|
|
Studium funkce AtAFB4
Zelinka, Radim
Despite the fact, that research on apical dominance started before more than 100 years ago, this economically important phenomenon is not fully understood yet. One of the genes involved in this regulation in the model plant Pisum sativum is PsMS2, that was cloned recently and it came out i tis an ortholog of the auxin F-box receptor AtAFB4. The aim of this bachelor thesis was to prepare hybrids of the Arabidopsis mutant afb4 with plants expressing the GFP gene for green fluorescent protein fused to genes of the PIN family (1,2,3,4,7) under native promoters. T1 plans from all five crosses were test-ed preliminary using the selection gene for resistance to the herbicide sulphadiazine contained in the genome of male plants and more than 50 % of T1 plants from all lines survived without any symptoms of damage. In further cultivation of the surviving plants strong phenotype resembling the afb4 mutation of the male plants was observed in all plants. From the above-mentioned facts we can conclude that we have obtained hybrids bearing the intended genetic information. These prepared plants will be used in further studies on the influence of the afb4 mutation on the changes of PIN membrane transporters and further characteristics, like changes in auxin transport, reaction to exogenous application of auxin and strigolactone, etc.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
Characterization of Arabidopsis thaliana FLOTILLINs and HYPERSENSITIVE INDUCED RESPONSE proteins - dynamics, interactions and functions
Daněk, Michal ; Martinec, Jan (advisor) ; Baluška, František (referee) ; Ovečka, Miroslav (referee)
This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...
|
|
|
Studying dimer formation and effectors of Arabidopsis thaliana nascent polypeptide-associated complex
Klodová, Božena ; Fíla, Jan (advisor) ; Robert Boisivon, Helene (referee)
The development of plant flowers represents a complex process controlled by numerous mechanisms. The creation of double homozygous mutant of both β subunits (sometimes also referred to as basic transcription factor 3) of nascent polypeptide associated complex in Arabidopsis thaliana (further referred to as nacβ1 nacβ2) caused quite a strong defective phenotype including abnormal number of flower organs, shorter siliques with a reduced seed set, and inferior pollen germination rate together with a lower ovule targeting efficiency. Previously, NAC complex was described to be formed as a heterodimer composed of an α- and β-subunit, which binds ribosome and acts as a chaperone in Saccharomyces cerevisiae. In plants, NACβ is connected to stress tolerance and to plant development as a transcription regulator. However, little is known of NAC heterodimer function in plants. In this thesis, yeast two hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays were used to verify the NAC heterodimer formation in A. thaliana and to establish any potential interaction preferences between both NACβ paralogues and five NACα paralogues. To deepen the understanding about molecular mechanisms behind the nacβ1 nacβ2 phenotype, flower bud transcriptome of the nacβ1 nacβ2 double homozygous mutants...
|
|
| |
|
|
AHL genes in plant development
Škrabálková, Eliška ; Širl, Marek (advisor) ; Ortmannová, Jitka (referee)
Plant development is a complex process where different factors come into play. The expression of certain genes, which determine basic plant structure and its signaling pathways, is also of great importance. The AHL gene family, which in case of Arabidopsis thaliana includes 29 members, is one of those determinators that have an impact on plant development on several levels. As far as structure is concerned, the AHL proteins are typically composed of two parts - the DNA-bonding AT-hook motif and PPC domain, which defines nuclear localisation and eventually enables oligomerisation. In terms of phylogenetics, the AHL proteins are divided into clades A and B on the basis of number and type of these domains. When it comes to affecting plant development, these nuclear proteins are capable of bonding with the corresponding DNA, and in cooperation with other factors influencing gene expression. In the plant body the AHL proteins are involved in root and floral organogenesis and also cooperate with a number of signaling pathways of phytohormones, such as auxins, gibberellins, brassinosteroids or senescence hormones. Moreover, they take part in photomorphogenesis or control systematic immune responses of the plant. Keywords: AHL, AT-hook, PPC domain, nuclear protein, Arabidopsis thaliana
|
|
|
Inducible RNAi against essential genes of nitrogen metabolism as a tool for control of GM plants
Kobercová, Eliška ; Fischer, Lukáš (advisor) ; Tylová, Edita (referee)
Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...
|
guest ::
