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The evolution of auxin homeostasis mechanisms
Skokan, Roman ; Petrášek, Jan (advisor) ; Buschmann, Henrik (referee) ; Holzinger, Andreas (referee)
The evolution of auxin homeostasis mechanisms Ph.D. thesis Roman Skokan, 2021 Abstract The streptophyte lineage consists of land plants (embryophytes) and several groups of primarily freshwater green algae called charophytes. While the phytohormone auxin is a conserved regulator of land plant development, little has been known of the possible origins of auxin response mechanisms in charophytes. We found that one of these mechanisms, the cellular auxin efflux via the PIN family of transport proteins, is most likely a deeply conserved feature in streptophytes. Additionally, we investigated the state of conservation in the green lineage (Viridiplantae) of the gene families known to be involved in auxin transport in land plants. We revealed that some families are deeply conserved outside land plants, but not others. We also helped uncover a unique radiation within the PIN family in Charophyceae. Striving to uncover the native significance of auxin transport in charophytes, we discovered a growth response to exogenously-applied auxin in Closterium, though the effort to produce stable mutant lines in the native PIN homolog is still underway. Altogether, we brought important insights into the evolution of auxin transport and response in the streptophyte lineage, though many questions still remain.
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The role of auxin in streptophyte algae
Schmidt, Vojtěch ; Petrášek, Jan (advisor) ; Pleskot, Roman (referee)
The phytohormone auxin is an important morphogen with an essential role in the development of land plants, where mechanisms of its action are well described. However, its role in green algae is poorly understood. Land plants are part of the phylum Streptophyta together with six closely related groups of predominantly freshwater green algae (charophytes). So far, the knowledge about the evolutionary origins of auxin action mechanism is mainly based on genomic information, and much less on experimental findings. In this work, the presence of auxin, its precursor, and catabolism products were shown in representative species of charophytes with varying levels measured compounds both produced endogenously and into the culture media. Thus, we gained a comprehensive insight into the possible strategies of auxin homeostasis across the non-land plant streptophytes. Also, an effect of exogenous auxin on the cell morphology and culture growth of the desmid Closterium was investigated. Image analysis of IAA-treated cells revealed a rather pleiotropic effect on cell morphology. The culture growth was inhibited by IAA. Additionally, IAA induced malformations in cell shape, and the extent of this phenomenon across individual cultures was dependent on the culture growth status. Lastly, we optimized the method of...
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Mechanisms coordinating auxin metabolism and transport
Novotná, Lenka ; Petrášek, Jan (advisor) ; Serre, Nelson Bernard Calixte (referee)
Auxin is a small molecule that functions as a plant hormone, and it exists in several forms, of which indole-3-acetic acid (IAA) is the most studied one. IAA modulates cell elongation, division, and differentiation by generating local gradients, and it is essential for almost every aspect of plant growth and development. These gradients are established by the cooperation of IAA biosynthesis, metabolism, and transport. A plant responds to both local auxin maxima and minima; thus, it is necessary to regulate auxin metabolism and transport tightly. However, lots of studies show the roles and regulation of auxin metabolism and transport separately, providing quite rarely discussion on the cooperation of these two processes. Hence, this thesis aims to sum up and refer to mechanisms and regulation of auxin metabolism and transport as a whole, rather than separately, and underline the importance of the cooperation of both auxin metabolism and transport in the plant development.
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The evolution of auxin metabolism, signalling and transport in the green lineage (Viridiplantae)
Schmidt, Vojtěch ; Petrášek, Jan (advisor) ; Fendrych, Matyáš (referee)
Phytohormone auxin is an essential coordinator of growth and development within land- plants. Mechanisms of auxin action in algae is still poorly understood, given that related research has begun recently. Current progress is derived mainly from available sequence data, which are necessary for analysis of gene homology with higher plants. In this case with the genes related to auxin action mechanisms, e.g. metabolism, signaling and transport. Auxin biosynthesis predated the split of Chlorophyta and Streptophyta, but tighter regulation of auxin levels is the matter of land-plants. First auxin responses were probably non-transcriptional. Transcription-regulating nuclear signaling pathway assembled from more ancient domains, some of them were identified in Charophyta, but whole pathway was complete after colonization of land. Auxin transport out of the cell was one of the original mechanisms of regulating its levels. More sophisticated auxin carrier system was proposed to evolve in Charophyta, where polar auxin transport was identified. Auxin as a signaling molecule was present in primitive algae groups, but its response spectrum dramatically increased after colonization of land. Future research of auxin action evolution needs more model organisms to be established, especially from Charophyta....
