PID/WAG-mediated phosphorylation of the Arabidopsis PIN3 auxin transporter mediates polarity switches during gravitropism

Intercellular distribution of the plant hormone auxin largely depends on the polar subcellular distribution of the plasma membrane PIN-FORMED (PIN) auxin transporters. PIN polarity switches in response to different developmental and environmental signals have been shown to redirect auxin fluxes mediating certain developmental responses. PIN phosphorylation at different sites and by different kinases is crucial for PIN function. Here we investigate the role of PIN phosphorylation during gravitropic response. Loss- and gain-of-function mutants in PINOID and related kinases but not in D6PK kinase as well as mutations mimicking constitutive dephosphorylated or phosphorylated status of two clusters of predicted phosphorylation sites partially disrupted PIN3 phosphorylation and caused defects in gravitropic bending in roots and hypocotyls. In particular, they impacted PIN3 polarity rearrangements in response to gravity and during feed-back regulation by auxin itself. Thus PIN phosphorylation, besides regulating transport activity and apical-basal targeting, is also important for the rapid polarity switches in response to environmental and endogenous signals

Intenational uranium business

Tématem mé práce jsou vzájemné obchodní vztahy mezi vybranými regiony (Rusko, Čína, Kazachstán a Austrálie) na poli obchodu s uranem. Cílem mé práce je zjistit, jak faktory, jako jsou přírodní zásoby uranu, dostupné technologie, poptávka po uranu a obchodní regulace, ovlivňují vzájemné vztahy těchto států. V první části je nastíněna každá ze zkoumaných zemí, jakými výhodami disponuje, jak může do mezinárodního obchodu s uranem přispět a její interakci s ostatními zkoumanými zeměmi. Ve druhé části se zaměřuji na nejdůležitější regulace a rizika v obchodu s uranem a v poslední části nastíním pohled na uranový trh z pohledu českého prostředí.The theme of my thesis is international business relations among selected regions (Russia, China, Kazakhstan and Australia) in the context of uranium business. The main goal of my thesis is to inspect how factors such as natural supply of uranium, available technologies, demand after uranium and business regulations effect mutual business relations of these regions. In the first part (chapter) I outlined each of the examined regions, with which opportunities they dispose, how they can benefit to international uranium business and their interaction with other examined regions. In the second part I focus my work on the examination of the most important regulations and risks in uranium business and in the last part I outline the situation on czech uranium market

Generator of Cyber Attacks

The article deals with the security of computer networks based on TCP / IP protocol suite, in particular, testing the resistance to DDoS attacks and tools for generating malicious traffic. In the article the selected tools Hping3, Mausezahn and Trafgen are analyzed. For these tools the results of comparative measurements are presented. Based on the results of the experimental tests, the main contribution of this paper is design and implementation of the new tool for generation of DDoS attacks and its subsequent performance testing for individual attacks. The tool also has a unique web interface for its easier usage.Článek se zabývá bezpečností počítačových sítí založených na protokolové sadě TCP/IP, konkrétně testováním odolnosti proti DDoS útokům a nástroji na generování škodlivého provozu. V článku jsou analyzovány vybrané nástroje, Hping3, Mausezahn a Trafgen. Dále jsou pro tyto nástroje uvedeny výsledky porovnávacích měření. Na základě získaných výsledků z experimentálních testů, je hlavním přínosem článku návrh vlastního nástroje pro generování DDoS útoků a jeho následné výkonnostní testování pro jednotlivé útoky. Vytvořený nástroj také obsahuje unikátní webové rozhraní pro jeho snadnější ovládání

WRKY23 is a component of the transcriptional network mediating auxin feedback on PIN polarity.

Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17- and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain- and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development

WRKY23 acts downstream of the Aux/IAA—ARF auxin pathway and marks developing vasculature.

