OsLEA3-2 in Salt and Drought Tolerance Response Figure 9. Protection of LDH by protein protectants. (A) SDS-PAGE analysis of OsLEA3-2 protein expression and purification. M, protein molecular weight marker; 1, 3, uninduced precipitant and soluble supernatant of BL21 (DE3) cells, respectively; 2, 4, induced precipitant and soluble supernatant, respectively; 5, OsLEA3-2 protein after purification. (B) Protection of LDH by protein protectants from aggregation due to freezing and thawing. LDH aggregation on repeated freezing and thawing is indicated by light scattering at A340. Results are shown for LDH alone (open bar), for LDH in the presence of BSA (black bar) and for LDH together with OsLEA3-2 (grey bar). *P,0.05 and **P,0.01 shown above the bar reprent results significantly different from those for LDH alone. (C) Effect of desiccation on LDH activity. LDH activity after vacuum drying (open bar) in the presence of BSA (black bar) or OsLEA3-2 (grey bar). All samples were assayed in triplicate. doi:10.1371/journal.pone.0045117.g009 growth performance was observed under osmotic stress (sorbitol). S. cerevisiae cells expressing OsLEA3-2 displayed an improved growth under both ionic and osmotic stress conditions (Fig. 4). To elucidate the contribution of OsLEA3-2 to abiotic stresses, Arabidopsis transgenic plants with the OsLEA3-2 gene overexpressed were generated. The transgenic lines were shown to be more tolerant than the wild type plants to salt and osmotic stresses (Fig. 5C). This suggests that LEA proteins from Oryza sativa (a monocot), can function properly in Arabidopsis, a dicotyledonous plant. There was no significant growth difference between the Oryza sativa transgenic plants overexpressing OsLEA3-2 gene and wild type plants under field conditions with normal irrigation (Fig. S5). Three transgenic lines (L10, L20, and L30) exhibited the same phenotypes as wild type for the majority of morphological traits observed (e.g., plant height, leaf length, spike length, and 1000- seed weight). However, transgenic seedlings that overexpressed OsLEA3-2 showed a significantly stronger growth performance than control under salinity or osmotic stress (Fig. 6D, E). The wild type rice could not survive the drought stress conditions, while transgenic plants that overexpressed OsLEA3-2 recovered and grew until the flowering stage and set seeds (Fig. 7), indicating that the transgenic lines could tolerate 20 days of drought stress and the drought stress did not completely damage the fertility of the transgenic rice plants. These results demonstrate that transgenic rice plants that overexpress the OsLEA3-2 gene are suitable for practical applications and are capable of the production crops even if exposed to long periods of drought stress. LDH undergoes aggregation on freezing and inactivation on drying (Fig. 9). Both OsLEA3-2 and BSA are able to protect LDH from aggregation and inactivation due to stresses. BSA is a well- known cryoprotectant protein. Even though OsLEA3-2 protects LDH from aggregation on freezing and thawing to a lesser extent than BSA (Fig. 9B), it is more effective than BSA in protecting enzyme activity on desiccation (Fig. 9C). Other studies of LEA function have found various LEA proteins to have protective properties similar to or better than BSA on freezing [33,34]. LEA proteins are more effective than BSA in protecting enzyme activity [24,35] on desiccation. All those suggest that LEA proteins are able to maintain enzyme activity on dehydration. Supporting Information Figure S1 The OsLEA3-2 cDNA sequence and its deduced amino acid residues. The asterisk shows the stop code, the polyadenylation signal sequence (AATAAA) is underlined. The full length cDNA of OsLEA3-2 is 1,298 bp in size, with a 96 bp 59 untranslated region (UTR) and a 167 bp 39 untranslated region, and encodes an ORF of 344 aa. (TIF) Figure S2 Summation of amino acids in the 11-mer repeating units. The upper number indicates the position of the amino acid, PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45117