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Význam rostlinných proteinů z podrodiny ABCB pro transport auxinu
Kubeš, Martin ; Zažímalová, Eva (advisor) ; Hejátko, Jan (referee) ; Reinöhl, Vilém (referee)
Polar auxin transport provides essential directional and positional information for many developmental processes in plants. At the cellular level, it is realized by both passive diffusion and the active transport through the membrane proteins - AUX1/LAXes, PINs and ABCBs. The aim of this thesis was to characterize the role of ABCB1, ABCB4 and ABCB19 proteins in polar auxin transport using transformed tobacco BY-2 cell lines. It was shown that the plasma membrane (PM) localization of the ABCB1, 4 and 19 is not polar. The ABCB4 was also more stable on PM after the treatment with auxin influx inhibitors; making use of ABCB4-cell line helped to uncover new characteristics of markers of endocytosis - the FM- dyes. The induction of ABCB19 has led to a decrease in 3 H-NAA accumulation with characteristic auxin starvation phenotype, similar to PIN7 overexpressing cell line, that could be rescued in case of PIN7 cell line by application of the auxin efflux inhibitor NPA; however, the accumulation of auxin in ABCB19-overexpressing cell line was less sensitive to NPA and the rescue of the auxin starvation phenotype was ineffective. Importantly, unique property of the ABCB4 was demonstrated: It displayed dual, auxin-concentration-dependent auxin transport activity in Arabidopsis roots, tobacco BY-2 and yeast cells. The...
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Visualization of root apoplastic pH in plants
Wernerová, Daša ; Fendrych, Matyáš (advisor) ; Paris, Nadine (referee)
Plant oriented movements, or tropisms allow the plant to actively respond to environmental stimuli to get more light, better access to nutrients and to grow roots deeper into the soil. Gravitropism drives the growth of roots along the gravity vector. Perception of gravity is triggered by the sedimentation of statoliths in columella root cap, but the exact signalling pathway behind this process is not known. Perception of gravity results in an unequal redistribution of the phytohormone auxin in the outer cell layers which leads to different rate of growth on the root's upper and lower side and bending of the root. The changes in auxin redistribution are accompanied by changes in apoplastic pH. Knowing an exact pattern of these pH changes could shed light on the mechanisms laying behind the gravitropic response pathway. While microelectrodes can be used to measure pH precisely, they are not suitable for the long-term imaging of growing roots. In the past few years, several pH sensitive dyes and genetically encoded sensors emerged. These can be used for long-term live in vivo imaging of pH changes in growing roots. In this thesis, I analysed the performance of several published pH sensitive genetically encoded sensors and available dyes in the roots of Arabidopsis thaliana. I observed that dyes varied...
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Mechanizmus transportu cytokininů přes buněčnou membránu a jejich metabolizmus v buňkách tabákové suspenzní kultury BY-2
Klíma, Petr ; Zažímalová, Eva (advisor) ; Galuszka, Petr (referee) ; Procházka, Stanislav (referee)
MECHANISM OF CYTOKININ TRANSPORT ACROSS PLASMA MEMBRANE AND THEIR METABOLISM IN TOBACCO BY-2 CULTURED CELLS Mgr. Petr Klíma / Abstract of Ph.D. Thesis / Prague 2011 Cytokinins (CKs) are plant hormones that play a major role in a number of developmental processes in plants. Those include promotion of cell division, active growth and differentiation, and maintenance of sink-source relationships, as well as control of environmental stress responses. Native CKs are low-molecular derivatives of adenine which seem to act either as paracrine or as long-distance signals. Due to their numerous physiological effects, plants have to precisely control the occurrence of bioactive CK molecules on the levels of the whole plant, its organs, tissues as well as single cells. To achieve this, a concerted action of metabolism and transport processes is required. Studies of the kinetics of CK translocation across plasma membrane in BY-2 suspension-grown tobacco cells suggested the existence of energy-dependent, partially selective transport routes for CK bases and CK ribosides. HPLC analysis of the metabolites of accumulated CKs pointed at their fast degradation or metabolic conversion into physiologically inactive forms. The prevalent ways of inactivation were the degradation to adenine and phosphorylation or phosphoribosyl...
