Dubouzet et al. [24] demonstrated that overexpression of OsDREB1A in Arabidopsis and rice improves tolerance to drought, salinity and freezing although a few target genes were not up- regulated in Arabidopsis, which were induced in rice. Significant genetic diversity exists among plant species for stress adaptation; hence elucidating the transcriptome from abiotic stress adapted species has greater significance. Several reports have shown that genes from stress adapted species are functionally more efficient in imparting tolerance [25], [26], [27]. Although the studies with Arabidopsis dissected several abiotic stress pathways, Arabidopsis being a susceptible species the focus has now been on its related tolerant family or species, namely, Thellungiella halophila, Thlaspi caerulescens and Arabidopsis halleri [28–31]. Apart from these, other stress adapted species and resurrection plants with very high tolerance threshold may possess mechanisms and genes which make them survive extreme conditions. Two of our recent studies [32], [33] and also other reports [34], [35] support this concept. Though stress adapted crops such as sorghum and maize genomes have several NAC genes, there is no information on the functional relevance of these genes in imparting stress-tolerance and stress- responsive NACs are yet to be identified in these species. Hence, discovery and characterization of stress-responsive NAC tran- scription factors from stress adapted species would provide insight on the importance of these transcription factors. Figner millet [Eleusine coracana (L.) Gaertn], a crop which is widely grown in semi-arid and arid tropics of Africa and Asia is widely adapted to a broad range of abiotic stresses. Finger millet survives under severe water-deficit and osmotic stress and shows remarkable recovery on alleviation of stress [33], [36]. In this study, we report the identification of a putative NAC family transcription factor, denoted as EcNAC1, from a subtracted cDNA library of finger millet. We show that EcNAC1 is induced by various abiotic stresses and expression of this gene in susceptible species, tobacco, imparts abiotic stress-tolerance. Materials and Methods Construction of cDNA Library in Finger Millet and Identification of EcNAC1 Plant material and growth conditions. Finger millet (var. GPU28) seeds were sown in pots filled with mixture of soil, sand and farmyard manure (3:1:1 proportion). Seeds of Nicotiana tabacum [tobacco (var. KST 19)] were germinated, transplanted in soil at two or three leaf stage, and maintained in greenhouse conditions at a day/night temperature of 2561uC. Stress imposition. Twenty-five-days after sowing, water- deficit stress was imposed to finger millet seedlings simulating field condition. Rain-out shelter was used to protect plants from adverse weather; otherwise plants were exposed to natural vapor pressure deficit. Desired stress levels were achieved gradually by gravimetric approach [37], [38]. Progressive stress was applied for a set of plants over a period of 5 days to reach 80% field capacity (FC), likewise 8 days to reach 60% and 10 days to reach 35% FC. Leaf tissue was harvested on same day for all stress levels. To study the expression of native NAC gene in tobacco, dehydration stress was created on two weeks old seedlings by transferring them to Petri- plates (9 cm diameter) containing MS medium supplemented with 5% PEG-10000 (w/v) and tissue was harvested at 24 h after treatment. To create salinity stress, two weeks old seedlings were watered with 200 mM NaCl solution and tissue was harvested at 24 h. In all the treatments a set of control seedlings were maintained. cDNA library. Total RNA was isolated [39] from fully expanded young leaves of both control and stressed (80%, 60% EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco and 35% FC) finger millet plants and subtractive hybridization was performed according to Mishra et al. [40]. The differentially expressed subtracted poly(A+) RNA was used to synthesize double- stranded cDNA and uni-directionally ligated to a phage lambda- ZAP vector, in-vitro packaged and infected to XL1-blue Escherichia coli cells according to the manufacturer’s instructions using a Uni- ZAP XR cDNA library construction kit (Stratagene, CA, USA). The cDNA inserts from individual recombinant plaques were verified by PCR with universal M13 forward and reverse primers using 1 ml of recombinant phage suspension as a template in 50 ml reaction volume for 30 cycles with the following PCR conditions, 94uC for 1 min, 55uC for 1 min and 72uC for 2 min. PCR amplified cDNA inserts were sequenced using universal T7 primers. Identification of EcNAC1. A truncated NAC domain containing gene was identified from the sequenced clones of cDNA library by NCBI BLAST search and comparative analysis. The missing 59 end was amplified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) kit (Invitrogen Corporation, Carlsbad, CA, USA) according to manufacturer’s instruction. Bioinformatic analysis. The EcNAC1 was compared with 140 NAC’s of rice [12]. The full-length protein sequences were multiple aligned with deduced protein sequence of EcNAC1 and phylogenetic tree was constructed with the program ClustalW [41]. The program multiple EM for motif elicitation (MEME) [42] was used to predict the potential motifs in the sequences. All motifs discovered by MEME were searched in the InterPro database with InterProScan [43]. PSORT [44] and NetNES [45] tools were used to predict the nuclear localization and nuclear export signals, respectively. Construction of Plant Expression Vectors and Plant Transformation Constructs. Coding region of EcNAC1 was amplified using cDNA synthesized from finger millet leaf tissue, undergoing water- deficit stress, with specific primers (Table S1A). The expression cassettes, CaMV35S:EcNAC1:NOS and 4xABRE:EcNAC1:NOS (4xA- BRE is a synthetic promoter with CaMV35S minimal promoter), were inserted into binary vector pGreen0179 [46] containing a hygromycin resistance selectable marker. The binary vector pGreen0179 with EcNAC1 expression cassettes and pSoup plasmids were co-mobilized into Agrobacterium tumefaciens strain LBA4404. Transformation. In-vitro regeneration approach was fol- lowed to generate transgenic tobacco plants [47]. Leaf discs from healthy tobacco plants (var. KST 19) were co-cultivated for 15 min with A. tumefaciens harbouring the recombinant binary vectors and cultured on Murashige and Skoog (MS) [48] agar medium supplemented with 6-benzylaminopurine (2.25 mg/l), naphthalene acetic acid (0.1 mg/l), cefotaxime (400 mg/l), and hygromycin (30 mg/l). The regenerated shoots were rooted on MS basal medium containing hygromycin (30 mg/l). Profusely rooted plantlets were transferred into pots and maintained in greenhouse following acclimatization for two weeks. Molecular Analysis of Tobacco Transgenic Plants Gene expression by RT-PCR and quantitative RT- PCR. First-strand cDNA was synthesized from total RNA (2 mg) primed by oligo (dT)15 using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT; MBI Fermentas, Han- over, MD, USA) according to the manufacturer’s instructions. For expression analysis, cDNA pool was used as a template. Thirty cycles of PCR (with 3 min of initial denaturation at 95uC, 94uC for 1 min, 50–58uC for 1 min, 72uC for 1 min) amplification, with a PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40397