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Creating and testing bioluminescence sensors for cytokinins in Arabidopsis thaliana
Novotná, Lenka ; Petrášek, Jan (advisor) ; Vosolsobě, Stanislav (referee)
Advances in microscopy and the sensitivity of novel biosensors have enabled the development of new methods for monitoring phytohormones with low invasiveness and relatively high resolution, providing greater insight into phytohormone function in plants. Genetically encoded indirect biosensors for cytokinins, one of the main plant hormones, are widely used for the tracking of dynamic changes in their levels during plant development and growth. The cytokinin-sensitive synthetic promoters used as cytokinin biosensors are being continuously improved, providing better insight into cytokinin function. However, the reporter systems used for the visualizations are either based on the enzymatic reaction producing colour or on the usage of fluorescence proteins. Luminescence-based biosensors are often used to visualize signaling outputs of plant hormones, but they are based on exogenous addition of luciferin substrate. Therefore, in this thesis, I have introduced a new technique for the generation of cytokinin signaling output sensors based on autonomous bioluminescence. This autonomous bioluminescence represents a non-invasive technique that depends on the expression of the whole autonomous bioluminescence system from fungi Neonothopanus nambi, in particular, the caffeic acid cycle with the fungal...
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Characterization of the role of cytokinins and abscisic acid during abiotic stress response
Přerostová, Sylva ; Vaňková, Radomíra (advisor) ; Havel, Ladislav (referee) ; Doležal, Karel (referee)
Abiotic stresses significantly reduce crop yield, causing serious problems in agriculture. Understanding the mechanisms of plant stress responses could contribute to the improvement of their stress tolerance. Phytohormones play an important role in plant stress defence as well as in regulation of growth and development. This thesis summarizes the results published in four articles focused on the evaluation of the effects of phytohormones during abiotic stresses, namely salinity, drought, ZnO nanoparticle treatment and cold stress. The main emphasis is put on abscisic acid as the key regulator of water status and stress defence, and on cytokinins, which regulate plant growth and stabilize photosynthetic machinery. Cytokinins act antagonistically to abscisic acid. Our results showed that abscisic acid is a general abiotic stress response regulator. Stress- tolerant plants (halophyte Thellungiella salsuginea or winter line of einkorn wheat Triticum monococcum) had a higher basal level of this hormone, especially in shoot meristematic tissues (apices, crowns), than stress-sensitive plants. Stress-tolerant plants reacted faster and in a more flexible way to stress. Active cytokinins were negatively affected by stress, which was associated with growth suppression. The drought stress study showed that...
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Transport and Metabolism of Radio-Labelled Cytokinins in Plant Cells and Tissues
Nedvěd, Daniel ; Ryšlavá, Helena (advisor) ; Vaňková, Radomíra (referee)
Cytokinins are a large group of phytohormones. Since their discovery in the 1950s, they have shown to play a pivotal role in plant physiology. Most studies so far focused on cytokinin action mechanisms and their metabolic regulation. Identification of AtABCG14 and AtPUP14 as cytokinin-specific membrane carriers brought researchers' attention to cytokinin membrane transport, too. In this thesis, we performed experiments with radio-labelled cytokinin tracers. We show that trans-zeatin and isopentenyladenine, two major biologically active cytokinins, are readily transported across the plasma membrane in tobacco BY-2 cell suspension. Making use of mathematical modelling, we show that BY-2 cells possess a membrane transport system with an affinity toward cytokinins. Next, we show that atabcg14 and atpup14 mutations affect cytokinin metabolism in Arabidopsis thaliana plants. Keywords: cytokinin, Arabidopsis thaliana, tobacco BY-2 cell lines, membrane transport, purine permease, ATP-binding cassette, radio-labelling
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Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level.
