Overexpression of <i>VrUBC1</i>, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in <i>Arabidopsis</i>

<div><p>The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean <i>VrUBC1</i> gene (<i><u>V</u>igna <u>r</u>adiata <u>UBC</u> 1</i>). RNA gel-blot analysis showed that <i>VrUBC1</i> mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast <i>ubc4/5</i> by <i>VrUBC1</i> revealed that <i>VrUBC1</i> encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed <i>VrUBC1</i> in Arabidopsis (<i>Arabidopsis thaliana</i>). The <i>VrUBC1</i>-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in <i>VrUBC1</i>-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (<i><u>A</u>. <u>t</u>haliana</i><u>V</u>rUBC1 <u>B</u>inding <u>P</u>artner <u>1</u>), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that <i>VrUBC1</i> plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.</p></div

Subcellular localization of GFP-fusion proteins and BiFC visualization of the interaction between VrUBC1 and AtVBP1 in <i>Agrobacterium</i>-infiltrated tobacco (<i>Nicotiana benthamiana</i>).

<p>(A) The <i>35S:GFP</i>, <i>35S:GFP-AtVBP1</i>, and <i>35S:AtVrUBC1-GFP</i> constructs were transformed into tobacco leaves via <i>Agrobacterium</i>-infiltration. Protoplasts were isolated from the infiltrated leaves after 36 h. Localization of fusion proteins was visualized by confocal microscopy. (B) YFP^N fusions of VrUBC1 and YFP^C fusion of AtVBP1 were coexpressed in tobacco leaves as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066056#pone.0066056-Walter1" target="_blank">[66]</a>. Epifluorescence from the interaction between YFP^N fusions of VrUBC1 and YFP^C fusion of AtVBP1 was observed in the nucleus of the protoplasts (scale bar = 20 µm). BiFC of Arabidopsis bZIP63 dimerization is shown as an interaction control as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066056#pone.0066056-Walter1" target="_blank">[66]</a>. Coexpression of <i>YFP^N-VrUBC1</i>/<i>YFP^C-bZIP63</i> is shown as a negative control.</p

Yeast complementation and thioester formation of VrUBC1.

<p>(A) For the <i>ubc4/5</i> complementation test, the yeast <i>ubc4/5</i> double mutant was transformed with pYES-GFP or pYES-VrUBC1, and selected on uracil-lacking SD medium plates (SD/−Ura). The wild-type (WT) and the <i>ubc4/5</i> transformants harboring pYES-GFP or pYES-VrUBC1 were grown to an OD600 of 1.0, and 10 µl aliquots of different dilutions (10⁻¹, 10⁻², 10⁻³) were spotted onto SD/−Ura plates and grown for 3 d at 30°C. (B) Thioester formation of VrUBC1. VrUBC1 forms DTT-sensitive ubiquitin adducts. <i>In vitro</i> ubiquitination reactions after 5-min at 37°C were treated with DTT or 4 M urea (-DTT). Reactions were resolved by SDS-PAGE and western blots were performed with with anti-His₆ antibodies.</p

Northern blotting of <i>VrUBC1</i> in mung bean.

<p><i>VrUBC1</i> RNA expression in mung bean leaves exposed to low temperature (4°C), dehydration, wounding, ABA (100 µM), or salt stress (100 mM NaCl). Twenty micrograms of total RNA was loaded in each lane<b>.</b> Following electrophoresis, RNA was transferred to a nylon membrane and hybridized with a probe specific for <i>VrUBC1</i>. Equal loading of the total RNA (20 µg) was confirmed by EtBr staining, shown as rRNA below the signal panel.</p

Real-time qRT-PCR analysis of drought-stress maker genes.

<p>Total RNA was extracted from the treated tissues and analyzed by real-time qRT-PCR. Light-grown, 4-week-old plants were dehydrated in a vinyl bag for 6 h. Induction patterns of various ABA- and drought-responsive genes (<i>ABF2</i>, <i>ABF3</i>, <i>ABF4</i>, <i>ABI5</i>, <i>ADH1</i>, and <i>KIN2</i>) were analyzed by real-time qRT-PCR. Data represent the fold induction of each gene by dehydration (6 h) relative to the control treatment (0 h). Mean values from three independent technical replicates were normalized to the levels of an internal control, <i>actin</i> mRNA. Asterisks indicate the significance of the difference from the values between the wild-type and the <i>35S:VrUBC1</i> Arabidopsis transgenic plants as determined by Student’s <i>t</i> test (*0.01 ≤ P<0.05, **P<0.01).</p

Tolerance tests of wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic plants under osmotic stress or ABA conditions.

<p>(A) Ten-d-old seedlings of the wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic lines (L19 and L23) were transferred to MS medium containing 2% (w/v) sucrose and 0.8% (w/v) phytoagar supplemented either mannitol (200 mM) or with NaCl (150 mM). (B) Root length was monitored after 10 days. The values are the means ± SD (n = 3). This experiment was carried out three times with consistent results.</p

Interaction between E3 ligases and VrUBC1 in yeast and <i>in vitro</i> self-ubiquitination of AtVBP1.

