RAN1 is involved in plant cold resistance and development in rice (Oryza sativa)

Summary OsRAN1 can improve cold tolerance in rice. It maintains cell division under cold conditions. Overexpression of OsRAN1 increases tiller number and alters root development.


Introduction
Ran is a small GTPase that is essential for nuclear transport, nuclear assembly, mRNA processing, and cell cycle control, and is also the only known member of the family of small GTP-binding proteins primarily localized inside the nucleus (Ciciarello et al., 2007;Di Fiore et al., 2004).Ran can switch between a GDP-and GTP-bound state, and the transition from RanGDP to RanGTP can occur by nucleotide exchange.Although the intrinsic rate of nucleotide exchange and hydrolysis of Ran is slow, it is stimulated by the regulator of chromosome condensation 1 (RCC1) and the GTPase-activating protein (RanGAP1) (Gorlich and Kutay, 1999;Sazer and Dasso, 2000).As RanGAP and RanBP1 (Ran binding protein 1) are excluded from the nucleus, they cooperate in the cytoplasm to deplete RanGTP (Bischoff and Ponstingl, 1991;Bischoff and Ponstingl, 1995;Ramdas et al., 1991).In plants, the identification of RanBP1 and RanGAP enabled the study of Ran function (Ach and Gruissem, 1994;Rose and Meier, 2001).Owing to the high similarity in amino acid sequence and subcellular localization, plant Ran proteins are probably highly conserved with their mammalian and yeast counterparts in nucleo-cytoplasmic trafficking and mitotic processes (Wang et al., 2006).
Ran is encoded by a family of four genes in Arabidopsis and three genes in rice (Haizel et al., 1997;Vernoud et al., 2003).The temperature-sensitive mutants of Pim1 (premature initiation of mitosis 1) in fission yeast enter mitosis without completing chromosomal DNA replication, and overexpression of yeast Ran GTPase homologue Spi1 suppresses the pim1-46 mutant phenotype (Matsumoto and Beach, 1991).Wheat RAN1 is involved in regulation of cell division and alters primordial meristem, mitotic progress, and sensitivity to auxin in rice and Arabidopsis (Wang et al., 2006).Ran GTPase may be involved in the plant response to hormone or environment signalling.Decreased ATP levels induced by oxidative stress lead to decrease in Ran-GTP levels and disordered Ran distribution (Yasuda et al., 2006).The expression of OsRAN1 is induced by jasmonic acid in rice (Miche et al., 2006); overexpression of OsRAN2 affects the sensitivity to salt stress in rice (Zang et al., 2010).OsRAN2 also plays an important role in cold tolerance (Chen et al., 2011).Therefore, Ran protein not only plays an important role in plant development but also mediates plant response to the environment.
Rice is a cold-sensitive plant that has its origin in tropical or sub-tropical areas.Unpredictable cold snaps at the booting stage delay heading and result in pollen sterility owing to the failure of microspore development under lowtemperature conditions, which was thought to be one of the key factors responsible for reduced grain yield (Imin et al., 2004).Screening for genes involved in cold tolerance is an important initial step for crop improvement strategy using genetic engineering (Andaya and Tai, 2006;Dai et al., 2004).Maintenance of cell division is essential for plant survival and growth during cold stress.However, the cell cycle-associated cold response requires further research.In this study, we explored the role of OsRAN1 in the cell cycle and cold tolerance regulation in rice.We also observed the nuclear envelope integrity during cold stress.Our results suggest that plant Ran GTPase may have an important and conserved role in cold stress signalling in plants.

Plant materials
Rice plants (Oryza sativa L. ssp.japonica) were germinated and grown in Hoagland nutrient solution and soil substrate at a photo flux density of 350-400 μmol m −2 s −1 , 60-80% relative humidity, 12/12 h day/night cycle at 28 °C in a phytotron.Arabidopsis (ecotype Columbia) and tobacco (Nicotiana tabacum L. cv.Gexin No.1) were grown under long-day conditions (16/8 h light/dark cycle) with a fluence rate of 120 μmol m −2 s −1 of white light (produced by cool-white fluorescent lamps) at 22 °C.The pot volume was 12 × 12 × 10 cm.The soil substrate used in the experiments was obtained from the suburban area of Shanghai, China.