Lubovská, Zuzana ; Wilhelmová, Naďa (advisor) ; Prášil, Ilja (referee) ; Kočová, Marie (referee)
Charles University in Prague, Faculty of Science Department of Experimetnal Plant Biology Ph.D. study program: Plant physiology and anatomy Abstract Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level. Zuzana Lubovská Supervisor: RNDr. Naďa Wilhelmová, CSc. Supervisor-consultant: RNDr. Helena Štorchová, CSc. Praha, 2015 The response of the antioxidant enzyme system to drought, heat and a combination of these stresses was followed in tobacco plants overexpressing cytokinin oxidase/dehydrogenase1 (CKX1) under the root-specific WRKY6 promoter (W6:CKX1 plants) and under the constitutive 35S promoter (35S:CKX1 plants) and in the corresponding wild type (WT). CKX1 over-producing lines developed a bigger root system, which contributed to their stress tolerance. The high stress tolerance of 35S:CKX1 plants was also associated with a dwarf shoot phenotype and changed leaf morphology. The genes for chloroplastic antioxidant enzymes, stroma ascorbate peroxidase (sAPX), thylakoid ascorbate peroxidase (tAPX) and chloroplastic superoxide dismutase (FeSOD), which are responsible for scavenging of reactive oxygen species produced via electron transfer during photosynthesis, were all strongly transcribed in control conditions. All the tested stresses down-regulated expression of these genes...
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Transport and Metabolism of Radio-Labelled Cytokinins in Plant Cells and Tissues
Nedvěd, Daniel ; Ryšlavá, Helena (advisor) ; Vaňková, Radomíra (referee)
Cytokinins are a large group of phytohormones. Since their discovery in the 1950s, they have shown to play a pivotal role in plant physiology. Most studies so far focused on cytokinin action mechanisms and their metabolic regulation. Identification of AtABCG14 and AtPUP14 as cytokinin-specific membrane carriers brought researchers' attention to cytokinin membrane transport, too. In this thesis, we performed experiments with radio-labelled cytokinin tracers. We show that trans-zeatin and isopentenyladenine, two major biologically active cytokinins, are readily transported across the plasma membrane in tobacco BY-2 cell suspension. Making use of mathematical modelling, we show that BY-2 cells possess a membrane transport system with an affinity toward cytokinins. Next, we show that atabcg14 and atpup14 mutations affect cytokinin metabolism in Arabidopsis thaliana plants. Keywords: cytokinin, Arabidopsis thaliana, tobacco BY-2 cell lines, membrane transport, purine permease, ATP-binding cassette, radio-labelling
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Role cytokininů v regulaci vyrůstání pupenů hrachu
Vetter, Martin
The diploma thesis was aimed to explain the role of cytokinins in regulation of cotyledonary bud outgrowth in pea (Pisum sativum L.). Decapitation of the apex and subsequent application of IAA lanolin paste containing also BAP caused increased expression of genes PsDRM1 and PsBRC1 associated with dormancy. It suggests that cytokinins may have effect on the amount of transported auxin in the major stem. Application of cytokinin to the cotyledonary buds of intact plants activated their outgrowth. Imunolocalization showed polarization of PIN1 proteins in the treated buds, on contrary to untreated buds. It means that cytokinin directly influenced polarization of PIN1 proteins and the subsequent canalization of the polar auxin transport.
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Charakterizace jaderného proteomu rostlin
Svetláková, Anna
Nuclear proteome contains important regulatory proteins that are directly involved in gene regulation mechanisms. It is estimated that about 30% of genes encode for proteins that are destined into the nucleus, but a large dynamic range in protein concentration results in some nuclear proteins being present at concentrations orders of magnitude lower than others and these are thus hidden in standard total protein extracts. This thesis The characterization of the plant nuclear proteome briefly reviews present-day knowledge of nuclear proteomics and summarises extraction protocols. In the experimental part, two different fractionation protocols were evaluated. Analyses of nuclear protein extracts increased the number of identified proteins, but the quantitative comparison with the results of total protein extractions indicate that at least some nuclear proteins were lost or depleted in the process. Finally, the effect of plant hormone cytokinin on barley nuclear proteins was analysed. In total, 65 known nuclear proteins (10% of identified proteins in the nuclear fraction) were found and 15 of them were differentially abundant in response to the cytokinin treatment, including e.g. RNA helicase SUB2 and a salt-stress response protein.
