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Interakce teploty a hormonů v růstu Arabidopsis thaliana
Andrýsková, Kristýna
Abstract Due to global warming, plants are exposed to higher temperatures more often and for longer time periods. Exceeding the temperature optimum causes heat stress and affects plant growth and morphology. The most common plant reactions to high temperature are elongation of the hypocotyl, leaf hyponasty, or elongation of leaf petioles. All these morphological changes are collectively referred to as thermo-morphogenesis. The key modulator of this process is the transcription factor phy-tochrome-interacting factor PIF4. It is the main regulator of thermomorphogenesis through which the YUCCA8 gene is activated. YUCCA8 is a biosynthetic enzyme in auxin metabolism that induces auxin accumulation and activates cell elongation. The experimental part was focused on the influence of cytokinin hormones on the growth of the hypocotyl in Arabidopsis thaliana at standard (20 °C) and elevated (29 °C) temperatures. The increased temperature had a positive effect on the growth of the hypocotyl of control plants as well as transgenic lines with ectopic expression of the PIF4 gene. The effect of the application of 10µM trans-zeatin was temperature dependent. At standard temperature, cytokinins had a positive effect on the growth of the hypocotyl of control plants but did not have a significant effect in transgenic lines. However, transgenic lines had elongated hypocotyls compared to wild-type plants at standard temperature. Conversely, at a higher temperature, cytokinins only had an inhibitory effect. Automatic analysis of the abundance of the PIF4 pro-tein using GFP showed that its level within the hypocotyl is uneven with increasing abundance in the direction from the root to the cotyledons. Confocal microscopy confirmed that the inhibitory effect of cytokinins on hypocotyl elongation at higher temperatures is not due to a decrease in the PIF4 protein level.
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Proteom rostlin v reakci na abiotický stres
Čotková, Veronika
Plants as a sessile organisms have evolved complex mechanisms to cope with unfavorable environmental conditions. This diploma thesis summarizes current state of knowledge and focuses on light, temperature and phytohormones in plant abiotic stress responses. The practical part is devoted to proteomics and completes and extends the project started during my Bachelor program. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S> GR> HvCKX2) were profiled to elucidate proteome-wide responses to down-regulation of cytokinin levels under heat stress at standard (80 umol.m-2.s-1) and low light intensity (20 umol.m-2.s-1). In total, 2-DE analysis and mass spectrometry analysis revealed 63 differentially abundant proteins involved in diverse metabolic processes. These data provide evidence of a link between temperature, light and cytokinin signaling in Arabidopsis and will be used in the modeling of cytokinin-light-temperature interactions.
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Vliv modifikace receptoru ultrafialového světla na jeho funkci
Blechová, Veronika
Ultraviolet radiation has a negative effect on living organisms, but it is also signal modulating growth and development in plants. This thesis is focused on the effects of UV-B light in Arabidopsis thaliana mutants in UV-signalling pathway with modulated cytokinin level. Our data show that UV-B decrease the quantum yield of the PS II (Fv/Fm) in both mutant lines. Decrease of Fv/Fm was accompanied by growth retardation which could be reverted by exogenous application of 1 µM BAP. Determination of the pigments showed that cytokinins interact with UV-B light and this interaction is dependent on the functional UVR8. On the other hand, inhibition of the thermomorphogenesis by cytokinins is independent of the functional UVR8. Experimental work was also focused on the creation of the fusion genes containing modified UVR8 sequence with marker gene eGFP. The correct sequence of the recombinant GFP was confirmed by commercial sequencing.
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Inhibice regenerace prýtů na explantátech petunie (Petunia x hybrida) in vitro způsobená fluoranthenem
Czyžová, Kateřina
The aim of thesis was to study the effect of fluoranthene (FLT) on growth and biochemical processes of plant Petunia (Petunia hybrida cv. Lady blue) in in vitro conditions. In this experiment were used segments of the middle parts of the leaves, shoots from organogenesis in vitro and apical meristem of stem with four to five leaves from long-term in vitro culture. It was observed if fluoranthene influence organogenesis of shoots in vitro, contents of chlorophyll a and b, content of cytokinins and rating of shoots (from organogenesis in vitro) and apical meristem of stem with four to five leaves. It was used culture MS medium containing benzylaminopurin (1.5 mg/l), 1-naphtylacetic acid (0.18 mg/l) and fluoranthene (1 mg/l) or without fluoranthene (control). In the case of organogenesis in vitro, the experiment was performed at 4 different variations of media with subculturing to other media in triplicate. Variations of the media were chosen to allow period of cultivation with fluoranthene, which affects growth and biochemical processes. MS medium containing indol-3-butyric acid (0.5 mg/l) and FLT in different concentrations (0.1, 1, 3 a 5 mg/l) was used for monitoring rooting. The results showed that fluoranthen is able to affect organogenesis of shoots formed on the segments of leaves. There was lower frequency compared to the control organogenesis. It was found that fluoranthene more affects beginning of cultivation compared to later exposure of fluoranthene during cultivation. It was found the highest uptake of benzyladenine and its riboside from the culture medium. Fluoranthene affected significantly content of dihydrozeatin. Zeatin and its riboside were recorded in a minimal amount. Fluoranthene caused lower content of chlorophylls a and b in explantats. In the case of chlorophyll a were no significant differences between influence by fluoranthene at the beginning of the cultivation or during of cultivation. It was noted that fluoranthene influence the morphology of the roots subcultured shoots from organogenesis in vitro. Plant roots exhibited less branching and roots were shorter in culture medium containing higher content of fluoranthene (3 and 5 mg/l).
