final extension at 72uC for 5 min was performed. EcNAC1 was amplified with the primers specific to the 284 bp 39 terminal region of its open reading frame (ORF) (Table S1B). To study the expression of target genes of EcNAC1, RT-PCR was performed with 14 closest tobacco homologs of known SNAC1 target genes [17] (Table S1C & Table S2). House-keeping gene, either Actin or Elongation factor 1a, was used in all expression studies and treated as control. The RT-PCR products were loaded on a 1% agarose gel and bands were analysed using ImageJ software version 1.34 s (National Institutes of Health) to calculate the changes in relative density of bands keeping wild-type as one. To study the expression pattern of EcNAC1 under water-deficit stress and salinity, quantitative RT-PCR (qRT-PCR; Opticon 2, MJ research, USA) was performed using the fluorescent dye SYBR-Green (DyNAmo SYBR-Green qRT-PCR kit, Finnzymes, Finland) for 30 cycles following the manufacturer’s protocol. The relative expression levels under a given stress condition compared to its corresponding control condition was calculated using Relative Expression Software Tool (REST) [49]. The data was normalized to expression levels of Actin. Similarly, the expression pattern of NtNAC was studied under dehydration and salinity using specific primers (Table S1D) and the data was normalized to expression levels of Elongation factor 1a. Abiotic Stress-tolerance of EcNAC1 Transgenic Tobacco Plants Abiotic stress treatment at seedling stage. Transgenic tobacco (T1) plants expressing EcNAC1 (constitutive transgenic lines - S1, S3, S4, S7 and S8; and stress-inducible transgenic lines - A1, A2, A4, A5 and A6) were evaluated under various stress conditions. For growth analysis, 15-day-old seedlings selected on 30 mg/l hygromycin were transferred to Petri-plates (9 cm diameter) containing MS medium supplemented with 5% PEG- 10000 (w/v), and 5 mM MV, separately, and fresh weight was recorded. For examining root growth, 15-day-old seedlings were grown in Petri-plates (12 cm diameter) containing MS +200 mM NaCl. For long-term stress treatment, 15-day-old seedlings were transferred to culture bottles containing MS medium supplemented with various concentrations of mannitol (100, 200 and 300 mM), and NaCl (100, 200 and 300 mM) and performance was measured after 30-days. Imposition of water-deficit. For water-deficit stress treat- ment under greenhouse conditions, 15-day-old seedlings were transferred to pots filled with potting mixture of known weight. After seedling establishment, soil water status was monitored by gravimetric approach [37], [38] and soil FC was gradually brought down to 35% and maintained for 10-days. Relative humidity in the greenhouse ranged from 75–80% and temperature was maintained around 29–30uC during day time and 24–26uC during the night period. Stress effect was assessed at the end of stress period by quantifying ROS and malondialdehyde (MDA) content in leaf tissue. After re-watering the recovery response was studied after 15-days by measuring total biomass accumulated. Determination of superoxide radical (O.22). Leaf discs were incubated in 1 ml of K-phosphate buffer (20 mM, pH 6.0) containing 500 mM XTT (Polysciences Europe, Eppelheim, Germany) in darkness at 25uC on a shaker. The increase in absorbance (A470) in the incubation medium was measured using spectrophotometer [50]. Determination of hydrogen peroxide (H2O2). Leaf discs were pre-incubated for 30 min in 3 ml of K-phosphate buffer (20 mM, pH 6.0) to remove pre-formed H2O2 and were then incubated in 3 ml of the same buffer containing 5 mM scopoletin [7- hydroxy-6-methoxy-2H-1-benzopyran-2-one (Sigma Aldrich, EcNAC1 Confers Abiotic Stress-Tolerance in Tobacco St. Louis, MO USA)] [51] and 3 mg/ml horseradish peroxidase in darkness at 25uC on a shaker. The decrease in fluorescence (excitation: 346 nm, emission: 455 nm) in the incubation medium was measured using reagent blanks as reference. Determination of hydroxyl radical (.OH). Leaf discs were incubated in 1.5 ml of buffer containing 20 mM 2-deoxy-D-ribose (Sigma Aldrich, St. Louis, MO USA). The formation of the breakdown product MDA was determined by mixing 0.5 ml of centrifuged incubation medium with 0.5 ml of 2-thiobarbituric acid (10 g/l in 50 mM NaOH) and 0.5 ml of trichloroacetic acid (28 g/l). After heating in boiling water for exactly 10 min, cooling in tap water, and clarifying by centrifugation, the reaction product was measured fluorometrically (excitation: 532 nm, emission: 553 nm) against reagent blanks [52]. Estimation of MDA content. MDA content was quantified by thiobarbituric acid reactive substances assay [53] (Heath and Packer 1968). About 0.5–1 g of tissue was homogenized in 5 ml of 5% (w/v) trichloroacetic acid and the homogenate was centrifuged at 12000 g for 15 min at room temperature. The supernatant was mixed with an equal volume of thiobarbituric acid [0.5% in 20% (w/v) trichloroacetic acid], and the mixture was boiled for 25 min at 100uC, followed by centrifugation for 5 min at 7500 g to clarify the solution. Absorbance of the supernatant was measured at 532 nm and corrected for non-specific turbidity by subtracting the A600. MDA equivalents were calculated by the extinction coefficient of 155 M21 cm21. Values of MDA contents were taken from measurements of three independent samples. Results Isolation of Water-deficit Induced Finger Millet NAC gene, EcNAC1 To monitor the changes in expression profiles of transcripts in response to dehydration, finger millet plants were exposed to different levels of gradual water-deficit stress and maintained at different FC viz., 100%, 80%, 60% and 35% (Figure S1). cDNA library was constructed from the leaf tissue following subtraction between the stress-exposed pooled samples and control-samples. The mRNA populations from stress-exposed seedlings were mixed with approximately five-fold excess of complimentary first-strand cDNA from corresponding control seedlings. Double-stranded cDNA synthesized from the subtracted poly(A+) RNA was used in generating the library. The cDNA inserts of selected recombinant plaques were successfully amplified by PCR using universal M13 primers and used for sequencing. One of the cDNA clones showed maximum homology to a gene encoding a putative NAC domain containing protein. The identified cDNA was approximately 1 kb with an ORF that could encode a protein of 246 amino acids without the starting methionine suggesting that the EcNAC1 cDNA clone was truncated in the 59 region. The 39 end seemed to be intact with 215 bp 39 un-translated region followed by a poly(A+) tail. In order to identify and isolate the missing 59 end of EcNAC1 cDNA, we performed 59 RACE-PCR with gene specific primer (CAGTCATCCAACCTTAGCGATC) in the antisense orienta- tion using RLM-RACE kit and amplified a fragment of 690 bp, which was cloned and sequenced. The RACE product shared an expected 145 bp homology with the partial EcNAC1 cDNA clone as the gene specific primer used in the 59 RACE was designed to amplify a 145 bp overlap at the 59 end of the partial cDNA clone. This observation confirmed that the fragment of 690 bp was indeed a part of EcNAC1 cDNA. As revealed, total size of EcNAC1 cDNA clone was 1547 bp (GenBank accession EU439937) with an ORF of 1110 bp, encoding 369 amino acid polypeptide flanked by 186 and 215 bp 59 and 39 un-translated regions, respectively. PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40397