EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco Figure 3. Short-term osmotic stress response of EcNAC1 transgenic tobacco plants. 15-day-old T1 transgenic seedlings selected on hygromycin were inter-planted with wild-type seedlings on MS medium supplemented with 5% PEG and observations were taken after seven days of stress treatment. (A) Phenotype, (B) Reduction in fresh weights over control, and (C) Root elongation of transgenic tobacco plants under stress. Each bar value represents the mean 6 sd (n = 12) of triplicate experiments (student’s t test; *P,0.05 versus wild-type). doi:10.1371/journal.pone.0040397.g003 Fang et al. [12] have systematically analyzed protein sequences of 140 putative NAC or NAC-like genes in rice and classified them into five groups (I–V). Based on trait specificity, stress-related NAC genes fell into the group III stress-responsive NAC genes. Phylogenetic analysis of deduced amino acid sequence of EcNAC1 along with rice NAC proteins and also the closest accessions of maize and sorghum showed that this gene actually represents the group III, suggesting its stress-responsive nature. MEME-based prediction of putative motifs among this cluster showed that N- terminal regions were conserved in parallel with NAC domain structure and EcNAC1 appear to have unique motif 6 in the C- terminal region. In a recent study, it was shown that overexpres- sion of PINO1, from a halophytic wild rice Porteresia coarctata, in rice and tobacco has substantially improved salt tolerance compared to its domesticated rice homolog RINO1 which lacks a unique stretch of 37 amino acids [35]. The same group also identified PcIMT1 gene, from Porteresia coarctata, which is absent in domesticated rice and overexpression of this gene has resulted in accumulation of pinitol under salt stress in rice [60] suggesting stress adapted species might have novel candidate genes or unique mechanisms which could help genetic engineering of susceptible species for improved stress-tolerance. Expression analysis of EcNAC1 in our PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40397