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3D rekonštrukcia povrchu buniek hypokotylov Arabidopsis thaliana so zmenenými hladinami cytokinínov
Hubinský, Marcel
Plants are multicellular organisms, which tightly control their growth and morphogene-sis to successfully accomplish the final reproductive individual plant. At the cellular level, plant development consists of two main processes, cell proliferation and cell ex-pansion. Differences in the timing, rate and spacing of these developmental processes determine the final size and structure of plants organs, such as hypocotyl. Cytokinins (CKs) play a key role during many aspects of plant development, including hypocotyl development. We confirmed the important role of CK during expansion phase of hypocotyl cells. Hypocotyls with increased levels of CK growing under low light conditions were formed by elongated epidermal cells whereas hypocotyls lacking CK produced smaller cells. Using advanced confocal microscopy methods and transgenic plants with GFP-labeled cortical microtubules (proCaMV35S::MBD-GFP) enabled to design recon-struction of hypocotyl surface with altered levels of CK. The final 3D model of hypocotyl epidermal cells shed a light on detail morphological changes upon CK alterations
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Totální syntéza [15N4] cytokininů a jejich využití
BUČEK, Jan
Cytokinins (CKs) and their metabolites and derivatives are essential for cell division, plant growth regulation and development. They are typically found at minute concentrations in plant tissues containing very complicated biological matrices. Therefore, defined standards labelled with stable isotopes are required for precise metabolic profiling and quantification of CKs, as well as in vivo elucidation of CK biosynthesis in various plant species. In this work, eleven [15N]-labelled C6-purine derivatives were prepared, among them five aromatic (4, 5, 6, 7, 8) and three isoprenoid (9, 10, 11) CKs. Compared to current methods, optimized syntheses of 6-amino-9H-[15N5]-purine (adenine, 1) and 6-chloro-9H-[15N4]-purine (6-chloropurine, 3) were performed to achieve more effective, selective and generally easier approaches. The chemical identity and purity of prepared compounds were confirmed by physico-chemical analyses (TLC; HRMS; HPLC-MS; 1H, 13C, 15N NMR). The presented approach is applicable for the synthesis of any other desired [15N4]-labelled C6-substituted purine derivatives.
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Cytokinines and their role in plant cell division, with accent on G2/M transition
Prášilová, Jana ; Ševčíková, Hana (advisor) ; Bíšová, Kateřina (referee)
The eukaryotic cell cycle is well understood mainly in yeasts and animals. Basic regulatory mechanisms, with cyclin-dependent kinases (CDKs) playing crucial roles, are similar in all eukaryotes including plants. CDKs operate mainly at the key cell cycle checkpoints, G1/S and G2/M. Phosphorylation and dephosphorylation of CDKs by kinases and phosphatases have both negative and positive effect. Negative regulator at the G2/M transition is WEE1 kinase which phosphorylates conserved amino acid residues T14 and Y15 of CDK. Phosphatase CDC25 removes this inhibitory phosphate in yeasts and animals and forces cells into mitosis. Plant cell cycle exhibits remarkable differences. Importantly, it is controlled by phytohormones, and some key points of regulation remain obscure - a functional plant homologue of yeast CDC25 phosphatase has not been found in plants yet though Y15 inhibitory phosphorylation by WEE1 kinase blocks mitosis entry in plants as well. Thus, the regulatory mechanism of G2/M transition in plant cells is still to be found. Phytohormones play a key role, not only in the plant cell cycle, but in whole plant development. Interplay between the two groups of phytohormones: auxins and cytokinins, is crucial. Especially cytokinins significantly influence the regulation of G2/M checkpoint. It is...
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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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Vliv mírného teplotního stresu na expresi genů metabolismu cytokininů a kinetiku růstu klíčních rostlin Arabidopsis thaliana
Truong, Thanh Huong
Climate change and thermal stress poses a problem for sustainable agriculture. Research in the adaptation and acclimation of plants to the elevated temperature is therefore one of the current scientific issues. Plants are exposed to different ambient temperatures during their life. The response to adverse temperatures includes a number of signalling pathways affecting development and growth processes in plants. Coordination of develomental processes under elevated temperature and other external factors is primarily controlled by plant hormones. Here effect of cytokinins on plant morphology, growth kinetics, gene expression and quanification in combination with increased temperature was observed. By determination of the hypocotyl growth during increased temperature, cytokinins were found to play important role in this process. Cytokinins were found to inhibit temperature induced hypocotyl growth and inversly seedlings treated with higher temperatutre showed decreased level of cytokinins which was confirmed on the level of marker gene expression and determination of levels of cytokinins.
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