Plasmid constructions and generation of transgenic plants
The full-length cDNA of OsRAN1 was amplified from rice plant Zhonghua 11 (Oryza sativa L. ssp.japonica) according to GenBank accession No. AB015971.The modified green fluorescent protein (GFP) gene was amplified using the pCMBIA1302 vector.To construct the plasmid for gene overexpression.OsRAN1 or OsRAN1:GFP were cloned into a pHB vector (Mao et al., 2005) to generate double 35S:OsRAN1 and 35S:OsRAN1:GFP transgenics.Expression in rice was controlled by the CaMV 35S promoter.The transgenes were confirmed by DNA sequencing.All the reagents and enzymes used here for PCR amplification or restriction digestion were purchased from Takara, Japan.Arabidopsis plants (ecotype Columbia) were transformed with double 35S:OsRAN1 and 35S:OsRAN1:GFP transgenes using the floral-dipping method (Clough and Bent, 1998).Japonica rice cv.Zhonghua11 plants were transformed with the double 35S:OsRAN1:GFP using an Agrobacterium-mediated transformation as described (Hiei et al., 1994).Hygromycin (Roche)-resistance was used to screen positive transgenic plants.Genomic PCR was used to confirm the transgenic plants with specific primers for the hygromycin phosphotransferase (HPT) gene.Semi-quantitative RT-PCR and qPCR were conducted to detect gene expression level in transgenic Arabidopsis and rice plants.Primers used for plasmid constructions are shown in Supplementary Table S1 available at JXB online.

Subcellular localization study
Tobacco epidermal cells were injected with the construct of double 35S:OsRAN1:GFP construct and analysed by confocal microscopy (Zeiss LSM510; Jena, Germany).In addition, the GFP signal was also observed in roots of 1-week-old transgenic Arabidopsis seedlings.

RT-PCR and real-time PCR
The synthesis of cDNA using ReverTra Ace qPCR Master Kit (FSQ-201) was performed as described previously (Zang et al., 2010).An aliquot of 2 μl of tenfold-diluted cDNA was used as an RT-PCR template in a 20 μl reaction system.All PCR products were loaded onto a 1% agarose gel to visualize the amplified cDNAs.RT-PCR was repeated three times.OsUbiquitin was used as a control for 25 cycles.Fluorescence intensity of DNA bands was quantified using Bio-Rad's ChemiDoc MP System.The relative expression level of OsRAN1 before treatment was set at 1 in different stress condition.For real-time PCR, the cDNA samples were diluted to 2ng μl −1 .Triplicate quantitative assays were performed with 1 μl of cDNA dilution with the SYBR GreenMaster mix and an ABI fast sequence detection system according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA).The relative quantification method (Delta-Delta CT) was used to evaluate quantitative variation.The amplification of actin2 was used as an internal control to normalize all data.The primers for gene expression are listed in Supplementary Table S2 available at JXB online.

Stress treatment of rice seedlings
Homologous T2 transgenic rice (OsRAN1 overexpression) seeds were used for the stress tolerance assay.All seeds were germinated in a mixture of nutrimental soil and vermiculite (2:1).Transgenic and wild-type seedlings were grown under 12 h light/12 h dark (28 °C/25 °C).To search the expression pattern of OsRAN1, oneweek-old transgenic rice seeds were germinated in water (as control) or in water containing 150 mM NaCl, 10% PEG 6000 (polyethylene glycol, average Mn 6000), or 1 μM indoleacetic acid (IAA).For cold treatment, two-week-old seedlings at the trefoil stage were treated at 4 °C for 84 h under 12 h light/12 h dark.After treatment, the seedlings were moved to a greenhouse for recovery in 2 weeks.Photographs were taken at the indicated times.

Imaging of root cell size
To examine cell arrangement and size, root tips were stained with 100 μg μl -1 propidium iodide solution and observed under a confocal laser scanning microscope (Zeiss) with an argon laser.

