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Characterisation of Arabidopsis thaliana mutants psbo1 and psbo2
Nykles, Ondřej ; Duchoslav, Miloš (advisor) ; Hála, Michal (referee)
The PsbO protein is necessary for the function of the electron-transport chain of the thylakoid membrane in higher plants. In most of the angiosperms, including Arabidopsis thaliana, this protein has two isoforms termed as PsbO1 and PsbO2. Many authors tried to reveal the fundamental difference between the PsbO1 and PsbO2 with the help of the mutant lines which lack one of the isoforms. The problem is that the mutants in psbO isoforms do not possess the same level of PsbO as WT does. So we made psbo1isoL mutants. These lines contain only one isoform but their level of the whole PsbO is comparable to the level of the whole PsbO of WT. Results from these experiments suggest that if a psbo1isoL plant has the same amount of PsbO as WT does, there is no observable phenotype difference. Thus we were not able to identify, in the usual cultivation conditions, if there are any functional differences between PsbO1 and PsbO2 Following the above mentioned results, we would like to know conditions (if there are any) in which T-DNA insertion mutants psbo2 (respectively psbo2cr which are made with the use of the CRISPR/Cas9), which have only PsbO1 isoform, could be phenotypically distinguished from WT. With the use of usual cultivation conditions, we are unable to tell apart the psbo2 and psbo2cr from WT by the...
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Function of PsbO isoforms
Duchoslav, Miloš ; Fischer, Lukáš (advisor) ; Špunda, Vladimír (referee) ; Sobotka, Roman (referee)
(English version) Oxygenic photosynthesis is crucial for most forms of the life on the Earth. The splitting of water and evolution of oxygen is conducted by photosystem II (PSII), a multi-subunit pigment- protein complex embedded in the thylakoid membrane. PsbO is an indispensable subunit of PSII, bound to its transmembrane subunits from the luminal side. The main function of PsbO is to stabilise and protect Mn4CaO5 cluster where the water splitting occurs. However, it has probably also some auxiliary functions. These additional functions might be different for isoforms of PsbO proteins, as suggested for Arabidopsis thaliana, which expresses two genes encoding protein isoforms PsbO1 and PsbO2. This thesis studies auxiliary functions of PsbO with a focus on functional differences between PsbO isoforms. We found that besides Arabidopsis thaliana, also many other plant species express two psbO genes. Interestingly, the duplication of psbO gene occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the PsbO isoforms differ at similar sites in the protein structure, suggesting that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. Biochemical characterisation of PsbO from green alga Chlamydomonas reinhardtii and...
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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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Grana
Hlavsová, Kateřina ; Duchoslav, Miloš (advisor) ; Kutík, Jaromír (referee)
The evolution of life on Earth is closely connected to the ability of some organisms to oxidise water and reduce carbon dioxide using solar energy in a process called oxygenic photosynthesis. Substantial part of the photosynthetic reactions is located in the thylakoid membrane inside of chloroplasts and cyanobacteria. The main photosynthetic complexes in the thylakoid membrane are photosystems I and II with their light harvesting antennas, cytochrome complex and ATP synthase. All higher plants form grana (stacks of appressed thylakoid membranes) that are interconnected by stroma lamellae. Proper distribution of certain processes between grana and stroma lamellae seems to be crucial for smooth flow of photosynthesis. First part of this thesis is focused on the major photosynthetic complexes while the second part focuses on grana structure, dynamics and stabilising forces as well as proteins essential for grana formation. The last part of the thesis describes some differences between granal and agranal organisms and summarises hypotheses about grana functions.
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Communitiy level impacts of alien invasive plants and the role of native range habitats in plant invasions
Hejda, Martin ; Pyšek, Petr (advisor) ; Duchoslav, Miloš (referee) ; Tichý, Lubomír (referee)
Conclusions Categorization of the species studied according to their impact on invaded communities This study focused on neophytes, species introduced after 1500 A.D., because their impact on native vegetation is higher and more obvious than that of archaeophytes, introduced before that date, due to considerable invasion dynamics and relatively short residence time (Pyšek et al. 2002). The target neophytes, included in the study, differ strongly in their community level impacts. Impatiens glandulifera had a miniscule impact on species diversity and composition, both in the removal experiment and comparative study (Chapter I). This small impact is likely to be associated with this annual species' lower competitive strength, which has a modest root system and forms stands that are not homogeneous in terms of cover. The density of I. glandulifera populations is known to decrease during the vegetation season, as plants fall due the weight of climbers, which are common in invaded riparian communities (Calystegia sepium, Cuscuta europaea - see Beerling & Perrins 1993). Moreover, the character of communities in which I. glandulifera invades, mostly riparian nitrophilous vegetation, does not provide opportunities for competitively weak species even if they are not invaded. This vegetation is dominated by tall,...
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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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Proteomic and functional characterization of PsbO isoforms
Duchoslav, Miloš ; Fischer, Lukáš (advisor) ; Hála, Michal (referee)
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...
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The role of manganese-stabilizing protein of photosystem II
Duchoslav, Miloš ; Rothová, Olga (referee) ; Fischer, Lukáš (advisor)
Miloš Duchoslav The role of manganese-stabilizing protein of photosystem II Abstract The appearance of oxygenic photosynthesis was a key event in the evolution of life on the Earth. All molecular oxygen in the atmosphere likely comes from a water-splitting reaction catalysed by the oxygen-evolving center of photosystem II. Photosystem II - a multisubunit protein-cofactor complex with a phylogeneticaly highly conserved structure - is embedded in the thylakoid membrane of chloroplasts and cyanobacteria. The mechanism of the photosynthetic water-splitting reaction, which occurs on the manganese cluster, has been widely investigated; however, it has not yet been fully understood. An essential role in the stabilization of the manganese cluster and in the facilitation of oxygen evolution is played by photosystem II extrinsic proteins that occur in thylakoid lumen. The most important among them is a manganese-stabilizing protein (MSP) that is present in all known oxyphototrophs. This protein is believed to have many functions: besides stabilizing the manganese cluster, it is also carbonic anhydrase activity, GTPase activity and regulation of the turnover of the D1 protein. The functions of the MSP are probably regulated through changes in its dynamic structure. The MSP is likely to take part in the regulation of...
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