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Mechanisms of transmembrane auxin transport in a broader evolutionary context.
Rubešová, Magdaléna ; Petrášek, Jan (advisor) ; Tylová, Edita (referee)
Auxin, represented by the molecule indole-3-acetic acid (IAA), is one of the main phytohormones involved in the regulation of plant development. Its intercellular transport establishes concentration gradients in individual cells that control gene expression and a number of downstream processes. In plants, a complex mechanism for efficient IAA transport has evolved, involving both long-distance transport and intercellular transport within individual tissues. Because our understanding of the auxin transport mechanisms is still incomplete, this thesis attempts to summarize the literature data on all modes of auxin transport across cell membranes that have been recognized to date and places them in a broader evolutionary context. The presence of IAA in many prokaryotic and eukaryotic organisms, together with the similarly wide occurrence of carriers from "auxin efflux carrier" transporter family, evolutionarily related PIN-FORMED-like carriers, points to the possibility that IAA transport may also be evolutionarily very ancient and may functionally derive from more general mechanisms of ions or amino acids.
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Characterization of factors participating in regulation of intracellular dynamics of auxin carriers
Lacek, Jozef ; Zažímalová, Eva (advisor) ; Ibl, Verena (referee) ; Nodzynski, Tomasz (referee)
Plants are known to adjust the orientation of their organs, shoot and root, to ensure maximal energy generation and nutrient uptake, but also to avoid toxic growth conditions. Directional growth regulation depends on asymmetric plant organ growth and it is crucial to ensure plant survival. It is orchestrated on cellular level in concert with exogenous and intrinsic signals. Even though tropistic growth responses of plants were described by Darwin on macroscopic level already in 1880, now it is necessary to understand molecular mechanisms that underpin efficient modulation of directional plant growth. During my studies I focused on factors that modulate directional root growth regulation. The root is a complex, three-dimensional object, which continuously modifies its shape and growth path. Since the root needs to expand its surface to supply the plant with nutrients and water, it is important to understand how roots cope with changing growth conditions while exploring the soil. If the root cannot manage to grow through soil efficiently, mechanical impedance and lack of resources will also restrict shoot growth as well. Manifold signaling pathways coordinate the complex processes that underpin efficient root growth, including those modulated by phytohormones, sugars, flavonoids and other...
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Cellular determinants of the distribution of PIN auxin transporters in the plasma membrane
Stelate, Ayoub ; Petrášek, Jan (advisor) ; Mravec, Jozef (referee) ; Nodzynski, Tomasz (referee)
Asymmetric localization of auxin carriers has always attracted the attention of many scientists around the world. However, to address this topic while focusing on the plasma membrane (PM), requires advanced microscopy techniques and knowledge of biophysics and biology. This doctoral work bridges the two disciplines to contribute to our understanding of the dynamics and distribution of PIN-FORMED auxin carriers from tobacco (NtPINs) within the PM. I have developed a novel correlative light electron microscopy (CLEM) method using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). To my knowledge, this is the first effort to achieve a correlation between immunofluorescence and electron microscopy imaging of plant integral PM proteins. As I have shown, individual NtPINs are organized differently within the PM. Dynamic analyses that quantify individual nanodomains' diffusion rates allowed me to show that NtPINs have constraints behavior with different diffusion rates. I have investigated the role of the cell wall and cytoskeleton in the organization and dynamics of NtPINs. Using pharmacological treatments, I have shown that they differentially affect the mobility and organization of NtPINs within the PM. Complete removal of the...
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Analysis of Gretchen Hagen 3 gene family in tobacco BY-2 cell culture
Helusová, Lenka ; Müller, Karel (advisor) ; Pěnčík, Aleš (referee)
Auxin conjugation is one of the crucial metabolic processes regulating auxin activity in plant cells. Gretchen Hagen 3 (GH3) is a family of acyl amido synthetases that conjugates auxin with amino acids and belongs amongst important enzymes involved in auxin conjugation. Due to the existence of more sensitive methods to detect auxin metabolites and the current study of abiotic stress effects, research on GH3 enzymes is intensified these days. These enzymes are best known in thale cress (Arabidopsis thaliana), soya bean (Glycine max), rice (Oryza sativa). These models don't allow to study their activities in a biochemical way. Therefore, the aim of this work was to monitor the auxin metabolism in the established model tobacco BY-2 cell lines (Nicotiana tabacum). The NtGH3.1 and NtGH3.6 genes, which were shown to have a variability in their expression regulation by auxin, were targeted and mutated using tne CRISPR/Cas9 method. Mutations in the derived lines were detected by sequencing. In the derived lines, auxin metabolic profililing was analysed by LC/MS. Metabolic profiling showed a correlation between the NtGH3.6d form and the specific production of the metabolite oxIAA- Gln (N-(2-onindole-3-acetyl)-glutamine). The study of an eventual substitution of individual GH3 gene forms in mutant lines...