Measurement of Pro and soluble sugar contents
Transgenic plants at the four-leaf stage were used for biochemical analysis.Free Pro content in leaves was determined by established techniques (Troll and Lindsley, 1955).About 50 mg of leaves were homogenized in 10 ml of sulfosalicylic acid (3%) and the homogeneous mixture was centrifuged at 13 000 rpm for 15 min at 4 °C.The extract (2 ml) was transformed to a microcentrifuge and mixed with 2 ml of acid ninhydrin (0.1 g ninhydrin dissolved in 24 ml of glacial acetic acid and 16 ml of 6-mortho-phosphoric acid) and 2 ml of acetic acid.The reaction mixture was boiled in a water bath at 100 °C for 30 min and cooled down at 4 °C for 30 min.This was followed by addition of 4 ml of toluene to the leaf extract, which was then thoroughly mixed.Finally, 1.2 ml of the toluene phase was removed for absorbance measurement at 520 nm in a UV 2800 spectrophotometer (Unicon).Total soluble sugars in leaves were determined by the modified phenolsulfuric acid method (Dubois et al., 1951).About 0.1 g of leaves were homogenized in 8 ml of double-distilled water, and boiled twice in a water bath at 100 °C for 30 min.The extract (about 5 ml) was transferred to a new microcentrifuge, mixed with 1.5 ml of double-distilled water, 1 ml of 9% (v/v) phenol, and 5 ml of sulfuric acid added, and kept at room temperature for 30 min.The absorbance was measured at 485 nm in a UV 2800 spectrophotometer (Unicon).

Flow cytometry of cell cycle progression
T2 generation seeds of OsRAN1 transgenic rice were sterilized with 0.15% mercuric chloride and germinated on the filter with sterilized water at 28 °C in the dark for 7 d.All of the seedlings of wild-type or OsRAN1 transgenic rice were assigned in equal quantity and subjected to 28 °C or 4 °C for 12 h.Samples of cell nuclei were prepared as described by (Galbraith et al., 1999).Root apical tips (3 mm) were excised, immediately chilled on ice, and chopped with a singleedged razor blade in a glass petri dish (diameter, 10 cm).Chopping buffer (45mM MgCl 2 , 30 mM sodium citrate, 20mM 4-morpholinepropane sulfonate, and 1 mg μl −1 Triton X-100, pH 7.0) was used to release the cells from the chopped tissues.The DNA content of individual transgenic cells was determined by flow cytometry.Cell nuclei were stained with 2 μg μl -1 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Ma et al., 2009).Each sample was prepared three times and subjected to BECKMAN COULTER Moflo-XDP three times.A total of 10 000 nuclei were designed to be measured per analysis.

Nuclear envelope observation
The transgenic and wild-type seeds were germinated for 7 d and then treated for 0 and 3 h at 4 °C.The root tips (2-3 mm) were fixed for 5-6 h in the fixation buffer (3% glutaraldehyde in 0.1 m PBS, pH 7.2).The materials were washed three or four times with 0.1 m PBS, and fixed in 1% osmic acid under 4 °C overnight.The materials were washed three or four times with 0.1 m PBS, dehydrated with an ethanol series of 30, 50, and 70% (4 °C, overnight), and then 80, 90, 95, 100-100% (30 min for every concentration).Ethanol was replaced with acetone (1:1) and infiltrated with acetone and a mixture of resin:acetone:resin=2:1; 1:1; 1:2, for 3 h and 100% resin for 12 h.The materials were embedded, and polymerized under 60 °C for 24 h.Root tips were cut into 50-70 nm using an ultramicrotome, the nuclear envelope (NE) was observed by transmission electron microscopy (HITACHI H-7650, Japan).

