|
|
Unraveling the Enigmatic World of Auxin Signalling: Subcellular and Tissue- Specific Aspects in the Root of Arabidopsis thaliana
Dubey, Shiv Mani ; Fendrych, Matyáš (advisor) ; Robert Boisivon, Helene (referee) ; Kulich, Ivan (referee)
In this dissertation, my main focus was to advance our understanding of how the plant hormone auxin regulates root growth at the sub-cellular and tissue levels in Arabidopsis thaliana. Auxin controls gene transcription through the SCFTIR1/AFB - Aux/IAA coreceptor complex. Among the TIR1/AFBs auxin receptor family members, TIR1 is strictly localized in the nucleus, while AFB1 is particularly abundant in the cytoplasm but also present in the nucleus. I confirmed the dominant role of AFB1 in controlling rapid auxin responses, such as calcium ion influx, apoplastic alkalinization and rapid root growth inhibition; processes associated with root gravitropism. I discovered a novel AFB1-dependent cytoplasmic auxin perception, and I identified the N-terminal domains of AFB1 and TIR1 crucial in determining their subcellular localization. Furthermore, my research contributed to the discovery that the root surface pH gradient on the root's longitudinal axis is not only regulated by AHA H+ -ATPases, but instead, the rapid auxin response module, comprising the AUX1 auxin influx carrier, AFB1, and the CNCG14 calcium channel, controls the apoplastic pH in the root transition zone. Further, I participated in the discovery of a deeply evolutionarily conserved rapid auxin response pathway that involves the RAF-like kinase,...
|
|
|
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...
|
|
|
RNA interference in plants
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Kulich, Ivan (referee)
The process of RNA interference allows cells to regulate functions of their genes. This process is usually initiated by the presence of double-stranded RNA within a cell. Such double-stranded RNA is diced by a specific protein called Dicer into duplexes of small RNAs, usually 20-25 nucleotides long. Single-stranded small RNAs, released from the duplexes, are the heart of RNA interference and they can be categorize into several groups according to their biogenesis. There are two groups of small RNAs in plants: miRNA and siRNA. Small RNAs can associate with a protein called Argonaut and guide it to the target molecule on the bases of sequence complementarity. The Argonaut-small RNA complex can act on itself or it can interact with other proteins in a wide spectrum of processes. The complex can slice the target mRNA (which can be handled by the sole Argonaut and small RNA), it can suppress translation or it can direct chromatin modifications. The phenomena of RNA interference can be found in almost all Eukaryotes where it can serve many functions, for example it can control cell differentiation, participate in stress responses, direct changes in chromatin and defend the organism against viruses. A diverse set of operating modes of RNA interference can be found in plants, which we are only at the...
|
|
|
The Role of selected exocyst subunits in response of plants to pathogen
Sabol, Peter ; Kulich, Ivan (advisor) ; Burketová, Lenka (referee) ; Dagdas, Yasin (referee)
In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...
|
|
|
Mechanoreception in plants
Martinek, Jan ; Vosolsobě, Stanislav (advisor) ; Kulich, Ivan (referee)
Because of their sessile nature, plants are unable to change their location and thus they are forced to adapt as much as possible to the environment they grow in. Plants evolved the ability to sense many environmental cues, which enables them to perceive the conditions in their surroundings. One class of these stimuli are mechanical forces - from wind sways to contact with obstacles, herbivores or other plants - other mechanical stimuli are e.g. gravity or sound waves. Carnivorous or climbing plants have structures specialised for perception and rapid response to mechanical stimuli. Intriguingly, there is a less spectacular but maybe even more interesting and important response to mechanical perturbation in non-specialized plants. This thesis tries to summarize ubiquity of mechanoperception in plant kingdom and its adaptive importance for the plant life - from activation of traps, to morphological adaptation for growth at windy sites, tendril coiling in climbing plants and root navigation through obstacles in soil. In the following part, the thesis summarizes the recent knowledge of molecular processes accompanied with mechanoreception, signal transduction and integration, and response to mechanostimulation. In the last part I proppose a scheme of mechanosensing workflow from initial mechanical...
|
|
|
Secretory pathway of plants in pathogene defence
Sabol, Peter ; Kulich, Ivan (advisor) ; Burketová, Lenka (referee)
Plants are sessile organisms that have to cope with the changes of their ambient environment. These changes include abiotic disturbances and stresses as well as biotic interactions with other organisms. In many of the biotic interactions, plant cells are hurt or damaged by various bacterial and fungal pathogens. Moreover, plants lack mobile immune cells, which would otherwise render them susceptible to most of these pathogens. Plants have, however, developed other mechanisms for suppressing the infection. Activating the components of the secretory pathway during resistance response is crucial step for suppressing the penetration and growth of pathogens. Fungal pathogens, such as powdery mildews, usually attempt to penetrate the cell wall in order to gain the access to protoplast providing nutrients and to exchange their virulence factors. On the other hand, pathogenic bacteria inhabit extracellular spaces of plant tissues. In both cases, however, the deposition of papilla into the plant cell wall was observed. Papilla thus seems to be the common defence mechanism. There is an increasing evidence confirming the indispensable role of polarized secretion in formation of papilla. Recently, SNARE proteins participating in papilla-associated defence have been described. PEN1 and SNAP33 syntaxins are of...
|
|
|
Conventional and Novel Functions of the Exocyst Complex in Plants
Kulich, Ivan ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.
|
|
|
Mechanisms and regulation of callose deposition to the cell wall of Arabidopsis thaliana
Modráčková, Jana ; Kulich, Ivan (advisor) ; Šustr, Marek (referee)
Callose (β-1,3-glucan) is a significant plant cell wall polysaccharide, which participates in many developmental and stress responses. Despite the importance of this polymer, the knowledge on the causes of callose synthesis, its regulation and the meaning of callose deposition are still limited. The family of callose synthases was revealed thanks to molecular and genetic methods. They are responsible for callose deposition in diferent reactions and the diferent spaces of the plant body. This thesis summarizes the knowleadge about the processes, in which callose is involved, and it presents an overview of the individual proteins from callose synthase family including their phylogenetic analysis and the comparison with the cellulose synthase focusing on the model plant Arabidopsis thaliana. Key words: Arabidopsis thaliana, β-1,3-glucan, callose, callose synthase, plant cell wall polymer
|
|
| |
|
|
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...
|
guest ::
