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Plant exocyst dependent secretory pathway and iron nutrition
Batík, Adam ; Žárský, Viktor (advisor) ; Vaňková, Radomíra (referee)
Previous results from our lab suggested possible phenotypic deviation of a double T-DNA knock- out mutant of an EXOCYST subunit EXO70E2 and vacuolar transmembrane protein LAZ1H1 upon iron starvation. To confirm these preliminary results, single mutants were obtained, with EXO70E2 knock-out mutant being produced here using CRISPR/Cas9 technology. Result did not confirm the previous findings in neither of the mutants tested (exo70e2, laz1h1, exo70e2/laz1h1 and exo70h4). This discrepancy is attributed to improper iron starvation protocol and different storage age of seeds used in the previously conducted experiments. Coumarins are synthetized and secreted from roots during iron starvation; protocol for their visualization was adopted and WT compared with mutants. No phenotypic deviations were observed in mutants. Pharmacological treatments of iron starved WT plants using BFA, wortmannin and concanamycin A did not suggest that endomembrane vesicle trafficking affect accumulation or secretion of coumarins. Previously published susceptibility of T-DNA knock-out mutant of EXO70E2 to drought stress was tested using CRISPR/Cas9 mutant produced here, the reported susceptibility was not confirmed. Upregulation of EXO70E2 mRNA upon UV exposure reported in eFP browser was also tested and not confirmed under...
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ARP2/3 complex interaction with plant secretory pathway components
Voloshina, Mariia ; Schwarzerová, Kateřina (advisor) ; Oulehlová, Denisa (referee)
The ARP2/3 complex is an evolutionarily conserved protein complex involved in actin nucleation. In animals, the loss of function of ARP2/3 subunits is usually lethal. In contrast, arp2/3 plants are viable and exhibit certain morphological defects. One of the characteristic traits of arp2/3 plants is impaired cell adhesion. This suggests a role for the ARP2/3 complex in plant cell wall secretion, specifically pectins. However, the exact molecular mechanism of ARP2/3 function in this pathway has not been elucidated yet. Preliminary results suggest a possible interaction of ARP2/3 with the secretory pathway proteins GNL1, COPI, and COG. This work combines two methods - TIRFM/VAEM and co-immunoprecipitation - to study the interaction of ARP2/3 with selected markers. However, neither colocalization nor physical interaction was demonstrated. This work also characterises COPI, COG and GNL1 with TIRFM/VAEM and shows that there is an interaction between GNL1 and endocytic markers. Key words: ARP2/3, COPI, COG, GNL1, secretory pathway
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Function of phospholipases D and lipid phosphate phosphatases in the regulation of plant cell morphogenesis
Bezvoda, Radek ; Žárský, Viktor (advisor) ; Hašek, Jiří (referee) ; Vaňková, Radomíra (referee)
of the thesis The presented work explores the function and regulation of intracellular signaling that utilizes phospholipase D (PLD) and phosphatidic acid (PA), especially in the context of cellular morphogenesis of plants. PLDs cleave membrane phospholipids to phosphatidic acid, which has important biophysical and signaling role in many contexts, such as stress response, regulation of cytoskeletal dynamics and vesicular transport. Vesicular transport is essential in focused tip growth of plant pollen tubes and root hairs. Part of the work deals with NADPH oxidases, that are an emerging counterpart of PLD/PA signaling. Tobacco pollen tubes served as the main experimental model, as it enables assessing of changes in secretory pathway after pharmacological or genetic treatments. A technique utilizing antisense oligonucleotides was used for selective knock-down of PLD isoforms, NADPH oxidase and newly studied family of lipid phosphate phosphatases (LPPs) in pollen tubes. This enabled to assess functions of individual isoforms. For studying of selected gene families, various bioinformatic tool were utilized, such as dendrogram construction, analysis of available expression data and creating of virtual proteome. These tools together enabled to select potentially important genes for further experimental...
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Function of phospholipases D and lipid phosphate phosphatases in the regulation of plant cell morphogenesis
Bezvoda, Radek ; Žárský, Viktor (advisor) ; Hašek, Jiří (referee) ; Vaňková, Radomíra (referee)
of the thesis The presented work explores the function and regulation of intracellular signaling that utilizes phospholipase D (PLD) and phosphatidic acid (PA), especially in the context of cellular morphogenesis of plants. PLDs cleave membrane phospholipids to phosphatidic acid, which has important biophysical and signaling role in many contexts, such as stress response, regulation of cytoskeletal dynamics and vesicular transport. Vesicular transport is essential in focused tip growth of plant pollen tubes and root hairs. Part of the work deals with NADPH oxidases, that are an emerging counterpart of PLD/PA signaling. Tobacco pollen tubes served as the main experimental model, as it enables assessing of changes in secretory pathway after pharmacological or genetic treatments. A technique utilizing antisense oligonucleotides was used for selective knock-down of PLD isoforms, NADPH oxidase and newly studied family of lipid phosphate phosphatases (LPPs) in pollen tubes. This enabled to assess functions of individual isoforms. For studying of selected gene families, various bioinformatic tool were utilized, such as dendrogram construction, analysis of available expression data and creating of virtual proteome. These tools together enabled to select potentially important genes for further experimental...
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