EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco Figure 4. Short-term salt stress response of EcNAC1 transgenic tobacco plants. 15-day-old T1 seedlings selected on hygromycin were inter- planted with wild-type seedlings on MS medium supplemented with 200 mM NaCl, and the observations were taken after seven days. (A) Phenotype, (B) Reduction in fresh weights over control and (C) Root elongation of transgenic tobacco plants under salt stress. Each bar value represents the mean 6 sd (n = 6) of triplicate experiments (student’s t test; *P,0.05 versus wild-type). doi:10.1371/journal.pone.0040397.g004 studies also confirms its stress-responsiveness. EcNAC1 was induced in finger millet seedlings/plants exposed to water-deficit and salinity stress. Among several NAC genes identified in Arabidopsis and rice, only a few were shown to be stress-responsive [15], [17– 20], [22], [58], [61], [62] and are grouped under stress-responsive NAC genes. The promoter regions of some of these genes have DRE and ABRE sequences [58], [63]. The promoter character- ization of EcNAC1 which has highest homology with rice SNAC1 could help in understanding the pathway through which the gene is regulated. The major focus in this study was to identify genes from drought adapted crop, finger millet, and study whether genes from tolerant species can impart better stress-tolerance in a susceptible species. We used tobacco as susceptible species and tested the relevance of EcNAC1 for stress-tolerance. As tobacco was used as system to transform EcNAC1, the stress-responsive nature of endogenous NAC gene was also analysed to understand the regulation of related NAC genes in tobacco. The closest homolog of a partial fragment was identified from TIGR database (TIGR index# TC5942) and designated as NtNAC. qRT-PCR analysis showed more than 10 and 5 fold higher expression of the EST over control under dehydration and salinity, respectively, suggesting the stress induction of related NAC genes in tobacco (Figure S9). There are contrasting reports on the effect of constitutive overproduction of regulatory molecules, such as transcription factors, on growth under normal condition [24], [64–67]. However, overexpression of EcNAC1 in tobacco plants did not affect growth under normal condition. Similar results were observed in overexpression studies of other transcription factors. Oh et al. [68] reported that the transgenic plants expressing CBF3 (C-repeat binding factor 3) under Ubiquitin1 promoter exhibited no visible phenotypic alterations in rice. These reports and our present result suggest that the growth abnormalities observed in many transgenic plants constitutively expressing the regulatory genes is not a universal phenomenon. PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40397