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Vliv transportu auxinu na regulaci vyrůstání kotylárních pupenů hrachu
Macoszek, Ondřej
The diploma thesis was aimed on role of polar auxin transport in regulation of cotyledonary bud outgrowth in pea plants (Pisum sativum L.) in relation to competitive canalization of auxin sources. Decapitation of growth apex, as primary source of auxin, causes release of cotyledonary buds from inhibition. This was followed by increased expression of genes for auxin transport proteins PsPIN1 and PsAUX1 and decrease in PsDRM1 and PsBR1 gene expression that are related to dormant state of plant tissues. In the next phase, different types of exogenous auxin application on decapitated stumps and cotyledonary bud growth dynamic were carried out. Auxin application on decapiteded stump caused inhibition of cotyledonary bud growth, its application on right or left side of the stump inhibits cotyledonary bud on the side of application. If auxin on the both sides of stump was applied, cotyledonary bud growth was not inhibited, on contrary to their separation with a plastic sheet, which resulted in significant growth inhibition of both buds. In case of both-sided application, these sources of auxin prevents each other in canalization. If these two sources of auxin were separated with a plastic sheet, canalization of auxin flow was observed and two independent auxin channels were formed. Analogy of this competition can be expected in the case of cotyledonary buds, as two potential auxin sources.
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Plant hormones homeostasis: auxin and cytokinin crosstalk
Doležálková, Lucie ; Zažímalová, Eva (advisor) ; Dračínská, Helena (referee)
5 AbstractAbstractAbstractAbstract It has been since 19th century when the phytohormones and their crosstalk became one of the central themes in plant biology. Due to advances in molecular biology there is number of metabolic pathways linked to certain groups of plant hormones revealed by now, nevertheless much remains to be determined about their cooperation. The aim of this thesis is to evaluate the effect of exogenous cytokinin application on oat leaf segments (Avena sativa) - both specifically towards other phytohormones levels and generally in physiological context (in relation to their senescence). Our results confirmed increased auxin levels due to exogenous application of selected cytokinins and identified biological activity of trans-zeatin-9-glucoside (previously considered as irreversibly deactivated cytokinin type substance) in respective plant model. (In Czech) Key words: phytohormones, auxin, cytokinin, Avena sativa, senescence assay
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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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Production of auxin transporter PIN1 in HEK293 cell line
Petermannová, Romana ; Vaněk, Ondřej (advisor) ; Hýsková, Veronika (referee)
PIN1 protein belongs to a PIN family of transporters. This group of transporters is significantly involved in auxin transports outside of the cell. The auxin transport among plant cells gives an important role in plant growth, gravitropism, etc. The principle of auxin transport by this carrier protein is still not explored. This work deals with the production of PIN1 carrier protein by recombinant expression in human embryonic kidney 293 cell line. Production in these cells was checked by the accumulation of radioactive auxin and by the immunofluorescence.
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The secretory vesicles tethering complex exocyst and the auxin transport polarization
Janková Drdová, Edita ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
The polarization of exocytosis in yeast and animals is assisted by the exocyst - an octameric vesicle tethering complex and an effector of Rab and Rho GTPases. Recently, the exocyst was described as a functional complex involved in morphogenesis also in plants. Hála et al. (2008) described involvement of exocyst complex in pollen tube growth and hypocotyls elongation in dark grown seedlings, Fendrych et al. (2010) uncovered key role of exocyst in cell plate formation, Kulich et al. (2010) emphasized the participation of exocyst in seed coat generation and Pečenková et al. (2011) described the contribution of exocyst subunits in plant defense towards the pathogens. All these processes are intimately linked to polarized secretion. Here we show involvement of exocyst in auxin efflux carriers PINs recycling. Using direct auxin transport measurement and GFP-tagged proteins, we showed that the exocyst is involved in recycling and polarization of PIN proteins and polar auxin transport regulation. Rootward polar auxin transport is compromised in loss-of-function mutants in exocyst subunits EXO70A1. On the cellular level we have detected small portion of PIN2:GFP in the "BFA-like" FM4-64 labelled compartments distinct from VHAa1 labeled endosoms. Moreover recycling of PIN1 and PIN2 is retarded in roots of...
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Transcriptional regulation of PIN4 protein, membrane transporter of plant hormone auxin.