Analyses of auxin effects
Col-0 Arabidopsis seeds were surface sterilized in 75% ethanol for 1 min, followed by 50% (v/v) NaClO solution for 8 min, and rinsed in sterile water.Seeds were then placed on plates and vernalized for 72 h to synchronize germination.After vernalization, all plates were placed in the same growth chamber and allowed to grow for 10 d to determine the effect of various concentrations of auxin (IAA) on root length and lateral root production (Kim et al., 2001)

Identification and characterization of the OsRAN1 gene
The OsRAN1 gene was cloned using primers designed according to Accession no.AB015971.OsRAN1 cDNA was isolated as a full-length coding region and encoding the predicted protein of 221 amino acids.A nucleotide BLAST search (http:// blast.ncbi.nlm.nih.gov) was performed against the Oryza sativa genome matching the isolated gene to the Os01g0611100 gene.The predicted protein sequence of OsRAN1 was aligned with related sequences from Arabidopsis (AtRan1, AtRan2, AtRan3, and AtRan4), wheat (OsRAN1), human (Ran/TC4), mouse (MusRan), Zea mays (ZmRan), and rice (OsRAN2, OsRAN3) (Fig. 1A).The alignment showed 87% sequence homology between OsRAN1 and its human counterpart, and 95% homology between OsRAN1 and OsRAN2 at the amino acid level.The characteristic domains of the Ran proteins, which are known to be involved in GTP-binding and hydrolysis, in addition to the acidic C-terminal domain and the effector-binding domain, are highly conserved in most Ran proteins of various organisms (Ma et al., 2009).
The expression pattern of OsRAN1 in leaves, root, stem, sheath, and panicle was investigated using quantitative realtime PCR.The expression level was higher in panicle than in other organs examined (Fig. 1B).The subcellular localization of OsRAN1:GFP was traced to root cells of transgenic Arabidopsis steadily overexpressing OsRAN1 and tobacco epidermal cells expressing transiently.The green fluorescent signal of OsRAN1:GFP was detected mainly within the nucleus, with some signals in the hypocotyl cytoplasm (Fig. 1C), whereas the green fluorescent signal was randomly distributed in the cell under the GFP vector control.These findings were consistent with mammalian counterparts, which indicates that Ran is GDP-bound in the cytoplasm during interphase, and GTP-bound in the nucleus (Quimby and Dasso, 2003).In addition, this localization pattern was also observed through transient expression of OsRAN1:GFP in tobacco epidermal cells with identical results (Fig. 1D).

Expression pattern of OsRAN1 in response to cold, salt, polyethylene glycol, and indoleacetic acid treatment
We investigated the effects of abiotic stress on OsRAN1 expression using semi-quantitative RT-PCR to monitor the expression pattern of OsRAN1 in response to different stresses.The transcript level of OsRAN1 began to increase after 18 h of cold treatment and further accumulated up to the peak level at 48 h (Fig. 2A, B).Saline stress was induced with NaCl (150 mM) (Verslues et al., 2006) increasing the expression of OsRAN1 after 6h and only slightly thereafter (Fig. 2C, D).We then investigated the effect of osmotic stress on the expression of OsRAN1, using 10% polyethylene glycol (Mn 6000; PEG 6000) to mimic osmotic stress.The expression of OsRAN1 was nearly 2-fold higher than that in the control 48 h after treatment, when it decreased again to normal level (Fig. 2E, F).Approximately 1 μM indoleacetic acid (IAA) was used for 84 h, with OsRAN1 levels starting to increase after 24 h of treatment and peaking at 72 h, with a 5-fold increase (Fig. 2G, H).In conclusion, the data suggest that OsRAN1 predominantly responds to low temperature and IAA treatment compared with salt and drought stress.

