recycled chlorophyll to newly synthesized D1 protein during the PSII reaction centre formation, iii) providing evidence that the early steps of PSII assembly and the repair-related D1 synthesis occur in the thylakoid membrane of Synechocystis, and iv) revealing that the cyanobacterial PsbP orthologue, CyanoP, assists in the early phase of PSII biogenesis as an assembly factor facilitating the association of D2 and D1 assembly modules.
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