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Analysis of endomembrane markers in the cortical cytoplasm and their co-localization with Arp2/3 complex
Jelínková, Barbora ; Schwarzerová, Kateřina (advisor) ; Fendrych, Matyáš (referee)
ARP2/3 is an evolutionarily conserved heteroheptameric protein complex. Its main activity lies in the nucleation of dendritic actin filaments that are involved in membrane remodeling. ARP2/3 takes part in plasma membrane remodeling and the formation of cytoplasmic protrusions that serve in the amoeboid motion of mammalian cells and some protists and plays role in exocytosis and endocytosis of animal and yeast cells. The main objective of this work was to find a connection between the ARP2/3 complex and the regulation of the plant endomembrane system. Using TIRF microscopy we visualized the localization of the ARP2/3 complex in the cortical layer of plant cells and compared it to the localization of several endomembrane markers from the Rab family and an exocytotic marker Exo84b. In the vicinity of the plasma membrane, the ARP2/3 complex subunits localized to dynamic dots very similar to the localization of Exo84b protein. Colocalization analysis showed that a small portion of Exo84b marker and ARP2/3 complex signals colocalize and this result was seconded by the biochemical approach of coimmunoprecipitation. Key words: ARP2/3, endomembrane system, cortical layer, RabA1g, RabC1, RaD2a, Exo84b
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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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Dynamics and role of the Arabidopsis thaliana IAA17/AXR3 protein in regulation of root growth by auxin
Kubalová, Monika ; Fendrych, Matyáš (advisor) ; Glanc, Matouš (referee)
Auxin is phytohormone that regulates several developmental processes and environmental responses. One of the most well-described outcome of the auxin signalling pathway is regulation of gene transcription. Aux/IAA proteins play an important role in this process, acting as transcriptional repressors. Recent studies revealed that several root growth responses are too rapid to be explained by changes in the level of transcription. The correlation between the amount of Aux/IAAs and the root growth rate suggests that these proteins might be involved in root growth regulation, especially during rapid growth responses that are not associated with transcriptional reprogramming. This work is focused on one of the 29 Arabidopsis Aux/IAA proteins - the IAA17/AXR3 protein. First, we produced stable transgenic lines of Arabidopsis thaliana expressing different combinations of fluorescently labelled AXR3-1 proteins and/or fused to subcellular localization tags under the control of different tissue-specific promoters, in order to characterize the subcellular localization of the studied protein. Subsequent visualization by confocal microscopy methods confirmed information about the role of IAA17/AXR3 protein in root growth responses, its involvement in auxin signalling, and gravitropism. Next, we showed that the...
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Molecular mechanism of mechanoreception in plants
Jelínková, Barbora ; Martinek, Jan (advisor) ; Fendrych, Matyáš (referee)
Plant, as sedentary organism, does not have many possibilities to physically escape it's unpleasant surroundings, instead it adapts oneself. One of many plant senses that are crucial for tracking environment changes is mechanoreception. Plant senses and differentiates between many mechanical cues, some of them affecting plant immunity and morphogenesis. The whole plant cell reacts to mechanical cues and many cellular structures are involved in mechanoreception. Any change in cell wall - a borderline between the cell and it's surroundings - is transduced to plasma membrane, then to the cytoskeleton and potentially to other structures. Concept of this cell wall-plasma membrane-cytoskeleton continuum and it's use as an instrument to illuminate molecular mechanisms of mechanoreception in plants are the key topics of my thesis.
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The evolution of auxin metabolism, signalling and transport in the green lineage (Viridiplantae)
Schmidt, Vojtěch ; Petrášek, Jan (advisor) ; Fendrych, Matyáš (referee)
Phytohormone auxin is an essential coordinator of growth and development within land- plants. Mechanisms of auxin action in algae is still poorly understood, given that related research has begun recently. Current progress is derived mainly from available sequence data, which are necessary for analysis of gene homology with higher plants. In this case with the genes related to auxin action mechanisms, e.g. metabolism, signaling and transport. Auxin biosynthesis predated the split of Chlorophyta and Streptophyta, but tighter regulation of auxin levels is the matter of land-plants. First auxin responses were probably non-transcriptional. Transcription-regulating nuclear signaling pathway assembled from more ancient domains, some of them were identified in Charophyta, but whole pathway was complete after colonization of land. Auxin transport out of the cell was one of the original mechanisms of regulating its levels. More sophisticated auxin carrier system was proposed to evolve in Charophyta, where polar auxin transport was identified. Auxin as a signaling molecule was present in primitive algae groups, but its response spectrum dramatically increased after colonization of land. Future research of auxin action evolution needs more model organisms to be established, especially from Charophyta....