Overexpression of OsRAN1 increased tiller number and later flowering, and reduced apical dominance and abnormal roots in transgenic Arabidopsis
To analyse the roles of OsRAN1 in plants further, OsRAN1 was overexpressed in rice and Arabidopsis under the control of the double constitutive cauliflower mosaic virus (CaMV) 35S promoter.Stable inherited homozygous transgenic lines were obtained at the T 2 generation.In addition, semi-quantitative reverse transcription (RT-PCR) and realtime quantitative PCR (qPCR) were conducted to examine exogenous OsRAN1 expression in the transgenic Arabidopsis and rice lines.As expected, it was found that OsRAN1 was overexpressed in both transgenic Arabidopsis and rice plants (Fig. 6C and Supplementary Fig. S1 available at JXB online).Furthermore, we observed the development of the phenotypes.We obtained 25 highly overexpressed lines from 32 independent transgenic lines (Supplementary Fig. S1 available at JXB online).Compared with the wild type, all the highly overexpressed transgenic Arabidopsis showed distinct phenotypes, such as excess rosette leaves, increased tiller number, longer hypocotyl in the white light, weak apical dominance, and abnormal root development (Fig. 3A-J).The flowers emerged about 4 d later in OsRAN1 transgenic plants on long days (Fig. 3K).Arabidopsis plants overexpressing OsRAN1 were shorter, with more lateral floral branches, and partly abortive flowers compared with wild-type plants (Fig. 3C-H).Overall, the apical dominance of transgenic Arabidopsis was reduced.Other distinct phenotypes among OsRAN1 transgenic Arabidopsis seedlings related to root development (Fig. 3L-P).Many lines of transgenic seedlings showed a similar phenotype, such as greatly reduced number of lateral roots and stunted primary roots.The number of lateral roots was only 4.4 per plant on average in the transgenic seedlings.In contrast, the wild type showed 10.1 per plant under the same condition.Exogenous applications of IAA addressed the phenotype of fewer lateral roots (Fig. 4).These results demonstrated that OsRAN1 controls development of shoots and the roots probably by affecting IAA signalling in the transgenic Arabidopsis.

Overexpression of OsRAN1 increased cell division in rice roots
We further examined the phenotypes in the OsRAN1 overexpressed lines of rice.The transgenic rice plants showed a tiller number up to 9.3 per plant on average.In contrast, wild-type rice had fewer tillers, about 7.3 per plant (Table 1).The OsRAN1 overexpressed rice plants were shorter with more tillering branches.These results suggest that OsRAN1 expression affected tiller initiation in the shoot meristem.Furthermore, the roots of transgenic rice contain many small and tightly arrayed cells emerging from the meristem (Fig. 5).This information, combined with the results of the increased 4C DNA in the OsRAN1 overexpression line (Fig. 8A-D), indicates that OsRAN1 is involved in meristem cell proliferation in the root.OsRAN1 probably regulates cell division, particularly in the root tip meristem.

Overexpression of OsRAN1 increased cold tolerance in transgenic rice and Arabidopsis
To test the possible role of OsRAN1 overexpression in cold tolerance of rice, the seedlings of T2 transgenic lines and wild type at trefoil stage were exposed to cold stress at 4 °C for 84 h.The plants were then removed to the greenhouse to recover at 28 °C.After 14 d recovery under normal conditions, the survival rates of three transgenic lines were between 71% and 78%, whereas the survival rate for the wild type was only 26 % (Fig. 6).We further examined the freezing tolerance of Arabidopsis overexpressing OsRAN1.The results showed that transgenic lines 3, 5, and 15 have higher freezing tolerance after 3 d cold acclimation.The survival rate of these three lines after 2 weeks of recovery was 52%, 68%, and 60%, whereas the survival rate of the wild type was 32% (Supplementary Fig. S2 available at JXB online).Therefore, we concluded that OsRAN1 overexpression increases the freezing tolerance of transgenic rice and Arabidopsis.

Increased Pro and soluble sugar contents in OsRAN1 transgenic plants under cold stress
Plant adaptation to environmental stresses is often associated with metabolic adjustment, such as accumulation of Pro and soluble sugars (Abraham et al., 2003).To investigate the physiological basis for the improved stress tolerance in transgenic rice, we measured the Pro and soluble sugar content in plants that overexpressed the OsRAN1 gene and the wild type under normal growth and stress conditions.Under normal growth conditions (28 °C), the levels of cellular free Pro did   not differ between wild-type and transgenic rice, at quantities ranging between 92 and 98 mg fresh weight (Fig. 7A).In contrast, after cold treatment (4 °C), the levels of free Pro in OsRAN1 transgenic rice increased substantially, with more than 178 mg g -1 (207 mg g -1 ,198 mg g -1 ) fresh weight compared with 146 mg g -1 fresh weight in the wild-type plants.
The levels of free sugar in OsRAN1 transgenic rice increased to 1.1 mg g -1 (1. 6 mg g -1 ,1.4 mg g -1 ) fresh weight compared with 0.85 mg g -1 fresh weight in the wild-type plants after cold treatment, although the levels of cell-free sugar did not differ between the two types of rice (Fig. 7B).We further measured the expression levels of two putative carboxylate synthetase genes (AK102633 and AK101230) and two putative Pro transporter genes (AK067118 and AK0666298) in the OsRAN1 transgenic plants subjected to cold stress.As show in Figure 7C, AK067118, AK0666298, and AK101230 had higher expression level (3-to 5-fold) in the OsRAN1 overexpressed lines than in the wild type after cold treatment.We also measured the expression level of putative sugar synthetase relative genes Os01g0205700 and OsSPS1 and two putative sugar transporter genes (Os08g0178200 and Os12g0641400) in the OsRAN1 transgenic plants under cold stress.Genes in the OsRAN1 overexpressed lines had higher expression level (2-fold-4.5-fold)than the wild type (Fig. 7D).