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The role of actin cytoskeleton in the targeting of auxin carriers to the plasma membrane.
Kebrlová, Štěpánka ; Petrášek, Jan (advisor) ; Pernisová, Markéta (referee)
Auxin plays an important morphogenic role in plant development, mainly through its effect on gene expression, but also through a number of faster processes that are directly dependent on its concentration. Therefore, in many plant tissues, directional auxin transport using specific transporters in the plasma membrane, is important for the coordination of morphogenic stimuli. The amount of auxin carriers in the plasma membrane directly affects the resulting auxin concentration inside the cell. Although the localization of auxin transporters and their abundance in the plasma membrane could be determined primarily by the actin cytoskeleton and its involvement in vesicle transport processes, this relationship is currently still unclear. Therefore, in this study, we were interested in how the localization and function of auxin transporters is affected when the function of the actin cytoskeleton is affected in a given cell type. To this end, the localization of the auxin transporters PIN3, PIN4, PIN7, and AUX1 was studied in epidermal cells of cotyledons in young seedlings of Arabidopsis thaliana whose morphogenesis was affected by mutations in subunits of the actin nucleation complex ARP2/3. Crosses of mutants in the ARP2/3 complex subunits with marker lines carrying fluorescently labeled auxin carriers...
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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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Regulation of potato tuber onset: the role of auxin
Myslivcová, Jitka ; Mašková, Petra (advisor) ; Havelková, Lenka (referee)
Potato (Solanum tuberosum) is an essential component of food worldwide. The process of tuber formation is controlled by the interplay of external and internal conditions. Understanding of the molecular basis of tuberization, including the effect of photoperiod, pointed to a key role of mobile signals. Among these morphogenic signals StBEL5 and POTH1 transcripts are involved, which are translated in the stolon and form a heterodimer regulating target genes with TTGAC tandem motifs, further an FT homologue, StSP6A, mobile from leaves to stolons as a protein. BEL5/POTH1 and StP6A signaling pathways seem to overlap. Phytohormones also play an important signaling role, i.a. auxins. During tuber initiation, IAA levels in stolon tissues increase. PIN transporters are likely to be involved in the redistribution of auxin in the stolon, but also LAX and ABCB transporters and components of auxin signaling pathway, Aux/IAAs and ARFs are also involved in the process. Although auxins are proved to play an important role during stolon-to-tuber transition, their exact role in the process is not sufficiently clarified. Auxin signaling is interconnected with BEL5/POTH1 and SP6A-mediated tuber signaling, as its target genes in the stolon include those that encode proteins involved in auxin biosynthesis (YUCCA1),...
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Auxin transport in algae
Skokan, Roman ; Petrášek, Jan (advisor) ; Bíšová, Kateřina (referee)
Phytohormone auxin plays an important role in regulating plant development. Directional (polar) cell-to-cell auxin transport creates auxin gradients within plant tissues, which trigger a specific developmental response. The vast majority of available data concerns angiosperms. Lower land plants have been much less explored in this regard, but the important auxin-related mechanisms (including polar auxin transport) are already present in mosses. To uncover the origins of auxin action, one must focus on green algae, especially of clade Streptophyta, which are the direct ancestors of all land plants. In this study, the possible effects of auxins, both native and synthetic, were investigated on two algae: basal, unicellular Chlorella lobophora and advanced, filamentous Spirogyra sp. The latter received comparably more attention, since it belongs to a clade now acknowledged as a sister group to land plants. Chlorella lobophora culture growth was irresponsive to synthetic auxin NAA. The average Spirogyra sp. cell length was, however, changed by auxins at high concentrations. By conducting accumulation assays of radioactively labelled auxins and HPLC analysis, auxin metabolism and transport was investigated in Spirogyra sp. This alga was able to metabolize the plant-native IAA, but not synthetic auxins...
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New insights on auxin metabolism
Helusová, Lenka ; Müller, Karel (advisor) ; Ryšlavá, Helena (referee)
Auxin is the longest known phytohormone with many functions. It participates in the development of embryo, in vegetative and generative development, plant stress reaction, and also in their senescence and death. Activity of auxin in plant cells and tissues is quite often compared to the effect of morphogens, i.e. the effect of substances, whose concentrations induce a strictly defined response in tissue and organ formation. Apart from intercellular and intracellular auxin transport, the main determinants of auxin concentration gradients are its biosynthesis and metabolism. Regardless of the long-lasting study of auxin, the complexity of metabolic pathway regulation is still not well known. The aim of this thesis is to summarize current knowledge of the metabolism of auxin in plants, i.e. its biosynthetic pathways, conjugation and oxidation, and put them into a context with older results.
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