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Hormonal regulation of plant cell division and elongation
Petrášek, Jan ; Zažímalová, Eva (advisor) ; Griga, Miroslav (referee) ; Procházka, Stanislav (referee)
a I I T T T I I a I I a T I T I t r T T I Jml PnrnÁŠnx Suvnalnv SuuuuARyoF DtssERTATtoNTHEsts This thesisis focusedon thehormonalregulationof plantcelldivisionand growth.Both these processes are crucial in shaping sessile plant body. The manifestationof directionalityin ce||p|ateformationduringcýokinesisas we||as in celle|ongationcou|d be found in a form of various shapes of cells, organs and whole plants.Equal and inequal cell division,cell elongationand isodiametriccell growth- they all allow establishing,formingand maintainingcharacterof tissues,organsand whole bodies duringplantdevelopment. Scientificresearchhas alwaysbeenfocusedon regulatorycompounds,the actionof which induces detectable and observable responses. By uncovering the exact mechanisms of their mode of action, many intermediatemessengers were found. However,classicalconceptof "majorregulators'surviveduntilthesedays.lndeed,plant hormones, small organic molecules with wide range of effects, are typical representativesof such compounds,which are studiedalready around one hundred years.They are synthesisedand utilisedin varioustissuesthroughoutthe wholeplant and theycouldbe transportedin vasculatureto longdistances. Auxins and cýokinins - regu|atorsof plant development The researchof recentyears contributedremarkablyto the understandingof...
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Mechanism of auxin transport across plasma membrane through PIN auxin efflux carriers
Lefnar, Radek ; Petrášek, Jan (advisor) ; Nodzynski, Tomasz (referee)
Phytohormone auxin and its directional distribution plays an essential role in the regulation of numerous processes during vegetative and reproductive plant development. Regulation of the expression, localization and activity of the PIN-FORMED (PIN) proteins is important for proper polar auxin transport in plant tissues. PIN proteins have been described as the major auxin efflux carriers regulating auxin's directional flow to build up gradients that provide information for the coordination of plant development. PIN protein structure topology prediction through bioinformatic analysis is still insufficient to understand their transport mechanism. Experimental analysis of PIN protein domains can provide valuable insight into understanding their role in mediating auxin transport. In this study, the C-terminal part of PINs have been modified by gradual trimming to determine the existence of relevant functional domains, which could be important for auxin transport. Seven modified PIN proteins from Arabidopsis thaliana and Nicotiana tabacum were prepared. Transiently transformed tobacco cell line Bright Yellow-2 (BY-2) was used to monitor differences in PIN transport activity. This approach allowed indirect monitoring of intracellular auxin levels using the DR5 reporter system. Transiently expressed...
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The role of cytoskeleton in auxin transport
Kebrlová, Štěpánka ; Petrášek, Jan (advisor) ; Mašková, Petra (referee)
Auxins are a class of plant hormones (phytohormones) with their most frequently endogenously occurring representative indol-3-acetic acid (IAA). Because of their influence on division and elongation of cells, auxins play an important role in many developmental and physiological processes such as embryo development, vascular tissue patterning and tropisms. These effects are often mediated by polar auxin transport, which results in a wide variety of auxin concentrations in cells and entire tissues. Transport of auxin from cell to cell is partly mediated by diffusion, the prevalence of auxin transport is however mediated by auxin carriers located on plasma membrane (PM). Among such carriers belong AUX1/LAX (AUXIN RESISTANT 1/LIKE AUX1) transporter family, which helps with auxin influx and families of PIN-FORMED (PIN) and ABCB/PGP (ATP-binding cassette subfamily B/P-glycoprotein) transporters, which take part in auxin efflux. These proteins are in various degrees dependent on a system of membrane vesicles, trafficking along actin cytoskeleton, which ensures among others cycling of these vesicles among PM and endosomal cell space. Regulation of auxin transport is possible on multiple levels including influencing of gene expression for carrier proteins and regulation of their localization, activity and...
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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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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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