100 ml NADH and 2 mM pyruvate. Change in A340 was monitored every 5 s for 1 min in a NanoVue Plus spectropho- tometer. All values given are expressed as percentage of rate of reaction measured for undried samples. Assays were performed in triplicate. Results Cloning and Characterization of the Full-length cDNA of OsLEA3-2 The full-length cDNA was obtained by 59 and 39 Rapid Amplification of cDNA ends (RACE). The reverse specific primers oligo1 and oligo2, together with GeneRacer primers, Race 59 and Race 59 nested primer, were used to clone the 59 end of the cDNA sequence. The forward specific primers oligo3 and oligo4 together with Race 39 and Race 39 nested primers were used to clone the 39 end cDNA sequence (Table S1). The complete cDNA sequence was 1298 bp in size, with a 96 bp 59 untranslated region (UTR) and a 167 bp 39 untranslated region (Fig. S1). It consisted of two exons, encompassed one intron, and potentially encoded for an ORF of 344 aa with a predicted molecular mass of 36.8 kD and pI of 6.81. The deduced amino acid sequence shares 42 and 36% amino acid identities with LEA3 of Arabidopsis thaliana and Oryza sativa (Fig. 1A, B), respectively. This gene was termed OsLEA3-2 and its accession number at GenBank was JQ043381. Predicted proteins of Oryza LEA3-2 showed a preponderance of Ala, Thr, Asp, Glu, Lys, Arg and Gly that constitute 20.6%, 10.2%, 10%, 9.3%, 9.3%, 9.3%, and 7.3%, respectively, and lack Trp. OsLEA3-2 has 16 11-mer repeating units, TKDA(A/T)ADK(A/ T)RE (Fig. S2). Hydropathy analysis showed that OsLEA3-2 is a hydrophilic protein (Fig. 1C). OsLEA3-2 Only Expresses in the Embryo Under Normal Conditions The expression profile of OsLEA3-2 gene in various tissues was analyzed employing the semi quantitative RT-PCR method. The ubiquitin gene was used as an internal control. A product of approximately 146 bp was amplified from the late embryo stage, while no transcript was detected in the roots, shoot base, and leaves of another plant (Fig. 2A). These results suggested a similarly to the previous reported information on LEA proteins, and that OsLEA3-2 may play an important role in the maturation process of the embryo. Expression Analysis of OsLEA3-2 Under Abiotic Stresses A semi quantitative RT-PCR analysis with the ubiquitin gene as an internal control revealed that the OsLEA3-2 gene does not express in vegetative tissues under normal conditions, however, it can be induced by abiotic stresses. Treatment of the seedlings with mannitol, salt, or PEG induced OsLEA3-2 gene expression. (Fig. 2B). While no transcript was detected in the seedlings treated with low temperature stress or ABA, OsLEA3-2 expression was detected when the rice was grown in Hoagland with ABA (Fig S3). OsLEA3-2 was induced by the ABA, but assimilation was not possible through a topographical application on rice leaves. Mannitol and salt stress also induced OsLEA3-2 gene expression in the shoot base and leaves, while the PEG treatment induced gene expression in both roots and shoots. Subcellular Localization of OsLEA3-2 The OsLEA3-2 coding sequence was fused in frame to the 39 end of a GFP (Fig. 3A). The subcellular localization of the GFP- OsLEA3-2 was examined through a transient expression of GFP- OsLEA3-2 in Salt and Drought Tolerance Response OsLEA3-2 in onion epidermal cells. The construct with GFP alone was used as a control. An examination of green florescence by confocal laser-scanning microscopy showed that GFP alone localized at the nucleus and cytosol of onion epidermal cells, while the green fluorescent signal of GFP:OsLEA3-2 was detected exclusively in the nucleus of the onion epidermal cells (Fig. 3B). More than 30 GFP positive cells were detected. GFP:OsLEA3-2 exhibited nucleus localization in all those cells. Another construct with OsLEA3-2 coding sequence fused in frame to the 59 end of a GFP was also used to determine the OsLEA3-2 subcellular localization. Nucleus concentrated green fluorescent signal was detected (data not shown). These results demonstrated that OsLEA3-2 is a nucleus- localized protein. Growth Performance of Transformed S. cerevisiae Strains Under Abiotic Stresses The growth performance of yeast cells transformed with pYES2 or pYES2-OsLEA3-2 was tested under ionic and osmotic stress conditions. The control strain, Y001582/pYES2, was grown in a YNBG medium to an early stationary phase and then transferred to a YNBG medium supplemented with 1.2 M NaCl. The growth of the Y001582/pYES2 strain was initially arrested and exponen- tial growth resumed after a lag phase of 60 hours (Fig. 4A). The Y001582/pYES2OsLEA3-2 strain cells displayed improved growth as compared to the control strain, and the lag phase of Y001582/pYES2OsLEA3-2 was about 24 hours, shorter than that observed with the control when transferred to 1.2 M NaCl (Fig. 4A). Similar experiments were carried out to investigate the effect of high KCl concentration (1.2 M) or osmotic stress (2.0 M sorbitol) on growth characteristics. The Y001582/pYES2O- sLEA3-2 cells displayed a shorter lag phase (24 h) than the control strain (39 h) in YNBG medium containing 1.2 M KCl (Fig. 4B). A similar phenomenon was observed in a YNBG medium supple- mented with 2.0 M sorbitol. The Y001582/pYES2OsLEA3-2 strain again displayed a shorter lag phase (48 h) than the control strain (84 h) (Fig. 4C). OsLEA3-2 Plays a Role in Abiotic Stress Tolerance of Plants To analyze the roles of OsLEA3-2 in plants, OsLEA3-2 was inserted into a pHB vector (Fig. 5A) and was overexpressed in Arabidopsis and rice under the control of the double constitutive cauliflower mosaic virus (CaMV) 35 S promoter. Transformants were selected according to hygromycin-resistance. Hygromycin gene specific primers were then used to perform PCR on genomic DNA from the transgenic lines or the wild type. Transgenic lines showed a 576 bp specific amplicon, while no observed size specific amplicon was amplified from WT plants. Stable inherited homozygous transgenic lines were obtained at the T2 generation. Three rice transgenic lines (L10, L20, and L30) were selected for Southern blot analysis. A single specific band was observed in each transgenic line (Fig. 6A). No band was observed in the wild type control. These results suggested single integration was occurred in the genome of each transgenic line. RT-PCR assays revealed OsLEA3-2 was overexpressed in both the transgenic Arabidopsis and rice plants (Fig. 5B, 6B). To assess the role of OsLEA3-2 in tolerance to abiotic stresses, transgenic Arabidopsis plants that overexpressed OsLEA3-2 were compared with WT under abiotic stress conditions. No differences in seed germination percent were observed between the transgenic and WT plants under abiotic stress conditions, but a faster speed of germination was observed in the transgenic rice (Fig. S4). The OsLEA3-2 overexpressing transgenic seedlings showed significantly stronger growth performance than WT plants (P,0.01) on MS media supplemented with 150 mM mannitol or 100 mM NaCl PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45117