OsLEA3-2 in Salt and Drought Tolerance Response Figure 6. Stress or ABA sensitivity of wild type and transgenic rice seedlings. (A) Southern hybridization analysis of transgenic plants. M, molecular marker; WT, wild type; L10, L20, L30, three transgenic lines. (B) Expression level of OsLEA3-2 in transgenic rice, Zhonghua 11 was used as control; (C) photographs were taken after 5 d or 2 weeks of growth in water control. Photographs were taken after 5 d (D) or 2 w (E) of growth in water containing 10% or 20% PEG, 100 or 200 mM NaCl, or 10 mM ABA. Effect of 2 weeks abiotic-stress on root (F) and shoot (G) length of rice. For each treatment, 8 seedlings were measured. *P,0.05 and **P,0.01 shown above the bar reprent results significantly different from wild type control. doi:10.1371/journal.pone.0045117.g006 stress completely inhibited their growth. After 20 days without irrigation, the leaves appeared to be bleached and were senescent in both the wild type and the transgenic rice (Fig. 7B). However, new leaves appeared only in the transgenic plants after the plants were supplied with water and grown in optimal conditions for 10 days post stress (Fig. 7C). None of the wild type plants survived the stress conditions, while 83% of the plants from line 10 and 67% from line 30 survived. The transgenic rice plants grew until the flowering stage (Fig. 7D) and later set seeds (data not shown). About 20% wild type plants could survive on 18-day-drought stress. However, those plants produced less grains (with one third shriveled) per spike than transgenic line L10 (Fig. 8). OsLEA3-2 Protein Prevents LDH Aggregation Due to Freezing Stress LDH suffers marked aggregate formation when subjected to cycles of freezing in liquid N2 followed by thawing, as determined in a light-scattering assay. The degree of aggregation increases with the number of freeze-thaw cycles, but addition of OsLEA3-2 or BSA, a well-known cryoprotectant protein, prevents the aggregation (Fig. 9B). OsLEA3-2 protein prevents LDH aggregate formation due to rapid freezing in liquid N2. OsLEA3-2 Protein Preserves LDH Activity Under Desiccation In accordance with its anti-aggregation activity, OsLEA3-2 preserves LDH activity under desiccation. BSA preserves enzyme activity to a lesser extent than OsLEA3-2 after multiple cycles of desiccation (Fig. 9C). Discussion Plants have developed mechanisms to perceive and transmit stress signals to cellular machinery that active adaptive responses in response to abiotic stress conditions [25,26]. It has been shown that LEA proteins are involved in plants drought/desiccation tolerance. LEA proteins are members of a large group of PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45117