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Vazba paralogů EXO70 na ATG8 a funkční rozdělení rodiny EXO70 dle účasti v autofagii (Arabidopsis thaliana).
Semerádová, Hana ; Kulich, Ivan (advisor) ; Motyka, Václav (referee)
The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...
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Genetic variability in growth, reproductive and photosynthetic parameters of plants and its changes by exogenously applied steroids
Rothová, Olga ; Holá, Dana (advisor) ; Motyka, Václav (referee) ; Hradilík, Jan (referee)
While animal steroid hormones are very well known and have been studied for a long time, in plants no steroid substances were known until relatively recently. Only in the second half of the past century brassinosteroids were discovered; later on, their hormonal function in plants was confirmed. Still a lot of unknown remains as regards their function in plant cells. This paper presents in its first part the evidence that brassinosteroids control in maize (Zea mays L.) grown under field conditions not only its morphology and yield but also some developmental/reproduction characteristics like e.g. number of female inflorescences or speed of the development of male inflorescences. Particular response of a plant depends, however, on the type of applied brassinosteroid, its concentration, and last but not least also on a particular maize genotype and developmental stage of the plant during applicatin. Impact of brassinosteroids on primary photosynthetic processes in plants has not been proven under these conditions, neither on the activity of photosystem (PS) I nor on the Hill reaction. No statistically significant differences in the content of photosynthetic pigments have been found either. Another topic dealt with in this thesis is the possible protective influence of brassinosteroids on plants...
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Effectors of RAB GTPases and their role in plant secretion
Růžičková, Martina ; Hála, Michal (advisor) ; Motyka, Václav (referee) ; Klíma, Petr (referee)
Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...
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Characterization and significance of N-glucosyltransferase pathway as a tool for regulation of cytokinin homeostasis in plants
Doležálková, Lucie ; Motyka, Václav (advisor) ; Vaňková, Radomíra (referee)
Adenine derivatives called cytokinins (CK) are a group of plant hormones, which in cooperation with other plant hormones orchestrates almost every aspect of plant growth and development. Despite the rapid progress in plant hormone research, there are still many aspects we may shed light on due to the metabolic and signalling pathways redundancy and the network complexity with crosstalk hubs. CK N-glucosides (in this case trans-zeatin-7-glucoside, tZ7G, and trans-zeatin-9-glucoside, tZ9G) have been traditionally viewed as irreversibly deactivated products of CK active form metabolism (in this regard trans-zeatin, tZ). Nevertheless, the tZ9G antisenescent activity was shown in oat leaf senescence bioassays (Avena sativa cv. Abel) and the possibility of metabolic conversion to O-glucosides was hypothesized. The aim of this work was to test the hypothesis on the close model of oat (Avena sativa cv. Patrik) and to examine also the metabolic conversion of related substances - N 6 -(Δ2 -isopentenyl)adenine (iP) and its N7- and N9- glucosides (iP7G and iP9G). While the senescence retardation caused by exogenous tZ9G application was confirmed in the Avena sativa cv. Patrik, metabolic conversions to O-glucosides remain to be verified. Besides the effects of above-mentioned substances on the oat leaf...
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Effectors of RAB GTPases and their role in plant secretion
Růžičková, Martina ; Hála, Michal (advisor) ; Motyka, Václav (referee) ; Klíma, Petr (referee)
Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...
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Vazba paralogů EXO70 na ATG8 a funkční rozdělení rodiny EXO70 dle účasti v autofagii (Arabidopsis thaliana).
Semerádová, Hana ; Kulich, Ivan (advisor) ; Motyka, Václav (referee)
The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...
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Metabolic pathways involved in the regulation of bioactive cytokinin levels in plants
Žižková, Eva ; Motyka, Václav (advisor) ; Pernisová, Markéta (referee) ; Hoyerová, Klára (referee)
SummarySummarySummarySummary Cytokinins (CKs) are important group of plant hormones involved in a wide range of physiological and developmental processes. Endogenous levels of CKs as well as proportions of individual CK forms and derivatives are not constant and differ among plant species. The amounts of biological active CK forms (free bases and ribosides) are regulated through tangled machinery of metabolic conversions including biosynthesis, conjugation and degradation pathways. The main object of this thesis was to characterize the metabolic pathways involved in the regulation of bioactive CK levels in plants especially via CK biosynthesis with aspect to the environmental stimuli and via N- glucosylation pathway. It was shown, that light signal is an important input for modulating some CK-related genes and CK levels in Arabidopsis plants. The complex diurnal expression profiles of CK-biosynthetic genes (AtIPT1 - AtIPT9) in Arabidopsis plants indicated a strong dependence of AtIPT1 and AtIPT5 on light/dark phase in leaf rossetes. In contrast, no diurnal oscillation of AtIPT transcript levels was recorded in roots. Although the content of endogenous CKs was not constant in plants and varied during a day, no statistically significant correlation between light/dark cycle and oscillation in CK levels was...
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Genetic variability in growth, reproductive and photosynthetic parameters of plants and its changes by exogenously applied steroids
Rothová, Olga ; Holá, Dana (advisor) ; Motyka, Václav (referee) ; Hradilík, Jan (referee)
While animal steroid hormones are very well known and have been studied for a long time, in plants no steroid substances were known until relatively recently. Only in the second half of the past century brassinosteroids were discovered; later on, their hormonal function in plants was confirmed. Still a lot of unknown remains as regards their function in plant cells. This paper presents in its first part the evidence that brassinosteroids control in maize (Zea mays L.) grown under field conditions not only its morphology and yield but also some developmental/reproduction characteristics like e.g. number of female inflorescences or speed of the development of male inflorescences. Particular response of a plant depends, however, on the type of applied brassinosteroid, its concentration, and last but not least also on a particular maize genotype and developmental stage of the plant during applicatin. Impact of brassinosteroids on primary photosynthetic processes in plants has not been proven under these conditions, neither on the activity of photosystem (PS) I nor on the Hill reaction. No statistically significant differences in the content of photosynthetic pigments have been found either. Another topic dealt with in this thesis is the possible protective influence of brassinosteroids on plants...
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Biologické účinky a metabolismus cytokininů typu cis-zeatinu v rostlinách
Gajdošová, Silvia ; Motyka, Václav (advisor) ; Brzobohatý, Břetislav (referee) ; Doležal, Karel (referee)
Cytokinins (CKs) are plant hormones that affect a wide range of developmental processes. The most important group of isoprenoid CKs represent zeatin and its derivatives occurring in two, cis and trans, positional isomers. Whereas trans-zeatin (transZ) was found to be a highly bioactive substance, cis-zeatin (cisZ) has been viewed for years as inactive or weakly active adjunct to its corresponding trans counterpart playing only an insignificant physiological role in plants. The occurrence of cisZ-type CKs was found in a great number of plant species with especially high levels identified in species of family Poaceae. All tested derivatives of cisZ, surprisingly including also zeatin-N9-glucoside, delayed dark-induced chlorophyll degradation in oat and wheat leaf segments. Additionally, cisZs effectively induced cell division in CK-dependent tobacco callus. The most pronounced activity was exhibited by cisZ riboside (cisZR) in the two types of CK bioassays. Metabolism of both zeatin isomers differed in short-term as well as in long-term experiments, which was supported also by various affinity of CK degrading enzyme, CK oxidase/dehydrogenase (CKX), to individual cis and trans isomers. Primary root enlargement of Arabidopsis seedlings was inhibited by cisZR in the same or similar way as by transZR,...
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