Cell cycle progression in transgenic rice lines
We monitored the mitotic index of AtRAN1-overexpressed lines under cold conditions.Mitotic index is defined as the ratio between the number of cells in mitosis and the total number of cells, which is used as a measure for the proliferation status of a cell population.Flow cytometry showed that the 4C DNA content of OsRAN1-overexpressed lines increased in comparison with the wild type under normal (28 °C) and cold (4 °C) conditions (Fig. 8A-D).Under the normal conditions, the index for the transgenic lines was 61-78% compared with that of the wild-type 46%.Even at 4 °C, the index for the transgenic lines was 21-31% compared with that of the wild-type 14% (Fig. 8E), although both the transgenic lines and the wild type showed a significantly decreased index at this temperature.Thus, the overexpressing lines possessed more cells in the proliferation, especially under the cold condition.Therefore, we conclude that OsRAN1 overexpression increased mitosis.

Overexpression of OsRAN1 promoted the formation of intact nuclear envelope under cold stress
Next, we observed the nuclear envelope (NE) of root tip cells of the overexpressed transgenic line 47 and wild-type rice under normal and cold conditions.Under the normal condition (28 °C), NEs of the transgenic line and wild type were all intact with no obvious difference in morphology (Fig. 9A, B).After treatment at 4 °C for 4 h, about 70% cells in wild type showed obscured or partly dissociated double membranes (Fig. 9E, F).However, most cells in the overexpressed lines formed an intact NE under cold stress (Fig. 9C, D), with cells showing partially dissociated NE.The results suggested that overexpression of OsRAN1 may promote the formation of an intact NE under cold stress.

OsRAN1 in cell cycle regulation
Several studies indicate that the small GTPase called Rasrelated nuclear protein (Ran) is a central regulator of several cell cycle events.In the 1990s, scientists first discovered that Ran was an essential factor for protein import into the nucleus during interphase.In subsequent studies, they discovered that Ran played a role in spindle assembly and nuclear envelope formation during mitosis.Ran seems to modulate these vastly different cellular processes through a gradient mechanism, wherein active Ran is more concentrated around its primary site of action and less concentrated elsewhere (Clarke and Zhang, 2001).Overexpression of various wildtype Ran homologues from plants, including tomato and tobacco Ran, suppressed the pim1 mutant phenotypes in yeast (Lee et al., 1993;Merkle et al., 1994), which suggests that the role of Ran GTPase may be conserved in cell cycle regulation.The precise details associated with specific processes during cell cycle progression have yet to be delineated (Rossi and Varotto, 2002).Plant meristematic cells do not undergo cellular expansion and are relatively uniform in size.The size of the meristematic cells of the primary root tip was reduced on average in our OsRAN1-overexpressed plants.The root-tip meristem of OsRAN1-overexpressed plants contained smaller cells than those in the wild type, thus increasing the total number of meristematic cells.In brief, our study reveals that OsRAN1 was involved in cell proliferation in the root.OsRAN1 overexpression enhanced the root tip mitotic index of transgenic rice (Fig. 8E), further increasing the cold tolerance.Previous research demonstrated that OsRAN1 and TaRAN1, sharing high identity at the amino acid level, were also increased in cold tolerance in transgenic rice plants (Chen et al., 2011).Another study conducted by our laboratory in Arabidopsis showed that atran1 atran3 double mutants have increased freezing sensitivity, whereas the atran1 or atran3 signal mutant in Arabidopsis did not have increased freezing sensitivity (data not shown).These results indicate that Ran GTPase regulates cold stress redundantly.Thus, Ran homologues may have well-conserved functionality in plants, yeast, and animals during evolution.

