Expression of a Finger Millet Transcription Factor, EcNAC1, in Tobacco Confers Abiotic Stress-Tolerance Venkategowda Ramegowda1,2¤, Muthappa Senthil-Kumar2, Karaba N. Nataraja1, Malireddy K. Reddy3, Kirankumar S. Mysore2, Makarla Udayakumar1* 1 Department of Crop Physiology, University of Agricultural Sciences, Bangalore, Karnataka, India, 2 Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, United States of America, 3 International Centre for Genetic Engineering and Biotechnology, New Delhi, Delhi, India Abstract NAC (NAM, ATAF1-2, and CUC2) proteins constitute one of the largest families of plant-specific transcription factors and have been shown to be involved in diverse plant processes including plant growth, development, and stress-tolerance. In this study, a stress-responsive NAC gene, EcNAC1, was isolated from the subtracted stress cDNA library generated from a drought adapted crop, finger millet, and characterized for its role in stress-tolerance. The expression analysis showed that EcNAC1 was highly induced during water-deficit and salt stress. EcNAC1 shares high amino acid similarity with rice genes that have been phylogenetically classified into stress-related NAC genes. Our results demonstrated that tobacco transgenic plants expressing EcNAC1 exhibit tolerance to various abiotic stresses like simulated osmotic stress, by polyethylene glycol (PEG) and mannitol, and salinity stress. The transgenic plants also showed enhanced tolerance to methyl-viologen (MV) induced oxidative stress. Reduced levels of reactive oxygen species (ROS) and ROS-induced damage were noticed in pot grown transgenic lines under water-deficit and natural high light conditions. Root growth under stress and recovery growth after stress alleviation was more in transgenic plants. Many stress-responsive genes were found to be up-regulated in transgenic lines expressing EcNAC1. Our results suggest that EcNAC1 overexpression confers tolerance against abiotic stress in susceptible species, tobacco. Citation: Ramegowda V, Senthil-Kumar M, Nataraja KN, Reddy MK, Mysore KS, et al. (2012) Expression of a Finger Millet Transcription Factor, EcNAC1, in Tobacco Confers Abiotic Stress-Tolerance. PLoS ONE 7(7): e40397. doi:10.1371/journal.pone.0040397 Editor: Keqiang Wu, National Taiwan University, Taiwan Received February 17, 2012; Accepted June 6, 2012; Published July 11, 2012 Copyright: 2012 Ramegowda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by grants from the Department of Biotechnology (Transcription factor project grant number F.No.BT/PR/4946/Agr/16/ 421/2004, Centre of excellence grant number F.No. BT/01/COE/05/03), Government of India and Indian Council of Agricultural Research (Niche area of excellence grant number F.No.10-(6)/2005 EP&D), India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: udayakumar_m@yahoo.com ¤ Current address: Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, Arkansas, United States of America Introduction Plants maintain cellular homeostasis under abiotic stress by adjusting their metabolic systems. The early events of plant adaptation to the environmental stresses involve stress-signal perception and transduction leading to the expression of stress- responsive genes and activation of various physiological and metabolic responses [1], [2], [3]. The products of stress-responsive genes can either directly protect cells against abiotic stress or regulate the expression of other genes. The stress regulatory molecules, including mitogen-activated protein kinases, histidine kinases, Ca2+-dependent protein kinases, as well as transcription factors are well characterized [4], [5]. The expression of stress- responsive genes is largely governed by specific transcription factors. Stress-responsive transcription factors predominantly interact with promoter elements of stress genes often resulting in expression of many functional genes. This has been elegantly shown for DREB and bZIP transcription factors which recognize drought responsible element (DRE) and the abscisic acid responsive element (ABRE), respectively, and activate an array of stress genes [6]. Several families of transcription factors, including ERF/AP2, HSF, bZIP, MYB, MYC, NAC, WRKY, and Zinc-finger, have been well characterized for their regulatory roles in eliciting plant stress-responses [5], [7–10]. NAC family proteins are plant-specific transcription factors that are characterized by a conserved DNA-binding NAC domain located in the N-terminal region and a variable C-terminal domain [11]. Although the genomes of rice and Arabidopsis were predicted to contain 140 and 75 NAC genes, respectively, [12], [13], only a few members of this large family have been characterized and most of them were reported to be involved in plant growth, development, and hormone signaling [14]. Recently, a few NAC proteins have been described as mediators of biotic and abiotic stress signaling. Expression analysis showed that NAC genes, such as ANAC019, ANAC055, and ANAC072 from Arabidopsis [15], and BnNAC from Brassica napus [16], were induced by various environmental stresses. Furthermore, overexpression of a few NAC genes has been shown to improve abiotic stress-tolerance in Arabidopsis and rice through activation of several stress genes [17– 21]. NAC genes seem to have non-redundant functions though they are stress-induced [17], [18], [22]. In addition, the function of same transcription factor could behave differently between the species. Surprisingly, overexpression of seven stress-induced NACs did not give tolerant phenotype in Arabidopsis [23]. However, PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40397