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Srovnávací analýza působení exogenního působení cytokininů s inhibicí degradace jejich endogenních hladin u Arabidopsis thaliana
Koukalová, Vladěna
Agricultural production is negatively affected by stress factors like drought or rising ambient temperature. Genetic engineering and production of specific GMO seems to be very promising in protection of the crop yield however these are strictly limited for the use in the field. For this reason alternative approaches like application of growth regulators are in the focus of recent research. INCYDE is a new potent growth regulator discovered as inhibitor of cytokinin degradation. In this Diploma thesis we show that high levels of INCYDE could also activate AHK4, cytokinin receptor, and act like cytokinins. Comparison of trans-zeatin and INCYDE showed that application of these regulators results in spatial-specific pattern in activation of the cytokinin signaling. trans-zeatin and INCYDE also induce different changes in proteom mainly in proteasome pathway, gluconeogenesis and fatty acids metabolism. Application of INCYDE simultaneously with inducing drought stress revealed that INCYDE could decrease sensitivity to this stressor. This feature seems to be very perspective for its utilization in the agriculture.
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Ovlivnění odpovědí rostlin k teplotnímu stresu modulací hladin cytokininů a giberelinů
Kharkevich, Kristina
Plants are sessile organisms which are very much affected by global climate change. In order to prevent themselves by overheating Arabidopsis plants induce growth to cool themselves more efficiently. Growth response to increased ambient temperature is modulated by different classes of hormones. Despite their importance in proliferation, effect of cytokinins under increased ambient temperature haven been studied in details so far. Experiments with transgenic plants expressing bacterial gene ipt and Hordeum vulgare gene HvCKX revealed that increased and also decreased levels of cytokinins affect morphometric parameters of Arabidopsis seedlings and plants. Experiments with mutant lines revealed that cytokinin-induced growth responses are independent of light signalling and response regulators controlling circadian rhythm. Combine experiments with pharmacological substance and genetic mutants show that cytokinin-induced growth responses under heat stress are independent of gibberellin signalling pathway. Formula clause:I herewith request that my Diploma thesis (Cytokinins and gibberellins induced changes in growth response to heat stress) is excluded from the open public access granted in accordance with the Section 47b of Act No. 111/1998 Coll., on Higher Education Institutions and the alterations and amendments thereto. This work contains sensitive data that are prepared for publishing in an impacted scientific journal. The restriction should last for at least 24 months.
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Vliv mírného teplotního stresu na expresi markerových genů cytokininů
Horáková, Adéla
Optimal temperature is one of the key environmental factors necessary for regular plant development, growth and production. Since ambient temperature is increasing due to global warming, response of the plants to increased ambient tempareture is in the focus of the recent plant research. In this context, phytohormones like auxins, gibberellins, brassinosteroids and newly cytokinins were shown to modulate plant response to increased temperature. Despite of high cytokinin impact on plant development and growth, the effect of the temperature on cytokinin signalling haven´t been studied yet. Analysis of cytokinin marker line TCSn::GFP showed that long-time higher temperature treatment increase cytokinin signalling in root tips of model plant Arabidopsis thaliana. Detailed RT-qPCR analysis of genes participating in cytokinin signalling showed that temperature modulates expression of both ARR type A and B genes. Moreover, short-time temperature treatment was shown to decrease expression of cytokinin markers ARR5 and ARR7 in whole-seedling samples.
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Interakce giberelinů a cytokininů v růstu klíčních rostlin Arabidopsis thaliana
Horáková, Adéla
Recently global warming and increasing environmental temperature are discussed very frequently. Ambient temperature is an important factor regulating plant growth and global changes in temperature could significantly affect the yield of the agriculture. Our previous work revealed the role of plant hormones cytokinins in response to temperature and showed cytokinins as negative regulators of the thermomorphogenesis. Since gibberellins are known as positive regulators of the thermomorphogenesis this work was focused on their interaction in response to temperature in Arabidopsis thaliana. Analysis of the morphology of plant seedlings showed that the effect of cytokinins dominates over gibberellins in response to high temperature and is detectable soon after high-temperature treatment. Analysis of gene expression showed that cytokinins induce expression of gibberellin biosynthesis enzyme GA20ox1 and decrease the expression of gibberellin degradation enzyme GA2ox6. On the other hand, cytokinins have an inhibitory effect on the expression of the master regulator of thermomorphogenesis PIF4. Analysis of transgenic line overexpressing PIF4 suggests that cytokinins could affect the thermomorphogenesis by regulation of the PIF4 expression but this process is not the key mechanism of the regulation of the response to high temperature.
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Stabilita membrán jako měřítko odolnosti vůči chladovému stresu u kontrastních ekotypů Arabidopsis thaliana
Kopytko, Valeriia
Cold acclimation is a process of adaptation to suboptimal temperatures preparing the plants to efficiently reduce the upcoming damage that may be caused by sub-zero temperature. The early phase of cold acclimation is associated with stimulation of the protective mechanisms and fast suppression of growth. This work was focused on the comparison of the effect of normal and low light intensity on the stability of the membrane in Arabidopsis plants during the cold acclimation period. Ion conduc-tivity is a sensitive method that directly evaluates the impact of stress on plant fitness and thus, we used the electrolyte leakage method for assessing cell membrane stability to test the cold stress tolerance. Next, we will focus on the natural variation of stress responses in a broad spectrum of Arabidopsis ecotypes to dissect genetic traits confer-ring the plants with a tolerance to cold and freezing stress. During the monitoring of expression profiles of the cold signaling pathway genes for various resistant ecotypes it was found, that ecotype C24 is sensitive to low temperatures, although it shows sig-nificant tolerance to other stresses. The Tamm-2 ecotype, conversely, shows a high level of cold and frost tolerance and has a high expression of cold signaling genes. Cold tol-erance correlated with expression of genes that contribute to low temperature responses: CBF1, CBF2, CBF3, and COR47.