Functional analysis of OsRAN1 suggests a role in auxin signal transduction; exogenous application of IAA partly repairs the transgenic root defects
Response to IAA in the lateral root initiation involves multiple root and shoot phenotypes commonly associated with auxin mutants (Berleth et al., 2000).We tested whether OsRAN1 transgenic Arabidopsis root development defects were affected by auxin by supplementing the growth medium with exogenous IAA The results indicate that IAA typically promotes the lateral root initiation but have no obvious effects

Fig. 1 .
Fig. 1.Phylogenetic analysis of plant Ran GTPase, tissue specific expression of OsRAN1, and its subcellular localization.(A) Phylogenetic tree of plant Ran GTPases.The tree was constructed with the MEGA5.1 software with amino acid sequences of rice OsRAN1, OsRAN1, and other Ran GTPases isolated from wheat (TaRAN1), Arabidopsis (AtRAN1, AtRAN2, AtRAN3), human (RAN/TC4), and mouse (MusRAN).(B) Quantitative real-time PCR quantification of OsRAN1 expression across different rice plant organs.Data are means±SD (n=3-5).Panicles, leaves, stems, and roots were harvested from individual 8-9-week-old wildtype plants.Stem expression was set to 1. (C) Subcellular localization of OsRAN1 in transgenic Arabidopsis root cells and tobacco epidermal cells; (d).Transient expression of OsRAN1:GFP in tobacco epidermal cells; DIC, differential interference contrast, referring to brightfield images of the cells.

Fig. 2 .
Fig. 2. Semi-quantitative RT-PCR analysis of OsRAN1 expression in stress response.(A, B).Time course of OsRAN1 expression during cold treatment (4 °C).(C, D) Time course of OsRAN1 expression during treatment with 150 mm NaCl.(E, F) Time course of OsRAN1 expression during treatment with 10% PEG 6000, a mimic for drought stress.(G, H).Time course of OsRAN1 expression during treatment with 1 μM IAA.The rice seedlings were germinated and grew for 10 d before they were treated with cold, salt, drought, and IAA stresses.OsUbiquitin was used as an internal control.

Fig. 3 .
Fig. 3. T2 generation phenotypes of different lines in overexpressed OsRAN1 transgenic Arabidopsis.(A) Rosette leaves of wild-type Arabidopsis grown for 3 weeks.(B) Increased rosette leaves of transgenic Arabidopsis grown for 3 weeks.(C) Apical inflorescence of wild-type Arabidopsis.(D) Apical inflorescence including partial abortion of transgenic Arabidopsis.(E) Normal floral apical dominance of wild-type Arabidopsis.(F) Increased tillery number in 35S-sense OsRAN1 transgenic mature Arabidopsis.(G) Normal phenotype of wild-type Arabidopsis.(H) Two branches with rosette leaves in a transgenic plant.(I) Wild-type and transgenic 6-d-old seedlings with elongated hypocotyl in the light.(J) Wild-type and transgenic 6-d-old seeding's in the dark.(K) Time curve of development of rosette leaves in wide-type and transgenic Arabidopsis plants.(L) Normal 10-d-old seeding.(M, N) Transgenic Arabidopsis with much fewer lateral roots and shorter main roots.(O) Statistical analysis of wild-type and transgenic lines main root length.(P) Statistical analysis of lateral root number in wild-type and transgenic lines.Asterisk indicates significant difference P<0.01.

Fig. 4 .
Fig. 4. Effect of auxin on root development in transgenic Arabidopsis plants.(A) Effect of IAA (10 −9 or 10 −7 M) on growth of primary roots in transgenic plants of Arabidopsis.The transgenic plants treated with IAA affected primary root growth compared with wild-type plants.(B) Effect of IAA (10 −9 or 10 −7 M) on lateral root in transgenic Arabidopsis plant.Results are presented as average values±SE from three experiments.More than ten roots were used in each experiment.Asterisk indicates significant difference P<0.01.

