Overexpression of OsRAN2 in rice and Arabidopsis renders transgenic plants hypersensitive to salinity and osmotic stress

Nucleo-cytoplasmic partitioning of regulatory proteins is increasingly being recognized as a major control mechanism for the regulation of signalling in plants. Ras-related nuclear protein (Ran) GTPase is required for regulating transport of proteins and RNA across the nuclear envelope and also has roles in mitotic spindle assembly and nuclear envelope (NE) assembly. However, thus far little is known of any Ran functions in the signalling pathways in plants in response to changing environmental stimuli. The OsRAN2 gene, which has high homology (77% at the amino acid level) with its human counterpart, was isolated here. Subcellular localization results showed that OsRan2 is mainly localized in the nucleus, with some in the cytoplasm. Transcription of OsRAN2 was reduced by salt, osmotic, and exogenous abscisic acid (ABA) treatments, as determined by real-time PCR. Overexpression of OsRAN2 in rice resulted in enhanced sensitivity to salinity, osmotic stress, and ABA. Seedlings of transgenic Arabidopsis thaliana plants overexpressing OsRAN2 were overly sensitive to salinity stress and exogenous ABA treatment. Furthermore, three ABA- or stress-responsive genes, AtNCED3, AtPLC1, and AtMYB2, encoding a key enzyme in ABA synthesis, a phospholipase C homologue, and a putative transcriptional factor, respectively, were shown to have differentially induced expression under salinity and ABA treatments in transgenic and wild-type Arabidopsis plants. OsRAN2 overexpression in tobacco epidermal leaf cells disturbed the nuclear import of a maize (Zea mays L.) leaf colour transcription factor (Lc). In addition, gene-silenced rice plants generated via RNA interference (RNAi) displayed pleiotropic developmental abnormalities and were male sterile

A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease

Analysis of salt stress and ABA tolerance of RAN1 overexpression plant and <i>atran1 atran 3</i> double mutant.

<p>(A) <i>atran1-1 atran3</i>, <i>atran1-2 atran3</i> mutants, <i>AtRAN1</i>-OE1, and the wild-type grow on MS medium supplemented with 1% sucrose. Photos were taken 7 days after germination. (B) Plants were grown on MS medium supplemented with 1% sucrose and 0.5 μM ABA (upper panel) or 100 mM NaCl (lower panel), respectively. Photos were taken 5 days after treatment. (C) and (D) Dose response curve of root growth under ABA (C), or salt stress conditions (D). Plants were germinated on MS medium supplemented with 1% sucrose. Root lengths were measured 5 days after ABA or NaCl treatment.</p

Morphological Changes in Nuclear Envelope under Normal and Freezing Conditions.

<p>Nuclear envelope of the (A) WT, (B) <i>atran1-1 atran3</i> and (C) <i>AtRAN1</i>-overexpressing plants under normal conditions (22°C). After 4-day 4°C acclimation, Nuclear envelope of the (D) WT, (E) <i>atran1-1 atran3</i> and (F) <i>AtRAN1</i>-overexpressing lines were treated for 2h at -4°C. Six root tips were observed in every condition. The root tips were transversely cut in the meristematic zones. Arrows indicate the abnormal nuclear envelope. Bars = 100 nm. The error bars show SD, and are from three independent replications. The results were repeated three times.</p

Cold response Genes and Cell Cycle-related Genes expression Under Normal and Freezing Conditions.

<p>Expression levels of (A) <i>AtCBF1</i> (DREB1B), (B) <i>AtCBF2</i> (DREB1C) and (C) <i>AtCBF3</i> (DREB1A) genes and (D) COR15A, (E) COR47 and (F) RD29A downstream genes in 7-day old <i>AtRAN1</i>-OE, the <i>atran1-1 atran3</i> mutant and wild-type plants under before and after freezing treatment. Values are means and SD (n = 4). Expression of (G) <i>MCM2</i>, (H) <i>MCM5</i>, (I) <i>Cycb1;1</i>, (J) <i>Cyca3;1</i>, (K) <i>Cycd3;1</i>, (L) <i>Cdkb1;1</i>, (M) <i>Cdkb2;1</i> and (N) <i>Cyca2;1</i> cell cycle-related gene levels in wild-type, <i>atran1-1 atran3</i> mutant and transgenic plants before and after 0.5h freezing stress, after 4°C acclimation, The error bars show SD, and are from three independent replications. And the results were repeated three times. Asterisk (*) indicates significant difference (P < 0.05).</p

Expression Patterns and Subcellular Localization of <i>AtRAN1</i>.

<p>The results of qRT-PCR reveal. (A) Real-time PCR analysis the <i>AtRAN1</i> gene expression pattern. (B) Subcellular localization of the vector control and <i>AtRAN1</i> in transgenic <i>Arabidopsis</i> root cells. (C) Subcellular localization of the vector control, <i>AtRAN1</i> in tobacco epidermal cells. DIC, differential interference contrast, referring to bright-field images of the cells. Time course analysis of <i>AtRAN1</i>, <i>AtRAN2</i>, and <i>AtRAN3</i> expression during (D) cold acclimation (4°C). (E) Salt and (F) ABA treatment conditions. <i>Arabidopsis</i> seedlings were germinated and grown for 7 d before they were subjected to treatment. <i>Actin2</i> was used as an internal control. The error bars show SD, and are from three independent replications. And the results were repeated three times. Asterisk (*) indicates significant difference (P < 0.05).</p

Pleiotropic Phenotype of <i>AtRAN1</i> overexpressing Plants.

<p>Seven-day seedlings hypocotyls in Wild-type (A, C) and <i>AtRAN1</i>-OE1 plants (B, D) transgenic lines plants under white light and dark conditions; Analysis of (E) Rosette leaf number; (F) Root phenotype of WT and transgenic plants. (G) Plant height; (H) Flower number; Five-week seedlings of Wild-type (I) and <i>AtRAN1</i>-OE1 plants (J). (K) Hypocotyl cell length in dark grown seedlings. Flower number; Figures I, J Bars = 0.5 cm. The error bars show SD.</p

Seed production and root phenotypes in different genotype plants.

<p>Seed production and root phenotypes in different genotype plants.</p

The Small G Protein AtRAN1 Regulates Vegetative Growth and Stress Tolerance in <i>Arabidopsis thaliana</i>

<div><p>The evolutionarily conserved small G-protein Ran plays important role in nuclear translocation of proteins, cell cycle regulation, and nuclear envelope maintenance in mammalian cells and yeast. <i>Arabidopsis</i> Ran proteins are encoded by a family of four genes and are highly conserved at the protein level. However, their biological functions are poorly understood. We report here that <i>AtRAN1</i> plays an important role in vegetative growth and the molecular improvement of stress tolerance in <i>Arabidopsis</i>. <i>AtRAN1</i> overexpression promoted vegetative growth and enhanced abiotic tolerance, while the <i>atran1 atran3</i> double mutant showed higher freezing sensitivity than WT. The <i>AtRAN1</i> gene is ubiquitously expressed in plants, and the expression levels are higher in the buds, flowers and siliques. Subcellular localization results showed that <i>AtRAN1</i> is mainly localized in the nucleus, with some present in the cytoplasm. <i>AtRAN1</i> could maintain cell division and cell cycle progression and promote the formation of an intact nuclear envelope, especially under freezing conditions.</p></div