Tolerance of EcNAC1 Transgenic Tobacco to Osmotic Stress In all the selected transgenic lines short-term stress response was studied by exposing to stress for seven days. Whereas long-term stress experiments by exposing to stress up to 30 days was carried out only in a few lines, S4, S7, A4 and A7, which showed superior stress-tolerance in short-term experiments. During short-term osmotic stress response studies, all the transgenic seedlings exposed to 5% PEG maintained better growth than wild-type as indicated by significantly lower growth inhibition (Figure 3A & B). Transgenic lines showed 1.5–3 fold less reduction in fresh weight compared to wild-type plants. The root growth was also better in transgenic plants under stress (Figure 3C). Under long-term stress created by exposure to mannitol, transgenic plants showed less reduction in fresh weight at higher concentration (300 mM) when compared to wild-type plants (Figure S5A & S5B). In addition, a densely developed root system was observed in transgenic plants. The root mass was 2–3 folds more in transgenic plants when compared to wild-type roots (Figure S5C). Wild-type plants were dwarf and the leaves were pale green, however transgenic plants maintained the greenness. Thus, these results clearly demonstrate that EcNAC1 transgenic tobacco plants exhibit a significant level of tolerance to PEG and mannitol-induced osmotic stress. EcNAC1 Transgenic Tobacco Plants Showed Rapid Root Elongation and Improved Growth Rate during Salt Stress Under short-term salt stress, the transgenic seedlings maintained better growth and showed significantly less reduction in fresh weight than wild-type (Figure 4A & B). Root elongation was also significantly more in all the transgenic seedlings compared to that of wild-type even under high salt stress (Figure 4C). Similarly, under long-term exposure to different NaCl concentrations the reduction in growth was less in transgenic plants and they maintained better leaf growth and chlorophyll content than the wild-type plants even at 200 mM NaCl (Figure S6A & B), suggesting that the overexpression of EcNAC1 in tobacco confers a high degree of tolerance to salinity stress. EcNAC1 Tobacco Plants Show Improved Tolerance to MV- induced Oxidative Stress Under short-term stress, transgenic plants survived and maintained growth at 5 mM MV for seven days when all the wild-type plants were completely bleached (Figure S7A). Trans- genic plants accumulated distinctly higher fresh weight (Figure S7B) and showed significantly better root growth under stress compared to wild-type (Figure S7C). Even under long-term exposure transgenic plants expressing EcNAC1 constitutively, survived whereas the wild-type plants were not able to grow and died within 10 days (data not shown). However, none of the plants survived at higher concentration of MV (10 mM). EcNAC1 Transgenic Plants Show Better Water-deficit Stress Tolerance and Recovery Gradual water-deficit stress was imposed till the soil FC reaches to 35% and subsequently the plants were maintained at same FC for 10 days. To test the free radical scavenging activity of EcNAC1 2 overexpressed plants under water-deficit stress, the ROS (O2. , H2O2, and OH radical) levels were quantified. Transgenic plants showed 5–7 fold lower O2.2 content over wild-type plants (Figure 5A). The H2O2 content was 12.5–14.5% lower in transgenic plants compared to wild-type plants (Figure 5B). Even the levels of OH radicals were significantly lower in transgenic plants suggesting higher free radical scavenging activity under EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco water-deficit conditions (Figure 5C). The level of lipid peroxida- tion in both transgenic and wild-type plants was estimated in terms of amount of MDA accumulated. All transgenic lines tested, except line S7, showed significantly lower MDA than the wild-type under water-deficit stress (Figure 6). The lower MDA levels in transgenic plants suggest that the EcNAC1 tobacco plants have less lipid peroxidation. The recovery response of the stressed transgenic and wild-type plants was studied 15-days after re-watering. All the transgenic plants tested, except line S7, recovered faster and accumulated significantly higher fresh weight when compared to wild-type (Figure 7A). Representative recovery phenotype of S4 and wild- type plants is shown in Figure 7B. These results showed relevance of EcNAC1 gene under water-deficit stress. Overexpression of EcNAC1 Activates a Few Known NAC Target genes in Tobacco To further understand whether the stress-tolerance observed in EcNAC1 overexpressing tobacco plants was due to transcriptional up-regulation of downstream stress-responsive genes, we analysed the relative transcript levels of several stress-responsive genes. The transgenic line S4 was chosen to study the possible regulation of selected genes by EcNAC1, as this line showed less growth inhibition even at 300 mM NaCl, better root growth under 300 mM mannitol, superior ROS scavenging activity and also lower MDA under water-deficit stress. Apart from that, this line also survived under long-term exposure to MV-induced oxidative stress. Expression of 14 target genes of SNAC1, [17], was analyzed by RT-PCR. The transcripts of genes under study were observed in both transgenic and wild-type tobacco plants and the levels of WUSCHEL-related homeobox 13 (HB-13), MYB-CC type transcription factor (MYB), protein phosphatase 2C (PP2C), serine-threonine protein kinase (STPK), NADPH-cytP450 oxidoreductase (CytOR), Rop subfamily GTPase (ROP), and ATP-binding subunit, ERD1 were more in the transgenic line when compared to wild-type as shown by the increase in relative density of the RT-PCR products (Figure S8). Based on these results we suggest that EcNAC1 can induce a few stress associated genes there by bringing in improved stress- tolerance in transgenic tobacco plants. Discussion Stress-responsive transcription factors that might regulate unique signaling pathways are relevant in bringing the desired levels of tolerance to plants. NAC family transcription factors, specific to plants, have been implicated in plant developmental programs [55], [56], defense [16], [57] and abiotic stress responses [19], [20], [58]. Only a few stress-responsive NAC proteins have been characterized [59] for their relevance in imparting tolerance to abiotic stresses. From this context, characterizing other NAC family genes or the stress-responsive homologs from stress adapted species provides greater insight about this unique group of transcription factors. Several studies have shown that stress adapted species have structurally efficient genes and regulatory pathways which impart stress-tolerance. For example, overexpression of Na+/H+ anti- porter gene, ApNhaP, from a halo-tolerant Cyanobacterium has drastically improved the salt-tolerance of freshwater Cyanobacte- rium [34]. To identify stress-responsive transcription factors from a stress adapted plant species, finger millet, subtracted stress cDNA library was constructed and a gene encoding NAC protein was identified. Sequence analysis of EcNAC1 gene isolated from the library suggested that EcNAC1 is closely related to a few known stress-responsive rice genes that encode NAC proteins. Previously, PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40397