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Molecular basis for regulation of cell wall pH in Arabidopsis thaliana
Bogdan, Michal ; Fendrych, Matyáš (advisor) ; Retzer, Katarzyna (referee)
The cell wall is one of the defining parts of the plant cell. Cell walls resist turgor pressure, give plants their rigidity and still allow the cell to grow despite their stiffness. The plant cell wall is a composite material that consists of different elements; thus, the knowledge of the plant cell wall composition helps us understand the impact of the mentioned pH changes. Alterations of plant cell wall properties result in a change of plant cell growth rate. A prime example of this is the modification of wall properties by pH change, termed the acid growth. Although acid growth has been studied for a long time, we still do not fully understand the underlying mechanisms, as we have not yet identified all the agents involved in acid growth and our ability to determine apoplastic pH is limited. Local cell wall pH fluctuations arise also due to plant organs having different roles and being affected by a diverse range of stimuli. Despite the fact that new pH measurement techniques, such as genetically encoded fluorescent probes, were developed in the last two decades, there is still a need to provide a higher spatiotemporal resolution of pH analysis. Key words: Arabidopsis, apoplastic pH, cell growth, acid growth theory, cell wall, auxin, apoplastic pH measurement
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Plant tomosyns and their role in secretion
Dejová, Lilly ; Hála, Michal (advisor) ; Fendrych, Matyáš (referee)
Tomosyn is a protein belonging to the Lgl family and conserved across the animal and plant kingdom. Tomosyn is composed of N-terminal domain containing WD40 motif and Cterminal domain, where the R-SNARE motif is located at the end of the C-terminal domain. This motif is classified as homologous to R-SNARE motif of synaptobrevine, which is a protein located on the surface of the vesicles and participating in the formation of SNARE complex and subsequent fusion of the vesicles with the plasma membrane. Thus the role of this tomosyn is mainly the regulation of exocytosis. Apart from the animal tomosyn, its yeast homolog Sro7/Sro77 is also examined, however during the evolution it has lost its R-SNARE motif and therefore the plant tomosyns remain unexplored. The aim of this diploma thesis was to characterize both of the plant tomosyns: AtTYN1 and AtTYN2 in Arabidopsis thaliana plant. The experimental thesis included the bioinformatic analysis, the DNA construct creation, a search for interactors by yeast two-hybrid system and monitoring the localization using the confocal microscope. The bioinformatic analysis results, including the creation of phylogenetic tree, not only revealed the conservation of tomosyns across the different classes, but also the division of both tomosyns into different clusters. There was...
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Role of the exocyst complex in stomata dynamics
Drs, Matěj ; Pečenková, Tamara (advisor) ; Fendrych, Matyáš (referee)
The exchange of gases between the plant and the environment is mediated by guard cells, which, by changing their volume, regulate the size of the stomatal pore where the exchange takes place. Unique properties of stomata are given by a specifically designed cell wall, allowing asymmetric cell expansion. During the opening of stomata, there is a large increase in turgor pressure, accompanied by the uptake of considerable amount of water. For this reason, it is necessary to quickly change the inner surface of the guard cell. This process is ensured by the dynamic movement of the membranes inside the cell. During these processes, the vesicles are removed from the plasma membrane and reintegrated back during stomatal closure. One of the important regulators of the polarized transport of vesicles within the cell is a multiprotein complex exocyst. Due to the multiplication of exocyst subunits, the exocyst complex functions in plants are extensively diversified and involved in many cellular processes. In this thesis the effect of mutation in subunits exo70B1, exo70B2 and its combination exo70B1/exo70B2, on stomatal dynamics is studied. Both of these mutations affect the opening of the stomata during increased light intensity. The mutation of exo70B1 is in this case affected in greater manner. These...
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Interaction of ectomycorrhizal and ericoid mycorrhizal host plants via ectomycorrhizal, ericoid mycorrhizal and pseudomycorrhizal fungi
Fendrych, Matyáš ; Albrechtová, Jana (advisor) ; Gryndler, Milan (referee)
Abstract 9. Abstract Roots of ectomycorrhizal and ericoid mycorrhizal plants are believed to be colonized by fungi belonging to different taxonomic groups. However, both frequent isolations of ericoid mycorrhizal fungi from ectomycorrhizal root tips and a few recent studies (Vrålstad et al. 2000, 2002b, Piercey et al. 2002, Hambleton & Sigler 2005) indicate that there is a group of mycobionts colonizing both types of roots. Ectomycorrhizal morphotype Piceirhiza bicolorata was shown to be induced by Meliniomyces sp. belonging to the Rhizoscyphus ericae aggregate (Vrålstad et al. 2000). The ability to colonize roots of potentially ectomycorrhizal and ericoid plants simultaneously was proven in in vitro experiments in the case of Rhizoscyphus ericae (Pirecey et al. 2002) and Cadophora finlandica (VillarrealRuiz et al. 2004). DSE fungi ("dark septate endophytes", formerly termed pseudomycorrhizal) represent another group of mycobionts colonizing both ericoid and potentially ectomycorrhizal plant roots. In the present work, we inoculated roots of ericoid (Vaccinium myrtillus) and potentially ectomycorrhizal plants (Picea abies, Pinus sylvestris and Betula nana) with typically ectomycorrhizal and ericoid mycorrhizal fungi and...
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Role of exocyst complex in growth and development of moss Physcomitrella patens
Rawat, Anamika Ashok ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee) ; Fendrych, Matyáš (referee)
During the course of evolution the early land plants gained extensive innovations that can be seen in modern day plants. The polar growth is an ancient feature of eukaryotic cells and is one of preadaptations that helped plants in successful colonization of land. The polar growth in plants regulates not only the direction of cell expansion and structural properties of cell wall but especially also the orientation of cell division, and is governed by various factors, including the exocyst complex. The exocyst is a well conserved vesicle tethering multi-subunit complex involved in tethering of secretory vesicles to the target membrane. The essential role of the exocyst complex in regulation of various cellular processes in Angiosperms is now well documented. Here I present results of a doctoral project that contributed to phylogenetic analyses of the land plant exocyst complex and especially to uncovering functions of three moss exocyst subunits, namely EXO70 (isoform PpEXO70.3d), SEC6 and SEC3 (isoforms PpSEC3A and PpSEC3B) in the model organism Physcomitrella patens. Various knock-out (KO) mutants in several moss exocyst subunits (Ppexo70.3d, Ppsec6, Ppsec3a and Ppsec3b) show pleiotropic defects directly or indirectly linked to the cell polarity regulation. Cell elongation and differentiation,...
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