Other notable observation is that substantial superior root growth of EcNAC1 transgenic lines compared to the improved shoot growth. Recently a few studies have shown the role of NAC proteins in root growth thereby improving salt stress-tolerance. He et al. [70] have suggested the involvement of NAC genes in improving Arabidopsis root growth under salinity stress. Overex- pression of AtNAC1 and AtNAC2 resulted in altered lateral root development. In addition, the auxin responsive genes AIR3 (Auxin Induced in Root cultures 3) and DBP (DNA-Binding Protein) were identified in a screen for downstream targets of NAC1 [14]. Overexpression of GmNAC20 has promoted lateral root formation in Arabidopsis under salt stress [71]. However, there is no direct evidence to support the involvement of NAC transcription factors in root growth under osmotic/dehydration and oxidative stress. We observed that under PEG and mannitol-induced osmotic stress, high salinity as well as oxidative stress there was significant increase in the root growth of EcNAC1 transgenic plants when compared to wild-type tobacco plants. Transgenic plants showed not only improved root growth but also better lateral root formation. Understanding the phytohormone signaling pathway in NAC overexpressing plants may provide further insight in this direction. By in-vitro studies using transgenic EcNAC1 seedlings, we provided evidences that EcNAC1 imparts tolerance to MV-induced oxidative stress. One of the major factors when plants are exposed to stress under high light is the generation of cytotoxic compounds like ROS [72]. The levels of these cytotoxic compounds in pot grown transgenic plants subjected to gradual water-deficit stress under natural high light condition was relatively less. These results are further substantiated by lower lipid peroxidation in transgenic plants as shown by lower levels of MDA. Thus, it is conceivable that the ROS-scavenging and membrane-protecting properties of EcNAC1 accounts, at least partially, for the enhanced tolerance to oxidative stress in transgenic tobacco plants. In rice OsNAC6 overexpression plants, several biotic and abiotic responsive genes were up-regulated. A transient trans-activation assay also demon- strated the expression of peroxidase in OsNAC6 transformants. Further, in a recent study by Balazadeh et al. [73], it was demonstrated that the up-regulation of several downstream stress- responsive genes having consensus cis-elements [RCGTR(4– 5n)RYACGCAA; R = A or G; Y = C or T] is regulated by NAC transcription factors. Among them, many are oxidative stress associated genes. This signifies the roles of a few NAC transcription factors in regulation of oxidative stress-tolerance. We observed the expression pattern of closest tobacco homologs of a few SNAC1 target genes [17]; presuming EcNAC1 might also have common target genes to regulate because of its 57% identity at protein level with SNAC1. There was up-regulation of a few stress-responsive genes such as HB-13, MYB, PP2C, STPK, CytOR, ROP and ERD1. The involvement of some of these genes in stress- tolerance is well studied (HD proteins [74]; MYB related transcription factors [66]; PP2C [75]; serine-threonine protein kinase [76]; Rop subfamily GTPase [77]; ERD1 [62]). NAC transcription factor has been shown previously to activate several downstream functional genes involved in stress adaptation [17], [20], [58]. Even though different NAC transcription factors interact with core cis-acting sequences in the promoters of target genes but the actual set of target genes for each paralog vary in different plants. Both cis-acting DNA sequences and transacting transcription factor amino acid sequences have co-evolved to regulate the coordinated expression of several stress adaptive genes. Similarly, the architecture of promoter sequences within these stress-related genes are not simply collinear, they are multifunctional and modular, and interact with multiple indepen- EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco dently regulated transcription factors and display a transcriptional complexity to cope up with the cross-talk in response to various environmental stresses. Taken together these results indicate that transgenic overexpression of EcNAC1 in tobacco positively correlates with stress-tolerance. In conclusion, this study shows that EcNAC1 is stress-inducible and several known stress-responsive genes are up-regulated in tobacco transgenic plants expressing EcNAC1 thus conferring tolerance to various abiotic stresses including dehydration, salinity and oxidative stress. Our work suggests the possibility of engineering abiotic stress-tolerance in a crop plant by overex- pressing EcNAC1 gene from a heterologous plant species. Supporting Information Figure S1 Phenotype of finger millet plants grown at different levels of water-deficit stress. Gradual stress was imposed on 25-day-old pot grown plants following gravimetric approach. Stress was applied for a set of plants over a period of 5 days to reach 80% FC, likewise 8 days to reach 60% and 10 days to reach 35% FC. Samples were collected on the same day. Rain- out shelter was used to protect the plants from adverse weather; otherwise plants were exposed to natural vapor pressure deficit. Photographs were taken at the end of stress period. (TIF) Figure S2 PCR analysis of putative T0 tobacco trans- formants expressing EcNAC1 under 4xABRE stress- inducible promoter. Genomic DNA was isolated from tobacco plants transformed with 4xABRE::EcNAC1:NOS and PCR reactions were performed with (A) HPTII forward and reverse, (B) Gene- specific forward and NOS terminator reverse, (C) Gene-specific forward and reverse and (D) Promoter forward and gene-specific reverse primers to confirm the integration. M - marker; P - plasmid. (TIF) Figure S3 PCR analysis of putative T0 tobacco trans- formants expressing EcNAC1 under CaMV35S constitu- tive promoter. Genomic DNA was isolated from tobacco plants transformed with CaMV35S::EcNAC1:NOS and PCR reactions were performed with (A) HPTII forward and reverse, (B) Gene- specific forward and NOS terminator reverse, (C) Gene specific forward and reverse and (D) Promoter forward and gene-specific reverse primers to confirm the integration. M - marker; P - plasmid. (TIF) Figure S4 RT-PCR analysis of wild-type and EcNAC1 expressing tobacco plants. A. Expression of the transgene analyzed by RT-PCR. B. The corresponding increase in the relative density of bands over wild-type. M - marker; P - plasmid. (TIF) Figure S5 Long-term osmotic stress response of trans- genic tobacco plants expressing EcNAC1. 15-day-old T1 transgenic seedlings selected on hygromycin were transferred to MS medium supplemented with 100, 200 and 300 mM mannitol and observations were taken after 30-days. (A) Phenotype, (B) Root fresh weight of wild-type and transgenic plants. Each bar value represents the mean 6 sd (n = 6) of triplicate experiments (student’s t test; *P,0.05 versus wild-type). (TIF) Figure S6 Long-term salt stress response of transgenic tobacco plants expressing EcNAC1. 15-day-old T1 seedlings selected on hygromycin were transferred to MS medium PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40397