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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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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 toll-like receptors in the pathogenesis of liver diseases
Petrášek, Jan ; Jirsa, Milan (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Červinková, Zuzana (referee)
Identifikační záznam: PETRÁŠEK, Jan. ÚLOHA TOLL-LIKE RECEPTORŮ V PATOGENEZI JATERNÍCH ONEMOCNĚNÍ. [The role of toll-like receptors in the pathogenesis of liver diseases]. Praha, 2010. 198s., Disertační práce. Univerzita Karlova v Praze, 1. lékařská fakulta, Laboratoř Experimentální Hepatologie IKEM. Vedoucí práce: Milan Jirsa. Abstrakt Společným jmenovatelem nejčastějších onemocnění jater je aktivace mechanismů vrozené imunity, které přispívají k rozvoji zánětu a poškození jaterního parenchymu. Klíčovou úlohu v rozvoji jaterního poškození hrají Toll-like receptory, jejichž charakterizace v posledním desetiletí vedla přehodnocení patofyziologie některých jaterních onemocnění. Předkládaná práce studuje význam alelických variant v genech kódujících proteiny Toll-like receptorové signální kaskády a mezibuněčné signalizace v patogenezi alkoholické nemoci jater, přináší nový pohled na probiotika v léčbě nealkoholické steatohepatitidy a nové poznatky o protizánětlivém působení interferonů I. typu u některých jaterních chorob. Abstract Recent reports suggest that majority of chronic and acute liver diseases share a significant degree of liver inflammation and injury attributable to innate immunity, activated through Toll-like receptors. Detailed characterization of Toll-like receptor sigaling cascades in the last...
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Cytoskeleton and endocytosis, a dynamic system for the localization of auxin efflux carriers
Jelínková, Adriana ; Petrášek, Jan (advisor) ; Nick, Peter (referee) ; Hašek, Jiří (referee)
Thanks to its sessile life strategy, the polarity of plant body reflects the polarity of single cells. The polarity is maintained by asymmetric distribution of various molecules downstream from extra and intracellular signals. Directional transport of auxin plays an important role in the pattern formation, morphogenesis, and directional growth responses. The positioning of PIN auxin efflux transporters has been shown to be crucial in the setting of auxin gradients. It is dependent on the plasma membrane deposition of membrane vesicles and their constitutive cycling between plasma membrane and endosomal space. Although some evidences support the idea of differential actin and microtubular cytoskeleton dependence of PIN protein trafficking, there is a significant lack of the information on the role of cytoskeleton in this process. In this paper we use combination of live cell imaging and immunofluorescence techniques to search for the molecular players of actin filaments (AFs) and proteins proteins associated with AFs in the mechanisms of endocytosis and directed PIN1 protein targeting. Seedlings of Arabidopsis thaliana carrying mutations in actin genes (ACT1, ACT2, ACT7, ACT11), Arp 2/3 complex genes (ARP2, ARP3, ARPC2, ARPC5), WAVE complex components genes (BRK1, NAP1, SRA1) and actin monomer...
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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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