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. 2016 Jun 3;11(6):e0154787.
doi: 10.1371/journal.pone.0154787. eCollection 2016.

The Small G Protein AtRAN1 Regulates Vegetative Growth and Stress Tolerance in Arabidopsis thaliana

Peipei Xu  1 , Aiping Zang  1 , Haiying Chen  1 , Weiming Cai  1
Affiliations

The Small G Protein AtRAN1 Regulates Vegetative Growth and Stress Tolerance in Arabidopsis thaliana

Peipei Xu et al. PLoS One. .

Abstract

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. Arabidopsis 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 AtRAN1 plays an important role in vegetative growth and the molecular improvement of stress tolerance in Arabidopsis. AtRAN1 overexpression promoted vegetative growth and enhanced abiotic tolerance, while the atran1 atran3 double mutant showed higher freezing sensitivity than WT. The AtRAN1 gene is ubiquitously expressed in plants, and the expression levels are higher in the buds, flowers and siliques. Subcellular localization results showed that AtRAN1 is mainly localized in the nucleus, with some present in the cytoplasm. AtRAN1 could maintain cell division and cell cycle progression and promote the formation of an intact nuclear envelope, especially under freezing conditions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression Patterns and Subcellular Localization of AtRAN1.
The results of qRT-PCR reveal. (A) Real-time PCR analysis the AtRAN1 gene expression pattern. (B) Subcellular localization of the vector control and AtRAN1 in transgenic Arabidopsis root cells. (C) Subcellular localization of the vector control, AtRAN1 in tobacco epidermal cells. DIC, differential interference contrast, referring to bright-field images of the cells. Time course analysis of AtRAN1, AtRAN2, and AtRAN3 expression during (D) cold acclimation (4°C). (E) Salt and (F) ABA treatment conditions. Arabidopsis seedlings were germinated and grown for 7 d before they were subjected to treatment. Actin2 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).
Fig 2
Fig 2. Pleiotropic Phenotype of AtRAN1 overexpressing Plants.
Seven-day seedlings hypocotyls in Wild-type (A, C) and AtRAN1-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 AtRAN1-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.
Fig 3
Fig 3. Analysis of salt stress and ABA tolerance of RAN1 overexpression plant and atran1 atran 3 double mutant.
(A) atran1-1 atran3, atran1-2 atran3 mutants, AtRAN1-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.
Fig 4
Fig 4. Freezing Tolerance Analysis of atran Mutants and transgenic plants.
(A) Three-week-old AtRAN gene double mutants and WT plants were cold stressed at -14°C after 4-day 4°C acclimation and then transferred back to the normal condition for recovery. Photographs of representative seedlings of the WT and transgenic lines were taken after 14 d of recovery. (B) Survival rate of the mutants and transgenic plants after freezing stress. (C) Ion leakage assay of the mutants and transgenic plants after freezing stress. The error bars show SD, and were from three independent replications. And the results were repeated three times. Asterisk (*) indicates significant difference (P < 0.05).
Fig 5
Fig 5. Cold response Genes and Cell Cycle-related Genes expression Under Normal and Freezing Conditions.
Expression levels of (A) AtCBF1 (DREB1B), (B) AtCBF2 (DREB1C) and (C) AtCBF3 (DREB1A) genes and (D) COR15A, (E) COR47 and (F) RD29A downstream genes in 7-day old AtRAN1-OE, the atran1-1 atran3 mutant and wild-type plants under before and after freezing treatment. Values are means and SD (n = 4). Expression of (G) MCM2, (H) MCM5, (I) Cycb1;1, (J) Cyca3;1, (K) Cycd3;1, (L) Cdkb1;1, (M) Cdkb2;1 and (N) Cyca2;1 cell cycle-related gene levels in wild-type, atran1-1 atran3 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).
Fig 6
Fig 6. Morphological Changes in Nuclear Envelope under Normal and Freezing Conditions.
Nuclear envelope of the (A) WT, (B) atran1-1 atran3 and (C) AtRAN1-overexpressing plants under normal conditions (22°C). After 4-day 4°C acclimation, Nuclear envelope of the (D) WT, (E) atran1-1 atran3 and (F) AtRAN1-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.
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Grants and funding

This work was supported by the Strategic Priority Research Program on Space Science of the Chinese Academy of Sciences (grant No. XDA04020202-15, XDA04020415), the National Natural Science Foundation of China (No. 31570859, No. 31500236, No. 31070237 and No. 90917009) and the China Manned Space Flight Technology Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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