Hurný, Andrej ; Petrášek, Jan (advisor) ; Holá, Dana (referee)
PIN-FORMED (PIN) proteins are plant-specific secondary transporters acting in the efflux of plant signaling molecule auxin from cells. Their asymmetrical localization within cells determines the directionality of auxin flow and thereby influences plant development. The activity of PIN proteins is regulated at multiple levels; however the primary step in the regulation of PIN proteins takes place at the level of gene transcription. Therefore the main focus of this diploma thesis is the characterization of the transcriptional regulation of PIN proteins, namely PIN4 protein. The observation of plants carrying transcriptional fusion consisting of various lengths of PIN4 promoter and green fluorescent protein (GFP) showed which part of PIN4 promoter is essential for binding transcription factors and for the start of transcription. This part of PIN4 promoter was used as bait for transcription factors in yeast one hybrid screens. Altogether, 24 transcription factors were identified in which the most numerous were transcription factors from GATA and APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) families. To verify the interactions between identified transcription factors and PIN4 promoter, the protoplast transient expression assay was used. Protoplasts isolated from Arabidopsis thaliana leaves and tobacco BY-2 cell...
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Evoluce mechanismů transportu auxinu.
Skokan, Roman ; Petrášek, Jan (advisor) ; Cvrčková, Fatima (referee)
Auxin, the longest studied phytohormone, is distinguished from other phytohormones by its unique directional, so-called polar transport. This feature helps to facilitate the broad range of auxin action at all stages of plant development. The polar auxin transport has been evolving together with plant lineages. By studying the mechanisms of auxin transport, biosynthesis, metabolism and particularly signaling we can perhaps better elucidate many milestones of plant evolution, such as complex multicellularity or transition to land. This bachelor thesis summarizes the available data and gives a basic overview of auxin-related characteristics. As far as we know, the advanced mechanisms of auxin transport and signaling known from land plants are probably not very ancient and are absent in various algae. Auxin biosynthesis, however, is rather common and a lot of green algae contain orthologs of important biosynthetic enzymes from land plants. Based on the available data it seems that a complete auxin signalling pathway coupled with proteasomal degradation and affecting gene expression is not present in algae. The polar auxin transport, so far with the earliest evidence from moss sporophytes, was recently found in the gametophytic thallus of stonewort (Chara) from a green algal clade Streptophyta, which is...
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The role of AGC protein kinases in the regulation of auxin transport
Martincová, Marie ; Petrášek, Jan (advisor) ; Opatrný, Zdeněk (referee)
There are several members of the subfamily of plant AGC kinases (AGCVIII) suggested to play a role in the regulation of auxin transport, protein kinases PID, WAG1, WAG2 and D6. They all have been shown to perform regulatory phosphorylation of PIN auxin efflux carriers. It is the asymmetrical subcellular localization of PIN proteins that enables the auxin molecules to be transported through a tissue in a polar manner. Regulation of their expression, localization or activity can therefore affect the quantity and directionality of auxin transport. This thesis is focused on better understanding of the PID-mediated regulation of auxin transport. The auxin accumulation as well as the localization of PIN and PID proteins has been studied using stable and transient expression of Arabidopsis thaliana PID in tobacco cell line BY-2. As shown here, the activity of PID does not enhance the activity of PINs, but still it has a positive effect on auxin efflux by increasing the amount of PIN proteins on the plasma membrane. Results presented here suggest that PID-mediated phosphorylation of PIN proteins most likely promotes their exocytosis from endosomal compartments towards the plasma membrane. Using transient co-expression of PID kinase mutated in its ATP-binding site and PIN1-RFP it was shown that functional...
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Role RMS2 v regulaci apikální dominance
Hiršová, Veronika
The role of RMS2 in regulation of apical dominance was studied with the use of rms2 mutants and wild type cv. Torsdag. Phenotypic changes and changes in polar auxin transport (by imunnolocalization of PIN1) in rms2 mutants and after auxin application were observed. The phenotypic changes of rms2 mutants against cv. Torsdag are enhanced branching from the first two nodes but much less than branching of strigolactone deficient mutants. Auxin application both on intact and decapitated rms2 mutants led to enhancement of growth of the buds adjacent to the place of application in contrary to wild type in that the application inhibits the growth of adjacent buds. Auxin application to the cut surface of decapitated stem or from the side of the stem led in the primary application always to canalization. Secondary application 24 hours later led to canalization only in rms2. Originally proposed feedback regulation of RMS1 expression by RMS2 was changed to RMS2 as being a negative regulator of polar auxin transport.
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