Specifita vybraných podjednotek exocystu při vývoji trichomu

Trichomy jsou jemné struktury epidermálního původu, které pokrývají povrch nadzemních orgánů většiny rostlin. Jednobuněčné trichomy Arabidopsis thaliana jsou dobře zavedeným modelem v buněčné a vývojové biologii rostlin, přesto je o procesech ukládání buněčné stěny během finální fáze zrání trichomu známo jen velmi málo. V naší laboratoři byla nedávno objevena role podjednotky poutacího komplexu exocyst EXO70H4 při zrání trichomu. V této práci byly za použití analýzy obrazu, histochemického barvení a FT-IR spektroskopie studovány defekty v buněčné stěně u ztrátového mutanta exo70H4. Bylo zjištěno, že mutace vede ke změně v ukládání pektinu a možná též ligninu a hemicelulóz. Byly připraveny transgenní linie s paralogy EXO70 exprimovanými pod promotorem EXO70H4 a jejich analýza ukázala, že nejbližší paralog EXO70H3 dokáže komplementovat mutaci EXO70H4, na rozdíl od paralogů EXO70A1 a EXO70B1. Na základě těchto výsledků jsou diskutovány otázky týkající se složení buněčné stěny trichomů, role EXO70H4 ve zrání trichomu a funkcí komplexu exocyst u rostlin. Klíčová slova: Arabidopsis, trichom, buněčná stěna, sekreční dráha, komplex exocyst, EXO70H4, FT-IR spektroskopieTrichomes 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 spectroscopyKatedra experimentální biologie rostlinDepartment of Experimental Plant BiologyPřírodovědecká fakultaFaculty of Scienc

Úloha sekreční dráhy v biosyntéze polysacharidů buněčné stěny rostlin a FT-IR spektroskopické metody v analýze složení buněčné stěny

Všechny rostlinné buňky jsou obklopeny buněčnou stěnou, která ustavuje jejich velikost a tvar a je nezbytná pro mnoho zásadních buněčných procesů. Buněčná stěna streptofytních rostlin je tvořena převážně vysokomolekulárními polysacharidy. Celulóza, hlavní složka buněčné stěny rostlin, je syntetizována transmembránovými proteinovými komplexy přímo na povrchu buňky, zatímco hemicelulózy a pektiny jsou tvořeny v Golgiho aparátu. Bylo popsáno několik proteinů účastnících se syntézy těchto polysacharidů, nicméně stovky dalších teprve čekají na objevení. Proteiny nutné pro syntézu celulózy a molekuly hemicelulóz a pektinů syntetizované v Golgiho aparátu jsou transportovány na povrch buňky sekreční drahou, tato dráha tedy hraje důležitou roli v biogenezi buněčné stěny. Hypotézy o regulaci sekrece komponent buněčné stěny počítají s působením aktinového a mikrotubulárního cytoskeletu, vápníkového gradientu nebo PIP kináz; komplexní regulace tohoto procesu však zdaleka není popsána. FT-IR spektroskopie je metoda schopná detekce molekulárních vibrací, s jejíž pomocí je možné získat informace o chemickém složení prakticky všech látek. Tato metoda je úspěšně používána v identifikaci mutantů s abnormální buněčnou stěnou, ve studiu interakcí mezi jednotlivými složkami buněčné stěny a v dalších experimentech...All plant cells are encapsulated in a cell wall that determines the cells' shape and size and is essential to many of their vital processes. The cell wall of streptophyte plants is composed mainly of polysaccharides of high molecular weight. Cellulose, the main constituent of the plant cell wall, is synthesized by protein complexes bound to the PM, while hemicelluloses and pectins are synthesized in the Golgi apparatus. Several proteins that participate in the synthesis of cell wall polysaccharides have been identified, but hundreds of them remain to be discovered. Secretory pathway plays an important role in plant cell wall biogenesis as it transports cellulose synthase complexes and noncellulosic polysaccharide molecules to the cell surface. Some regulatory mechanisms that might be involved in cell wall material secretion, such as actin cytoskeleton, Ca2+ gradient or PIP kinases have been proposed; however, the regulation of this process is very complex and far from being understood. FT-IR spectroscopy is a method that can detect molecular vibrations and provide information about chemical composition of virtually all substances. It has been used successfully in screens for cell wall mutants, the study of interactions between cell wall polymers, as well as other areas of cell wall biology. FT-IR...Katedra experimentální biologie rostlinDepartment of Experimental Plant BiologyPřírodovědecká fakultaFaculty of Scienc

AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into howthese factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. Wethus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.Plant science

Specificity of selected exocyst subunits in trichome development

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 spectroscop

Mechanismy ustavení a udržení polarity PIN přenašečů v Arabidopsis

Buněčná polarita představuje v biologii rostlin klíčový koncept. Subcelulární lokalizace auxinových přenašečů rodiny Pin-formed (PIN) v kořeni "#%&'()*&* 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...Department of Experimental Plant BiologyKatedra experimentální biologie rostlinFaculty of SciencePřírodovědecká fakult

Secretory pathway in plant cell wall polysaccharide biosynthesis and FT-IR spectroscopy methods in plant cell wall composition analysis

All plant cells are encapsulated in a cell wall that determines the cells' shape and size and is essential to many of their vital processes. The cell wall of streptophyte plants is composed mainly of polysaccharides of high molecular weight. Cellulose, the main constituent of the plant cell wall, is synthesized by protein complexes bound to the PM, while hemicelluloses and pectins are synthesized in the Golgi apparatus. Several proteins that participate in the synthesis of cell wall polysaccharides have been identified, but hundreds of them remain to be discovered. Secretory pathway plays an important role in plant cell wall biogenesis as it transports cellulose synthase complexes and noncellulosic polysaccharide molecules to the cell surface. Some regulatory mechanisms that might be involved in cell wall material secretion, such as actin cytoskeleton, Ca2+ gradient or PIP kinases have been proposed; however, the regulation of this process is very complex and far from being understood. FT-IR spectroscopy is a method that can detect molecular vibrations and provide information about chemical composition of virtually all substances. It has been used successfully in screens for cell wall mutants, the study of interactions between cell wall polymers, as well as other areas of cell wall biology. FT-IR..

Mechanisms of establishment and maintenance of PIN polarity in Arabidopsis

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..

Specificity of selected exocyst subunits in trichome development

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 spectroscop

Mechanisms of establishment and maintenance of PIN polarity in Arabidopsis

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..

Secretory pathway in plant cell wall polysaccharide biosynthesis and FT-IR spectroscopy methods in plant cell wall composition analysis

All plant cells are encapsulated in a cell wall that determines the cells' shape and size and is essential to many of their vital processes. The cell wall of streptophyte plants is composed mainly of polysaccharides of high molecular weight. Cellulose, the main constituent of the plant cell wall, is synthesized by protein complexes bound to the PM, while hemicelluloses and pectins are synthesized in the Golgi apparatus. Several proteins that participate in the synthesis of cell wall polysaccharides have been identified, but hundreds of them remain to be discovered. Secretory pathway plays an important role in plant cell wall biogenesis as it transports cellulose synthase complexes and noncellulosic polysaccharide molecules to the cell surface. Some regulatory mechanisms that might be involved in cell wall material secretion, such as actin cytoskeleton, Ca2+ gradient or PIP kinases have been proposed; however, the regulation of this process is very complex and far from being understood. FT-IR spectroscopy is a method that can detect molecular vibrations and provide information about chemical composition of virtually all substances. It has been used successfully in screens for cell wall mutants, the study of interactions between cell wall polymers, as well as other areas of cell wall biology. FT-IR..