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

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

Seed production and root phenotypes in different genotype plants.

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

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

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

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

Microarray Analyses and Comparisons of Upper or Lower Flanks of Rice Shoot Base Preceding Gravitropic Bending

<div><p>Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (<i>Oryza sativa</i> sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base.</p></div

Changes of transcripts for GA metabolism at 6 h after gravistimulation.

<p>A. MapMan output was used to illustrate the significant transcriptional changes across the tissue in GA metabolism at 6 h after reorientation. The reported values are log transformed ratios of transcripts in the lower flank to that in the upper flank. Each transcript is indicated as up-regulated (red square; increase in the ratio of the lower to upper transcript values) or down-regulated (blue square; decrease in the ratio of the lower to upper transcript values). Squares arranged in rows and columns represent individual transcripts at a single time point in the process. An individual square in a given area has the same color as the guide bar in the same column. A filled circle indicates that no transcript was detected. B. qRT-PCR analysis of <i>OsGA20ox4</i> (Os05g34854), <i>OsGA2ox7</i> (Os01g11150) and <i>OsGA2ox9</i> (Os02g41954) expression after horizontal reorientation. Relative changes in transcript abundance after gravity stimulation compared to the vertical control at each time point were analyzed by qRT-PCR, and results were compared with the microarray data. Relative changes in transcript abundance of qRT-PCR were all determined using the non-reoriented control at the relevant time point as reference. 0.5 h down, 0.5 h up, 6 h down and 6 h up represent for lower flank at 0.5 h, upper flank at 0.5 h, lower flank at 6 h and upper flank at 6 h, respectively. The relative gene transcript abundance was calculated as the ratio between the control and gravistimulated samples at each time point. Values are means ± SD; n = 3; □ real-time PCR results; ▪ microarray data.</p

Venn diagrams constructed based on significantly different transcript abundance in gravistimulated rice shoot base.

<p>Venn diagrams show the distribution of transcripts that were significantly changed in abundance by at least 2-fold (<i>P</i> value <0.05) between the lower flank and upper flank of the rice shoot base at 0.5 h and 6 h after gravitropic stimulation.</p