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Morfogenéza epidermálnych buniek listov Arabidopsis thaliana v odpovedi na chladový stres a zníženú dostupnosť svetla
Tekulová, Veronika
The bachelor thesis was focused on the physiological, cellular and transcriptomic characterization of Arabidopsis thaliana treated with cold under different light conditions. To determine the role of the cold stress in leaf ontogenesis, the analysis included leaves in the different developmental stages. It was found that 4 weeks old plants have almost completely developed 6th leaf and cold treatment has no impact on its cellular morphology. In contrast, younger leaf number 14 which initiates a transition from proliferation to expansion stage was strongly affected by cold treatment. It is shown that exposition to a low temperature significantly reduces its cell area and a number of epidermal cells. Propidium staining confirmed that young proliferating leaves are also more sensitive to stress conditions than older leaves. The higher sensitivity of young leaves to cold stress was accompanied with lower expression of CBF genes that play an important role in cold stress tolerance. Younger leaves also showed higher expression of cytokinin responsive genes and genes related to cell cycle corresponding to ongoing developmental processes.
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Characterization and significance of N-glucosyltransferase pathway as a tool for regulation of cytokinin homeostasis in plants
Doležálková, Lucie ; Motyka, Václav (advisor) ; Vaňková, Radomíra (referee)
Adenine derivatives called cytokinins (CK) are a group of plant hormones, which in cooperation with other plant hormones orchestrates almost every aspect of plant growth and development. Despite the rapid progress in plant hormone research, there are still many aspects we may shed light on due to the metabolic and signalling pathways redundancy and the network complexity with crosstalk hubs. CK N-glucosides (in this case trans-zeatin-7-glucoside, tZ7G, and trans-zeatin-9-glucoside, tZ9G) have been traditionally viewed as irreversibly deactivated products of CK active form metabolism (in this regard trans-zeatin, tZ). Nevertheless, the tZ9G antisenescent activity was shown in oat leaf senescence bioassays (Avena sativa cv. Abel) and the possibility of metabolic conversion to O-glucosides was hypothesized. The aim of this work was to test the hypothesis on the close model of oat (Avena sativa cv. Patrik) and to examine also the metabolic conversion of related substances - N 6 -(Δ2 -isopentenyl)adenine (iP) and its N7- and N9- glucosides (iP7G and iP9G). While the senescence retardation caused by exogenous tZ9G application was confirmed in the Avena sativa cv. Patrik, metabolic conversions to O-glucosides remain to be verified. Besides the effects of above-mentioned substances on the oat leaf...
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Mechanism of cytokinin transport across plasma membrane
Nedvěd, Daniel ; Ryšlavá, Helena (advisor) ; Dračínská, Helena (referee)
Cytokinins are one of major groups of phytohormones, signaling molecules participating on the crucial physiological processes of plants. Regulation of their function is mostly associated with metabolic changes, recent identification of membrane proteins AtABCG14 and AtPUP14 as selective cytokinin transporters has brought up the possibility of the regulation of cytokinin signalization via polar membrane transport, though. Using the accumulation of radioactively labeled cytokinins in transgenic BY-2 cell culture, we studied the kinetics of cytokinin transport via AtABCG14 and AtPUP14 transporters at cellular level and possible changes of cytokinin metabolism linked to elevated expression of these transporters. Elevated expression of AtPUP14 transporter led to elevated accumulation of labeled cytokinins in form of both ribosides and free bases, while it had no effect on metabolism of labeled cytokinins. Elevated expression of AtABCG14 had no effect on accumulation of labeled cytokinins in BY-2 cells. (In Czech) 1
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