Bioinformatic Analysis of EcNAC1 Sequence homology search performed using the deduced amino acid sequence of EcNAC1 against translated non- redundant nucleotide database using tBLASTN and protein database using BLASTP showed an overall 57–59% sequence identity to sorghum (GenBank ID: XP002466217), maize (GenBank ID: ACL52720) and rice (Os03g60080) genes as well as many other plant genes encoding NAC proteins. The NAC domain containing proteins are highly conserved across all plants and encoded by multi-gene family. The gene duplications and their subsequent divergence were central to multiplicity of the NAC family. It was very difficult to establish the evolutionary relationship of EcNAC1 isolated in this study with other plant orthologs based on its NAC domain mediated trans- activation of several downstream genes. The availability of complete genome sequence of rice has provided a reference platform to identify the complete list of genes that encode NAC proteins in rice plants and allowed us to conduct phylogenetic analysis and establish the EcNAC1 relationship with other rice orthologs. A phylogenetic study was conducted using known 140 genes of NAC family full-length protein sequences from rice and also closest accessions from sorghum (GenBank ID: XP002466217) and maize (GenBank ID: ACL52720) along with EcNAC1 sequence by ClustalW program. The EcNAC1 was phylogenetically aligned to a cluster of 14 rice and also sorghum and maize NAC proteins and, particularly, evolution- arily very close to rice NAC ortholog Os03g60080 (SNAC1) (Figure 1A). In addition to the amino acid sequence homology, several structural motifs predicted based on the MEME program in EcNAC1 polypeptide are well conserved with the same order as in rice (Os03g60080) as well as sorghum and maize NAC proteins except for the motif 6 which is absent in EcNAC1, signifying the unique C-terminal region of the gene and also suggesting that these NAC proteins are evolutionarily evolved from a common ancestor (Figure 1B & Table S3). Amino acid sequence similarity of EcNAC1 with known stress- responsive NAC family genes suggests the plausible involvement of this gene in stress adaptation. When translated amino acid sequence was analyzed by PSORT for nuclear localization and nuclear export signals, EcNAC1 showed four residue pattern composed of three basic amino acids (RRR) and H (one residue at 190 and another at 122) with nuclear targeting certainty score of 0.98 for 1. Apart from that, the sequence also showed 7 residue pattern of nuclear targeting at position 176 with two basic residues, ten residues spacer and another basic region consisting of three basic residues out of five residues (KK NEWEKMQMKK GYRRR). In addition, NetNES tool predicted the residue ‘‘LAL’’ at 213 amino acid with calculated nuclear export signal score exceeding the threshold score suggesting the particular residue is expected to participate in a nuclear export signal. A comprehensive analysis of NAC family genes in rice and Arabidopsis (75 and 105 predicted NAC proteins, respectively) predicted conserved DNA binding domain at the N-terminal region and a variable C-terminal region which is known to be involved in transcriptional activation [11]. Analysis of EcNAC1 amino acid sequence using InterProScan predicted a NAC DNA binding domain from 24–176 amino acid. A conserved motif detected in the C-terminal region using the MEME program was also found in the SNAC1 [17]. It has been clearly shown that this C-terminal activation domain in SNAC1 had transactivation activity suggesting that EcNAC1 is also capable of transactivation. EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco Expression Pattern of EcNAC1 gene in Response to Abiotic Stress In plants, a multi-gene family encodes NAC domain containing proteins, with a very high sequence homology among the family members. Each of these genes responds differentially to environ- mental and/or developmental cues. Therefore, it is very difficult to monitor the expression of these genes individually by northern blot analysis due to cross hybridization. Therefore the relative expression of EcNAC1 gene was monitored in finger millet in response to various stresses in comparison to non-stressed control seedlings by quantitative RT-PCR analysis using gene-specific primers. The mRNA purified from the stress exposed finger millet seedlings and their corresponding control seedlings was reverse transcribed separately into first-strand cDNA and used as a template. The EcNAC1 transcript was detectable in both stress exposed and control seedlings. The up-regulation of EcNAC1 gene in response to different environmental stresses is shown in Figure 2. In an attempt to capture both rapid and long-term changes in EcNAC1 gene expression, we sampled the finger millet seedlings 12 and 24 h after exposure to NaCl. Under water-deficit stress the expression was seen at mild (60% FC) as well as severe stress (35% FC). The relative up-regulation of EcNAC1 was ,40 folds higher in response to mild water-deficit stress and down-regulated at severe water-deficit stress (Figure 2A). The relative up-regulation of EcNAC1 gene was observed not only in response to mild water- deficit stress but also in response to salinity (200 mM NaCl at 12 h) stress with ,110 fold increase (Figure 2B). Molecular Analysis of Transgenic Tobacco The NAC transcription factor-mediated transcriptional activa- tion of stress-responsive genes and their associated signaling networks regulate dynamic co-expression of several stress-respon- sive genes together to alleviate stress-induced cellular damage. To evaluate whether the ectopic transgenic overexpression of EcNAC1 confers any stress-tolerance, we expressed the full-length EcNAC1 cDNA cloned under the control of CaMV35S promoter as well as 4xABRE stress-inducible promoter in a heterologous plant system, tobacco a relatively susceptible species. 4xABRE is a synthetic promoter containing four copies of ABRE elements and is known to be induced by dehydration, high salinity, low temperature and ABA [54]. Both constitutive and stress-inducible constructs were transformed to tobacco following A. tumefaciens-mediated transfor- mation. The putative transgenic tobacco plants were regenerated and selected on hygromycin containing medium. These plants were further screened for transgene integration by PCR (Figure S2, S3 & Table S1E). The PCR positive ten independent T0 plants (five each from constitutively expressing EcNAC1 transgenic lines - S1, S3, S4, S7 and S8; and stress-inducible EcNAC1 transgenic lines - A1, A2, A4, A5 and A6) were selfed individually and seeds were harvested for further analysis. Approximately 70–80% of seeds from these selected putative T0 transgenic lines germinated and developed into T1 seedlings on MS medium supplemented with hygromycin (30 mg/l) suggesting a 3:1 ratio of Mendelian segregation. The RT-PCR analysis of stressed leaf tissue from transgenic plants confirmed the transgene expression (Figure S4A). Expression of the gene was not observed in wild-type tobacco plants, as there was no homology found in tobacco for the selected primers used for RT-PCR analysis. The relative density of the bands was determined to show the increase in transcript levels over wild-type (Figure S4B). Transgenic overexpression of EcNAC1 under the constitutive CaMV35S promoter as well as 4xABRE promoter did not show any phenotypic abnormities either during stress or non-stress growth conditions. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40397