<p>(A) Protein interactions of VrUBC1 with five C3HC4-type RING E3 ligases and three positive regulators for osmotic tolerance such as RHA2a, SDIR1 and XERICO were analyzed by Y2H system. For the strong positive interaction control, Krev1 (Rap1A, a member of the Ras family of GTP binding proteins) and RalGDS-wt (the Ral guanine nucleotide dissociator stimulator protein) was used. RalGDS-m1 has weak interaction and RalGDS-m2 has no interaction with Krev1. Yeast MaV203 strains containing the indicated plasmid combinations were grown in SD medium without Leu and Trp to an OD600 of 1.0, and 10 µl aliquots of different dilutions (1, 10⁻¹, 10⁻², 10⁻³) were spotted onto selective and non-selective plates (non-selective medium, SD/−Leu/−Trp; selective medium, SD/−Ura/−Leu/−Trp). The combination of plasmids is indicated on the left and dilution series are indicated at the top. β-galactosidase activity was determined in the MaV203 yeast cells cotransformed with the BD/AD plasmids. Data represent means ± SD from three independent experiments. All the experiments were carried out at least in three replications. (B) Purified GST-AtVBP1 was incubated at 30°C for 2 h with VrUBC1 (E2), yeast E1, Ub, and ATP. Samples were separated by SDS-PAGE, and ubiquitinated proteins were detected by immunoblot analysis using anti-GST antibodies.</p

Germination rates of the wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic plants under osmotic stress or ABA conditions.

<p>(A) Photographs of seedlings at 4 d after the end of stratification. Seeds were germinated on MS medium containing different concentrations of ABA (0, 0.5, 1, or 5 µM), NaCl (0, 100, 150 or 200 mM), or mannitol (0, 100, 200, or 300 mM) and were incubated at 4°C for the stratification (3 d). (B) Seed germination percentage of the indicated lines grown on different concentration of NaCl, mannitol, or ABA, was recorded at 4 d after the end of stratification. Data show the mean ± SD of three replicates. At least 100 seeds per line were measured in each replicate. Asterisks indicate the significance of the difference from the values between the wild-type and the <i>35S:VrUBC1</i> Arabidopsis transgenic plants as determined by Student’s <i>t</i> test (*0.01 ≤ P<0.05, **P<0.01).</p

Identification and phenotypic characterization of <i>atvbp1</i> mutant plants.

<p>(A) Schematic structure of the <i>atvbp1</i> allele with the T-DNA insertion. The triangle indicates the T-DNA, white bars indicate coding regions, and the solid line represents introns. Gene-specific (F1, F2, and R1) and T-DNA specific (LB) primers used in genotyping and RT-PCR are indicated with arrows. (B) Genotyping PCR of the <i>atvbp1</i> loss-of-function mutant plant. The T-DNA-specific and gene-specific primer sets used for genomic PCR are shown at right of the agarose gel. (C) RT-PCR analysis of <i>AtVBP1</i> in the wild-type and <i>atvbp1</i> plants. Two primer sets for RT-PCR are indicated at right on the agarose gel. <i>Actin</i> transcript levels were used as loading controls. Primers used in genotyping PCR and RT-PCR are listed in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066056#pone.0066056.s001" target="_blank">File S1</a>. (D) Five-d-old seedlings of the wild-type and <i>atvbp1</i> plants were transferred to MS medium containing 2% (w/v) sucrose and 0.8% (w/v) phytoagar supplemented with either mannitol (200 mM) or NaCl (150 mM) and incubated under short day conditions in a growth chamber for 5 days. (E) Seed germination percentage of the indicated lines grown on different concentrations of NaCl, mannitol, or ABA, was recorded at 4 d after the end of stratification. Data show the mean ± SD of three replicates. At least 100 seeds per line were measured in each replicate. Asterisks indicate the significance of the difference from the values between the wild-type and <i>atvbp1</i> plants determined by Student’s <i>t</i> test (*0.01 ≤ P<0.05, **P<0.01).</p

Overexpression of <i>VrUBC1</i> enhances drought tolerance and stomatal closure in response to ABA and osmotic stress in Arabidopsis.

<p>(A) Four-week-old, soil-grown wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic plants were kept in a growth chamber without watering for 14 d. Photographs were taken 3 d after rewatering. (B) Differential transpiration water loss between the wild-type and the <i>35S:VrUBC1</i> Arabidopsis transgenic plants. Detached leaves from 3- to 4-week-old plants grown on soil were incubated at room temperature and fresh weight (FW) was measured at the time intervals indicated. Water content was calculated from percentage of FW compared with weight at zero time. Error bars, mean ± SD of three replicates. (C) Stomatal aperture of the wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic plants. Stomatal guard cells were observed by light microscopy in epidermis from wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic plants treated with NaCl (300 mM) and ABA (100 µM) for 3 h. Bar = 10 µm. (D) Measurement of stomatal aperture of wild-type and <i>35S:VrUBC1</i> Arabidopsis transgenic plants. Data are mean ratios of width to length ± SD of three independent experiments (n = 60).</p