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ALBA-family proteins and their role in male gametophyte development
Náprstková, Alena ; Honys, David (advisor) ; Rothová, Olga (referee)
Alba family proteins are highly conserved in all domains of life. They are involved in RNA metabolism in Archae and Eucarya, while they are involved in the chromatin organisation in Crenarchaea. In animals, ALBA proteins were identified to associate with RNase P/MRP subunits. The objective of my thesis was the characterization of ALBA family proteins in a model plant Arabidopsis thaliana. The Arabidopsis genome contains six genes with close homology, three from Rpp20-like subfamily and three of Rpp25-like subfamily. Here I present the localization of GFP-fused proteins in Arabidopsis stable lines harbouring constructs cloned by Gateway® Technology. ALBA proteins were localized in the cytoplasm and undefined particles in root differentiation zone and in mature pollen. The characterization of the respective T-DNA insertion lines did not reveal significant phenotype defects in growth and development of sporophyte and gametophyte in comparison to Columbia-0 plants, probably because of likely functional redundancy od the paralogs. Expression profiles and localization of ALBA proteins suggest their possible role in differentiation and dehydration stress response in Oryza. They were also observed to associate with repressed mRNA transcripts in storage EPP particles. Collectively, I propose the likely role...
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Characterization of selected RAB GTPase activating protein (RAB GAP) of Arabidopsis thaliana
Metlička, Jáchym ; Hála, Michal (advisor) ; Eliáš, Marek (referee)
8 ABSTRACT Rab GTPases (Rabs) are the most populous branch of eukaryotic Ras GTPase superfamily. In active GTP-binding conformation, they serve as key instruments in defining transient membrane identity and through various effectors regulate formation, transport, conversion, and fusion of membrane vesicles. This is important for upkeep of compartmentalized structure of eukaryotic cells and for facilitating both endo- and exocytic processes. Rabs are converted into GDP-binding conformation by interactions with Rab GTPase activating proteins (Rab GAPs) that possess ability to significantly speed up weak intrinsic GTP hydrolytic activity of Rabs. Through this process, Rab GAPs can limit scope of the Rabs' activity and lay out spatiotemporal boundaries for varying Rab populations. In this thesis, I tried to characterize a Rab GAP, GAP2, seemingly necessary for standard development of thale cress plants. Besides TBC catalytic domain, GAP2 (product of At2g39280 gene) possesses a C-terminal coiled-coil motif, which was previously found to interact with Rab GTPases. Experiments aiming to complement T-DNA insertion mutant in GAP2, elucidate GAP2 intracellular localization, novel interacting partners, and character of interaction with the Rabs discovered in the pilot study were undertaken. The results suggest that...
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Role of formins in the organization and dynamics of intracellular structures in Arabidopsis thaliana
Rosero Alpala, Elvia Amparo ; Cvrčková, Fatima (advisor) ; Baluška, František (referee) ; Malcová, Ivana (referee)
On the basis of detailed phenotypic examination of fh1 and fh2 mutants we observed that the main housekeeping Arabidopsis thaliana formin AtFH1 (At3g25500) and its closest relative, AtFH2 (At2g43800) are involved in both actin filaments and microtubule dynamics. fh1 mutants showed increased sensitivity to the actin polymerization inhibitor Latrunculin B (LatB). Formin mutants had cotyledon pavement cells which exhibited more pronounced lobes compared to the wild type, and alterations in vascular tissue patterning were found. The double fh1 fh2 homozygote was not obtained, suggesting that at least one functional formin gene is required for proper gametophyte development. Methods used to observe and quantify both architecture and dynamics of the cortical cytoskeleton from confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM) were standarized and allowed to find that mutants exhibited more abundant but less dynamic F- actin bundles and more dynamic microtubules than wild type seedlings, fh1 mutant phenotype observed in roots was further aggravated by a (heterozygous) fh2 mutation. The formin inhibitor SMIFH2 mimicked the alterations observed in fh1 mutants in plants, it has been the first report of this inhibitor in plants. Defects in membrane trafficking were...
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Subunits of exocyst complex in the development of Arabidopsis epidermis
Vojtíková, Zdeňka ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
Exocyst is protein complex evolutionary conserved in yeasts, animals and plants, which plays a role in control of cell morphogenesis and polarity. It is a tethering complex whose function is to attach secretory vesicles to specifi c foci on plasma membrane. Complex exocyst is formed by eight subunits. Subunit EXO70 is encoded by 23 paralogue genes in genome of Arabidopsis thaliana. Mutation in paralogue subunit EX070H4 causes defect in trichome maturation. Mutant trichomes have thin, not reinforced cell wall, making them soft and elastic. Transcription of EXO70H4 gene is induced by UV radiation, therefore observations of plants cultivated on UV-B radiation were done. Analysis of mutants cultivated on UV-B radiation revealed hyperaccumulation of vesicules in cytoplasm, which were visible by light microscope. Hyperaccumulation was not observed in control plants cultivated on UV-B radiation, but thickening of cell wall was induced. Th is reaction to UV in trichomes hasn't been described yet. Analysis of cellular localization made with YFP tagged constructs revealed that EXO70H4 localizes into mobile corpuscules associating with Golgi apparatus. It was found with yeast two hybrid system that EXO70H4 interacts with TRS120, subunit of tethering complex TRAPPII which is active in Golgi apparatus....