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Specificity of selected exocyst subunits in trichome development
Glanc, Matouš ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy
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Molecular mechanism of transport of plant hormone auxin into cells
Laňková, Martina ; Zažímalová, Eva (advisor) ; Stiborová, Marie (referee) ; Opatrný, Zdeněk (referee)
The non-uniform distribution of the plant growth regulator (phytohormone) auxin is known to mediate many fundamental processes in plant development. Auxin is transported through the plant body either via vascular pathways or from cell to cell by specialized polar auxin transport machinery. This machinery consists of a balanced system of passive diffusion combined with the activities of auxin influx and efflux carriers. This work is focused on the processes that are involved in the uptake of auxin into plant cells. On the basis of molecular-biological and biochemical characterization, the function as an auxin influx carrier was confirmed for PaLAX1 protein from wild cherry (Prunus avium). The sequences of isolated cDNA of the PaLAX1 gene and of its protein product are highly similar to both the cDNAs and the corresponding protein products of the AUX1/LAX-type genes, coding for putative auxin influx carriers in model plant A. thaliana. On the level of organs and single cells, we have shown that the overproduction of PaLAX1 in transgenic lines resulted in an increase of the content of native auxin indole-3-acetic acid as well as of the uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid. Further, the mechanism of action of putative auxin influx inhibitors 1-naphthoxyacetic acid (1-NOA),...
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Mechanisms of establishment and maintenance of PIN polarity in Arabidopsis
Glanc, Matouš ; Friml, Jiří (advisor) ; Grebe, Markus (referee) ; Luschnig, Christian (referee)
Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...
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Visualization of root apoplastic pH in plants
Wernerová, Daša ; Fendrych, Matyáš (advisor) ; Paris, Nadine (referee)
Plant oriented movements, or tropisms allow the plant to actively respond to environmental stimuli to get more light, better access to nutrients and to grow roots deeper into the soil. Gravitropism drives the growth of roots along the gravity vector. Perception of gravity is triggered by the sedimentation of statoliths in columella root cap, but the exact signalling pathway behind this process is not known. Perception of gravity results in an unequal redistribution of the phytohormone auxin in the outer cell layers which leads to different rate of growth on the root's upper and lower side and bending of the root. The changes in auxin redistribution are accompanied by changes in apoplastic pH. Knowing an exact pattern of these pH changes could shed light on the mechanisms laying behind the gravitropic response pathway. While microelectrodes can be used to measure pH precisely, they are not suitable for the long-term imaging of growing roots. In the past few years, several pH sensitive dyes and genetically encoded sensors emerged. These can be used for long-term live in vivo imaging of pH changes in growing roots. In this thesis, I analysed the performance of several published pH sensitive genetically encoded sensors and available dyes in the roots of Arabidopsis thaliana. I observed that dyes varied...
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FUNCTIONAL ANAYSIS OF SELECTED EXO70 EXOCYST SUBUNITS IN PLANTS
Kubátová, Zdeňka ; Kulich, Ivan (advisor) ; Grossmann, Guido (referee) ; Lichtscheidl-Schultz, Irene (referee)
Arabidopsis thaliana trichomes are large unicellular epidermal outgrowths with a specific development and intriguing shape, which makes them an excellent cell type for our research of cell polarization mecha- nisms. Cell polarity is essential for plant development and the exocyst complex is one of its key regulators. It is an octameric protein complex that mediates polarized exocytosis and growth by targeted tethering of secretory vesicles to the plasma membrane. Its EXO70 subunit functions as a landmark for exocytosis site and physically binds the target membrane through interaction with phospholipids. A remarkable multipli- cation of EXO70 subunit paralogs in land plant genomes is well documented, but the functional diversity of these paralogs remains to be described. In trichomes we revealed the specific role of the EXO70H4 paralog in secondary cell wall deposi- tion, especially in callose synthase delivery. We documented formation of a thick secondary cell wall during the maturation phase of wild type trichome development and a lack of it in the exo70H4 mutant. Moreover, we showed evidence for silica deposition dependency on callose synthesis. Further, we unveiled the formation of apical and basal plasma membrane domains, which differ in their phospholipid compo- sition and ability to bind...
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