Materials and Methods Plant Material and Treatments The rice cultivar ZH11 was used in the transformation. Rice plants were grown in the glasshouse at 28uC and the Arabidopsis plants were grown in growth chambers at 21–25uC, 16 h/8 h (day/night). To impose the salt and osmotic stresses, one month old seedlings of ZH11 were irrigated with 200 mM NaCl and 25% PEG-6000 or 400 mM mannitol in half strength Hoagland solution, respectively. For the ABA treatment, 100 mM ABA in 0.02% Tween-20 was sprayed on the ZH11 seedlings. One month old seedlings were incubated at 4uC for the cold treatment trials. All abiotic stress treatments, samples were harvested after 6 hours to analyze the expression of OsLEA. RNA Extraction and Reverse PCR Total RNA was extracted from the embryo, root, shoot base, and leaf of the rice cultivar ZH11 using a TRIzol reagent (Invitrogen). Total RNA was also extracted from the control and stress-treated roots, shoot bases, and leaf tissues. The RNA was then reverse-transcribed using an oligo(dT)18 primer and AMV reverse transcriptase (Toyobo) according to the manufacture’s protocol. RT-PCR was conducted with oligos OSLEA2F and OSLEA2R (Table S1) using a Taq DNA polymerase (Dingguo) supplemented with 5% dimethyl sulfoxide (DMSO) to analyze the gene expression. Cloning and Sequencing of the Full-length OsLEA cDNA The total RNA isolated from 200 mM NaCl treated seedlings was used to clone the OsLEA gene. The full-length OsLEA cDNA was cloned by 59- and 39-Rapid Amplification of cDNA ends (RACE), using a GeneRacer kit (Invitrogen) according to the manufacture’s protocol. The primer sets used in the 59- and 39- rapid amplification of cDNA ends experiments are listed in Table S1. PCR was performed using a PrimeSTAR DNA polymerase (TaKaRa) with a GC buffer, and the products of which were then cloned into a pMD18-T vector (TaKaRa) and sent to be sequenced. Subcellular Localization of OsLEA3-2 Onion epidermal cells were bombarded with the construct of a double 35 S: GFP: OsLEA3-2 transgene using a particle gun- mediated system (PDS-1000/He; Bio-Rad) and were observed with a confocal microscope (Zeiss LSM510; Carl Zeiss Micro- Imaging GmbH, Jena, Germany). Overexpression of OsLEA3-2 in Saccharomyces Cerevisiae OsLEA was amplified with the following primers, OsLEA-F: CCC AAG CTT AAA ATG GCG TCG AGG CAG GAC A and OsLEA-R: TGC TCT AGA TCA CAT CTT GCC GTG GCG primers. Hind III and Xba I restriction sites are underlined in the forward and reverse primers, respectively. The amplicon was digested with Hind III and Xba I and placed under the control of a GAL1 promoter in the plasmid pYES2. To achieve high levels of expression of OsLEA in Saccharomyces cerevisiae, the resulting plasmid, pYES2OsLEA, was transferred into the S. cerevisiae strain Y001582 using the lithium acetate method. Cells were cultured in a YNPD medium (0.67% yeast nitrogen base without amino acids, and 2% glucose) or in a YNBG medium (0.67% yeast nitrogen base without amino acids, and 2% galactose). For the ionic and osmotic stress assay, S. cerevisiae cells that contained pYES2OsLEA were grown in YNBG medium for two days. Two hundred microliters of the culture was inoculated into OsLEA3-2 in Salt and Drought Tolerance Response YNPG (20 ml) supplemented with 1.2 M NaCl, 1.2 M KCl, or 2.0 M sorbitol. At each time point, 50 microliters of culture was removed, and the OD600 was measured with a spectrophotometer. Growth was measured three times. Overexpression of OsLEA3-2 in Arabidopsis OsLEA3-2 was cut from pYES2-OsLEA3-2 with Hind III and Xba I and cloned into modified binary vector pHB [21]. The binary vector pHB-OsLEA3-2 was transformed into the Agrobacter- ium strain EHA105 and was used for transformation into Arabidopsis thaliana ecotype Columbia according to a previously described method [22]. The transgenic plants were then selected on hygromycin. The T1 plants were confirmed by PCR with specific primers for the hygromycin phosphotransferase (HPT) gene: 59- TGTCCTGCGGGTAAATAGC-39 and 59-TGCTCCATA- CAAGCCAACC-39 (AY836546). To evaluate the abiotic stress tolerance of transgenic plants over expression of OsLEA3-2 in the germination stage, wild type (WT) and transgenic (T4) seeds were sown on a MS, MS+150 mM mannitol or an MS+100 mM NaCl media. Ectopic Expression of OsLEA3-2 in Oryza Sativa The binary expression construct pHB-OsLEA3-2 was intro- duced into japonica rice Zhonghua 11 by an Agrobacterium mediated transformation as previously described [23]. Southern blot was used to analyze the transgenic plants. 20 mg of total genomic DNA from leaf tissue of transgenic plants and wild type plants was digested with appropriate restriction endonuclease Hind III. DNA fragments were separated by electrophoresis on a 1% (w/v) agarose gel and then transferred to a nylon membrane (Amersham Bioscience) according to standard protocols. Dig-high DNA labeling kit I (Roche) was used to label the Hygromycin DNA probe. Transgenic rice (OsLEA3-2 overexpression) seeds were used for stress tolerance assays. Rice seeds were germinated in water (as control) or in water containing 100, 200 mM NaCl, 10%, 20% PEG 6000, or 10 mM ABA. Photographs were taken after five days and 14 days. Drought Stress Treatment Both the wild type and transgenic rice plants were placed in soil and allowed to germinate and grow for one month in a glasshouse at 28uC61uC. The one month old seedlings were then treated for drought stress (without irrigation) for 20 or 18 days. The plants were then irrigated with water and grown for one month and ten days. The plants that survived developed new leaves were counted. Aggregation Assay LDH was obtained from SIGMA and diluted in 100 mM sodium phosphate buffer (PBS, pH6). Aggregation of LDH was monitored by reading absorbance A at 340 nm in a NanoVue Plus spectrophotometer. 170 ml of 0.2 mg/ml LDH with or without 0.4 mg/ml protectant proteins in polystyrene tubes was flash- frozen by immersion in liquid N2 for 30 s and then thawed at ambient temperature. All samples were assayed in triplicate. LDH Activity LDH enzyme activity was measured according to Goyal, K. [24] with modifications. 2 ml of 0.1 mg/ml LDH was added to a total of 50 ml of 25 mM Tris/HCl (pH7) with or without stabilizing agents in a microfuge tube. Tubes were vacuum-dried for 1 hour at 25uC in an Eppendorf vacuum dryer and rehydrated immediately in 50 ml of water at room temperature. 2 ml of LDH/ stabilizer solution was added to 1 ml of 100 mM PBS (pH6) with PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45117