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Size matters - siRNAs biogenesis and function in Arabidopsis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Honys, David (referee)
RNA interference (RNAi) play a key role in various biological processes including regulation of gens and transposons, phylogenetic of part plant body, stress response, chromatin remodeling and antiviral mechanism. The ground of RNAi is short RNA molecules (small RNA, sRNA). In plants they are produced in range from 21 to 24 nucleotides (nt) and on the basis of being complementary they recognize target molecule of RNAi. It is possible to divide small RNA in two basic classes: microRNAs (miRNA) and small interfering RNAs (siRNA). To product and put small RNA into activate needs proteins from several gene family. DICER-LIKE (DCL) proteins create small RNAs from double-strand RNA precursors, which are often created by RNA dependent RNA polymerase (RDR) activity. With these small RNAs interact ARGONAUTE (AGO) proteins and together create RNA-Induced Silencing Complex (RISC). Those complexes play a key role in recognizing target molecule in active phase of RNAi. Structure and biogenesis of sRNAs has decisive influence on RISC complex and its next way in biogenesis. RNAi cause effect on post-transcriptional level (PTGS), as degradation of target molecule or repression of translation. And on transcriptional level (TGS) as sRNA intermediate histone and DNA methylation.
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The proteomic study of abiotic stress of plants.
Barabášová, Kamila ; Podlipná, Radka (advisor) ; Smrček, Stanislav (referee)
Keywords: Arabidopsis thaliana, phytoremediation, abiotic stress, ibuprofene, doxorubicin, two-dimensional electrophoresis Nowadays, develop of the pharmaceutical industry is very fast. Reason of this trend is ever-increasing number of diseases, lifestyle and still increasing demand for the drugs. With this trend growing interest in the analysis of the residues of pharmaceuticals in the environment which is result of incomplete wastewater treatment. This diploma thesis is studying effect of cytostatic drugs, specifically doxorubicin and one of the most widely used analgesics - ibuprofen, at the proteome level of the model plant Arabidopsis thaliana. Proteins isolated from plants exposed to the drugs were separated by two-dimensional electrophoresis. Comparing of protein maps by PDQest program (Bio-Rad, USA) was found several proteins whose expression was affected by the presence of drugs in the culture medium. Selected proteins were identified by LC - MS / MS.
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Cloning and characterisation of selected Class II formins
Stillerová, Lenka ; Cvrčková, Fatima (advisor) ; Rösel, Daniel (referee)
Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...
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Mechanisms of epidermal cells polarization in plants.
Vojtíková, Zdeňka ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
Plant epidermal cells form contact area of the plant, they protect it from impacts of surrounding environment and they mediate the communication with its neighbourhood. In the epidermis there are evenly distributed several cell types with quite specialized morphology (pavement cells, trichomes and guard cells) due to the polarization mechanisms. The cytoskeleton and signal molecules of ROP GTPase family promote the polarized growth. Thanks to polarized growth the cells reach their shapes. The cytoskeleton responds to the signal by expanding the cell, helps with targeting of the secretion to the sites of active growth and mediates polarized formation of the cell wall. On the upper side of the epidermis the cuticle and layer of epicuticular waxex is secreted. The secretion of cuticular components is baso-apicaly polarized. This work summarizes the mechanisms of the polarization in plant epidermal cells discoverd untill now.
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Characterization of membrane protein DREPP
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Cvrčková, Fatima (referee)
Proteins of DREPP family (20-25 kDa, syn. PCaP1 in Arabidopsis thaliana) first appeared in ferns and we have shown that several independent duplications of DREPP protein occurred during evolution of large families (Poaceae, Brassicaceae, Solanaceae and Asteraceae) and in group Coniferophyta. Secondary losses of one paralogue occurred in subfamilies Pooideae and Solanoideae.We have also detected two large-scale modification of DREEP protein in Asparagales and Brassicaceae (this divergent paralogue was previously described as MAP18 protein). We have examined colinearity of chromosome fragments in vicinity both PCaP1 and MAP18 paralogues in Arabidopsis thaliana and we hypothesize that MAP18 gene arose during genome duplication on the origin of Brassicaceae family. DREPP protein was previously identified in detergent-resistant membrane microdomain fraction and a myristyl anchor was shown to be necessary for their membrane localization. Membrane association was shown to be modified by the interaction of unique N-terminal domain with PtdInsPs, which was inhibited by binding of Ca-calmodulin (Nagasaki et al., 2008). The mutation of Gly2 by Ala in the myristilation site, or C-terminal GFP-fusion (GFP-DREPP), affect membrane association in Arabidopsis thaliana (Nagasaki et al., 2008). Several DREPP paralogues in...
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