Flavonol-induced changes in PIN2 polarity and auxin transport in the Arabidopsis thaliana rol1-2 mutant require phosphatase activity

The phytohormone auxin is a major determinant and regulatory component important for plant development. Auxin transport between cells is mediated by a complex system of transporters such as AUX1/LAX, PIN, and ABCB proteins, and their localization and activity is thought to be influenced by phosphatases and kinases. Flavonols have been shown to alter auxin transport activity and changes in flavonol accumulation in the Arabidopsis thaliana rol1-2 mutant cause defects in auxin transport and seedling development. A new mutation in ROOTS CURL IN NPA 1 (RCN1), encoding a regulatory subunit of the phosphatase PP2A, was found to suppress the growth defects of rol1-2 without changing the flavonol content. rol1-2 rcn1-3 double mutants show wild type-like auxin transport activity while levels of free auxin are not affected by rcn1-3. In the rol1-2 mutant, PIN2 shows a flavonol-induced basal-to-apical shift in polar localization which is reversed in the rol1-2 rcn1-3 to basal localization. In vivo analysis of PINOID action, a kinase known to influence PIN protein localization in a PP2A-antagonistic manner, revealed a negative impact of flavonols on PINOID activity. Together, these data suggest that flavonols affect auxin transport by modifying the antagonistic kinase/phosphatase equilibrium

Ubiquitin-related modifiers of arabidopsis thaliana influence root development

Ubiquitins are small peptides that allow for posttranslational modification of proteins. Ubiquitin-related modifier (URM) proteins belong to the class of ubiquitin-like proteins. A primary function of URM proteins has been shown to be the sulfur transfer reaction leading to thiolation of tRNAs, a process that is important for accurate and effective protein translation. Recent analyses revealed that the Arabidopsis genome codes for two URM proteins, URM11 and URM12, which both are active in the tRNA thiolation process. Here, we show that URM11 and URM12 have overlapping expression patterns and are required for tRNA thiolation. The characterization of urm11 and urm12 mutants reveals that the lack of tRNA thiolation induces changes in general root architecture by influencing the rate of lateral root formation. In addition, they synergistically influence root hair cell growth. During the sulfur transfer reaction, URM proteins of different organisms interact with a thiouridylase, a protein-protein interaction that also takes place in Arabidopsis, since URM11 and URM12 interact with the Arabidopsis thiouridylase ROL5. Hence, the sulfur transfer reaction is conserved between distantly related species such as yeast, humans, and plants, and in Arabidopsis has an impact on root development

Properties and activities of URM11 and URM12.

<p>(A) Bulk tRNA was extracted from wild-type (WT<i>)</i>, <i>Δurm1</i> and <i>Δurm1</i> yeast strains complemented with <i>URM11</i> or <i>URM12</i>. Thiolated tRNAs (arrow) show slower migration than non-thiolated tRNAs (bottom of gel) in an acrylamide gel containing APM. A band of unknown nature (arrowhead) occasionally occurred. URM11 and the GFP-URM fusion proteins of Arabidopsis are functional in yeast, resulting in tRNA thiolation in the otherwise thiolation-defective <i>Δurm1</i> mutant. A representative result of several repetitions is shown. (B) Western blotting of total protein extracts and purified nuclei of <i>urm11-1 urm12-2</i> double mutants expressing <i>HA-URM11</i> or non-transgenic double mutants. Immunolabelling was done with an anti-HA (upper lane) and an anti-histone H3 (lower lane) antibody. The experiment was performed twice with comparable outcome. (C) A representative result of the yeast-two-hybrid experiment (performed three times independently) revealed the interaction of URM11 and URM12 with ROL5, resulting in yeast growth on selective medium and blue staining of the cells due to β-galactosidase activity. Transformation with only one of two constructs with the empty second plasmid did not result in yeast growth and β-galactosidase activity.</p

Effects of <i>urm11-1 urm12-2</i> on root development.

<p>(A) Lateral root density is reduced in the <i>urm11-1 urm12-2</i> double mutant compared to the wild type. Complementation with a <i>35S:HA-URM11</i> or <i>35S:HA-URM12</i> construct restores lateral root formation. Error bars represent the standard error, the asterisk indicates the only value significantly different from the others (two-sided t-test; p = 0.01; n≥25). (B) In contrast to the wild type (Col), <i>lrx1</i> mutants frequently have collapsed root hairs. While <i>urm11-1</i> or <i>urm12-2</i> have no effect on <i>lrx1</i>, an <i>lrx1 urm11-1 urm12-2</i> triple mutant shows suppression of <i>lrx1</i> and develops wild type-like root hairs. (C) Suppression of <i>lrx1</i> by <i>urm11-1 urm12-2</i> is complemented with either <i>35S:HA-URM11</i> or <i>35S:HA-URM12</i>, resulting in the <i>lrx1</i> root hair phenotype. Col: wild-type Columbia. Bar = 0.5 mm.</p

<i>urm11 urm12</i> double mutant fails to thiolate tRNAs.

<p>(A) Schematic structure of <i>URM11</i> and <i>URM12</i>. Black boxes represent exons and white boxes introns. T-DNA insertions are highlighted by black arrows and are located in the first intron for <i>urm11-1</i> and <i>urm12-2</i>, which were further analyzed. (B) RT-PCR on total RNA of entire seedlings revealed absence of <i>URM11</i> and <i>URM12</i> mRNA in the corresponding mutants. The <i>ACTIN2</i> gene was amplified as a control for comparable RNA extraction efficiency. PCR on genomic DNA reveal larger products due to introns. (C) The <i>urm11-1 urm12-2</i> double mutant is impaired in tRNA thiolation. In the presence of APM, thiolated tRNAs show slower migration in an acrylamide gel, non-thiolated tRNAs migrate faster (bottom of the gel). In contrast to the wild type, <i>rol5-1</i> and <i>urm11-2 urm12-2</i> mutants lack thiolated tRNAs (arrow). Bands of unknown nature (arrowhead) occasionally occurred. Representative examples of several independent experiments are shown. Col: wild-type Columbia.</p

Expression patterns of <i>URM11</i> and <i>URM12</i>.

<p>The expression pattern of both genes was investigated by promoter <i>GUS</i> fusion constructs in transgenic Arabidopsis. The <i>URM11:GUS</i> construct led to a homogeneous GUS staining at the seedling stage and the adult stage. <i>URM12:GUS</i> is predominantly active in vascular tissue. Shoots (A) and roots (B) of seedlings and cauline leaves of adult plants (C) are shown. Bars = 2.5 mm.</p

Homology between URM proteins of Arabidopsis and yeast.

<p>Alignment of the Arabidopsis URM11, URM12, and the yeast Urm1p. Identical positions are indicated in black, colons indicate conservative amino acid substitutions, and periods indicate similar amino acids.</p