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Vliv cílených mutací receptoru cytokininů CRE1/AHK4 z Arabidopsis thaliana na jeho odezvu vůči přirozeným a syntetickým cytokininům
Horáčková, Kamila
The Arabidopsis thaliana cytokinin receptor CRE1/AHK4 is a part of the signalling pathway by which cytokinin regulates the transcription of target genes via transcription factors. To further elucidate its molecular function, a transgenic E. coli strain KMI001 baring a ß-galactosidase-based reporter system can be employed. The theoretical part of this thesis describes the AHK4 receptor, including its structure, function and previously characterized mutations, and outlines the bacterial reporter system for AHK4 activity assays. The experimental part summarizes the results of ß-galactosidase activity assay optimization, PCR mutagenesis, the transformation of mutagenized plasmids into the E. coli strain KMI001 and receptor activity assays. In accordance with previous reports, the receptor mutant forms exhibited a higher sensitivity to trans-zeatin than the corresponding AHK4 wild-type. Further, the V471A mutant showed a significant decrease in the half maximal effective concentration for 6-benzylaminopurine and thidiazuron.
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Význam glutathionu v hormonální regulaci odpovědi na stres u Arabidopsis thaliana
Šlapáková, Martina
Tripeptide glutathione plays a key role in combating stresses such as heavy metal or xenobi-otic exposure. Recent studies have shown that glutathione content is negatively modulated by cytokinin. Thus, glutathione may be intimately involved in enhanced stress tolerance displayed in cytokinin-deficient plants. To probe the physiological relevance of the crosstalk between cytokinin and glutathione, a response to a glutahione-depleting xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) was analysed in the model plant Arabidopsis thaliana. Here we show that CDNB causes decrease of cytokinin status. We found that transgenic plants with reduced endogenous cytokinin levels showed a CDNB-tolerant phenotype. Cy-tokinin depletion provoked the accumulation glutathione. Glutathione-deficiency abolishes CDNB tolerance of cytokinin-deficient plants supporting an essencial role of glutathione for CDNB sequestration. Our findings indicate that cytokinin is an important regulatory factor in plant adaptation to CDNB stress.
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Vplyv kyseliny jasmónovej na rast a vývoj rastlín v odpovedi na zmenené hladiny endogénnych cytokinínov
Perašínová, Nikola
Plant hormones act as messengers of intracellular and extracellular signals in the plant cell that regulate various developmental processes. The main plant hormones participating in intracellular and extracellular signal transduction pathways are, among the others, cytokinins (CK) and jasmonic acid (JA). The role of both hormones in plant development is well-known, whereas their mutual interactions remain rudimentary. In this thesis, we have found that elevated JA levels contribute to enhancing CK sensing by plants. On the other hand, the high levels of CK were found to stimulate expression of transcription factors that play a key role in JA signaling pathway. Our results suggest interesting interaction network of two plant hormones, which participate in abiotic and biotic stress responses. These findings offer a novel universal method for the sustainable agriculture that raises demands for stress-tolerant crop species.
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Stimulace růstu rostlin změnou teplotních podmínek
Mašlaňová, Anna
Ambient temperature is the key factor that may positively or negatively regulate the growth and development of land plants. For instance, the change in temperature conditions determines the leaf development, flowering period and the fruit ripening. In this thesis, we investigate the role of cytokinin (CK) in the response of plants to higher soil temperature. Using genetic and transcriptomic analysis, we have identified several components of CK signaling pathway that orchestrate the increase growth rate of plants growing in the warm (35 °C) soil. The cellular analysis of epidermal leaf cells showed that this higher growth rate is caused by 2-fold higher cell expansion. This pattern of changing cell morphogenesis correlates with decreased levels of active forms of CK that act as a key factor in the maintenance of cell proliferation phase in the leaf.
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Role cytokininů při teplotou indukovaném přechodu rostlin z vegetativní do generativní fáze
Malých, Veronika
Recent numerous studies have been focused on the impact of the climate changes on the crop production. The response of plants to increased temperature include hypocotyl and petiole elongation, leaf hyponasty, alternations in timing of the flowering and even the inhibition of the flower bud development which may lead to reduced crop yields. Acclimation and stress responses are co-regulated by levels of plant phytohormones such as auxins, gibberellins, brassinosteroids and cytokinins. Although cytokinins play important role in physiological and developmental processes the effect of the interplay of temperature and cytokinin in growth and developmental processes has not been studied till now. This thesis is focused on the impact of cytokinins on thermomorphology and time of flowering of Arabidopsis thaliana in combination with increased temperature under long day conditions. It was observed that modulation of cytokinin levels reduce biomass and area of leaf rosette and this effect is dependent on temperature. As next, it was confirmed that interactions of cytokinins and temperature play important role in transition to generative developmental stage of plant Arabidopsis.
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