Fig. 5 .
Fig. 5. Comparison of cell numbers between transgenic plants and wild-type rice.Primary root meristem zone of primary roots of wild-type (A) and transgenic (B) rice stained with propidium iodide.The number of transgenic rice cells in the meristem was increased over those in the wild type.Bar=50 μm.(C) Cell number in primary roots meristem elongation.Results are presented as mean±SE from three experiments (n=5-10).Bar=50 μm.Asterisk indicates significant difference P<0.01.(This figure is available in colour at JXB online.)

Fig. 6 .
Fig. 6.Cold tolerance analysis of transgenic rice overexpressing OsRAN1.(A) Two-week-old OE transgenic and WT plants (top photographs) were cold stressed at 4 °C for 84 h and then transferred back to the normal condition for recovery.(B) Photographs of representative seedlings of WT and three transgenic lines were taken after 14 d of recovery.(C) Real-time RT-PCR analysis of the expression of OsRAN1 in transgenic rice relative to that of actin.Data represent means and SE of three replicates.OE3, OE5, OE7, OE9, OE11, OE13, OE15, OE18, OE23, OE27, OE34, OE38, OE47, OE53, OE55, OE57 represent transgenic lines overexpressing (OE) OsRAN1.(D) Survival analysis of WT and OsRAN1 transgenic plants 14 d after cold treatment.Error bars indicate standard deviation and results are from three independent replications of the same experiment.The phenotype was confirmed by further experiments that were repeated more than three times.Asterisk indicates significant difference P<0.01.(This figure is available in colour at JXB online.)

Fig. 7 .
Fig. 7. Contents of soluble sugars and free Pro in transgenic rice plants overexpressing OsRAN1 compared with the wild-type plants under normal and cold stress.Free Pro (A) and soluble sugar (B) contents in the OsRAN1-overexpressing plants under cold stress (4 °C) for 3 d.Values are means±SD (n=4).FW, fresh weight.(C) Expression levels of putative Pro synthesis genes (AK102633 and AK101230) and Pro transporter genes (AK067118 and AK0666298) in cold-stressed (4 °C for 1 d) OsRAN1 transgenic plants by real-time PCR.(D) Expression levels of putative sugar synthesis relative genes (Os01g0205700 and OsSPS1) and sugar putative transporter genes (Os08g0178200 and Os12g0641400) and two putative sugar transporter genes (Os08g0178200 and Os12g0641400) in cold-stressed (4 °C for 1 d) OsRAN1 transgenic plants by real-time PCR.The bars represent three repeats.Asterisk indicates significant difference P<0.01.

Fig. 8 .
Fig. 8. Cell cycle progression and mean root tip mitotic index of wild-type and transgenic rice overexpressing OsRAN1 during cold stress.(A) The wild-type at 28 °C.(B) The wild-type at 4 °C.(C) Overexpressing line 47 (OE47) of OsRAN1 transgenic rice at 28 °C.(D) Overexpressing line 47 at 4 °C.Seedlings 7 d after germination were treated with low temperature (4 °C) or room temperature (control, 28 °C) for 12 h.Cell nuclei (10 000) taken from the root apical meristem were stained with DAPI and analysed by flow cytometry.2C and 4C represent the DAPI signals that correspond to nuclei with different DNA contents.(E) Cell mitotic index in root apical meristem (RAM) in rice.The error bars show SE and are from three independent replications of the same experiment.Six root tips were analysed in every replicate.Asterisk indicates significant difference P<0.01.(This figure is available in colour at JXB online.)

Fig. 9 .
Fig. 9. Morphologies changes of the nuclear envelope in the wild type (WT) and transgenic line under cold stress.Nuclear envelope of the WT (A) and OsRAN1-overexpressing line 47 (B) under normal conditions (28 °C); nuclear envelope of the WT (E, F) and OsRAN1-overexpressing line (C, D) after 3 h treatment at 4 °C.Six root tips were observed in every condition.The root tips were transversely cut in the meristematic zones.Arrows indicate the nuclear envelope.Bars=500 nm.

Table 1 .
Tiller number in 35S-sense OsRAN1 transgenic mature rice