<p>(A) Schematic depiction of WRKY23 promoter; AuxRE and AuxRE-like response elements are shown as triangles (B and C) <i>WRKY23</i> transcript levels depend on auxin dose and treatment time. qRT-PCR analysis of <i>WRKY23</i> expression after a 4 h treatment with different concentrations of NAA (B) and after different treatment times with 10 μM NAA (C). <i>TUB2</i> and <i>SLR/IAA14</i> are shown as negative and positive controls, respectively. Values represent relative fold change of expression. Error bars represent standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007177#sec010" target="_blank">Materials and Methods</a> for detailed description). (D and E) <i>WRKY23</i> expression depends on the SCF^TIR1-Aux/IAA-ARF signalling pathway. qRT-PCR confirmation of the microarray experiment showing the expression of <i>WRKY23</i> and genes previously connected to PIN polarity in <i>HS</i>::<i>arx3-1</i> (D), and in <i>arf7 arf19</i> double mutant plants (E). Values represent relative fold change. Error bars indicate standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007177#sec010" target="_blank">Materials and Methods</a> for detailed description). (F, G) Expression of <i>WRKY23</i>::<i>GUS</i> in the shoot apical meristem (SAM) and in the presumptive leaf vasculature (G). Besides strong activity in the SAM, GUS staining overlaps with, and partly precedes, the appearance of differentiating vascular strands in young leaves. Two representative plants in consecutive developmental stages are shown. Patchy expression of <i>WRKY23</i>::<i>GUS</i> in the vasculature of young developing true leaves (G). Arrowheads in F and G depict areas with GUS activity presumably coinciding with future vascular strands that are not morphologically discernible yet.</p

Isolation and characterization of <i>wrky23</i> mutants.

<p>(A) Schematic representation of the <i>WRKY23</i> locus. Exons are represented by boxes, while introns are shown as lines. Coding regions are filled with dark grey. Exact locations of the T-DNA insertions are depicted. (B) qRT-PCR analysis of <i>WRKY23</i> expression in the isolated mutant lines. Relative expression values are normalized to the level detected in WT Col-0. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007177#sec010" target="_blank">Materials and Methods</a> for more details. (C) Evaluation of cotyledon vasculature defects in <i>WRKY23-SRDX</i>, <i>35S</i>::<i>WRKY23-SRDX</i> and <i>wrky23</i> mutants. A One-Way ANOVA test compared marked sets of data (* p<0.05; *** <i>p</i><0.001; <i>n</i>>50 cotyledons). (D) Schematic representation of cotyledon vasculature pattern. l1, first loop; l2, second loop; mv, midvein. Yellow and red box delineate UD and BD zone of evaluating. (E) Representative images of analysed vasculature defects. (F) Representative images of immunolocalization analysis of PIN1 in developing young first leaves. In the WT, PIN1 shows typical polarization, whereas in <i>wrky23-2</i> mutant this polarization is abolished. At least 50 leaves per genotype were analysed.</p

Putative transcriptional components of the auxin-mediated PIN polarization.

<p><b>(</b>A) Simultaneous immunolocalization of PIN1 and PIN2 in <i>HS</i>::<i>axr3-1</i> plants. Heat shock-induced overexpression of <i>axr3-1</i> abolishes lateral PIN relocation after auxin (4 h, 10 μM NAA) treatment, confirming dependence on the SCF^TIR1-Aux/IAA-ARF signalling pathway. Arrowheads highlight representative examples of PIN localization in the respective tissues and treatments (PIN1 in endodermis and PIN2 in cortex). Bar = 10 μm. epi, epidermis; co, cortex; en, endodermis. (B) Quantitative evaluation of (A), confirming reduced auxin-dependent relocation of PIN1 (top) and PIN2 (bottom) in the induced <i>HS</i>::<i>axr3-1</i> line. Graph shows mean ratio of lateral-to-basal signal intensity of PIN1 in endodermal and PIN2 in cortex cells. Error bars indicate standard error. A One-Way ANOVA test compared marked sets of data. (** <i>p</i><0.01; *** <i>p</i><0.0001; <i>n</i>>35 cells corresponding to a minimum of 10 roots per treatment and experiment were imaged under comparable conditions). Experiments were carried out at least 3 times; one representative experiment is presented. (C) Scheme of the microarray experiment and analysis strategy.</p

Developmental roles of Auxin Binding Protein 1 in Arabidopsis thaliana

International audienceAuxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain-and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy