Cloning and characterization of Na + /H + antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance Muhammad Rauf • Khurram Shahzad • Rashid Ali • Moddassir Ahmad • Imran Habib • Shahid Mansoor • Gerald A. Berkowitz • Nasir A. Saeed Received: 27 June 2013 / Accepted: 30 December 2013 Ó Springer Science+Business Media Dordrecht 2014 Abstract Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na ? / H ? antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid resi- dues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved func- tional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L -1 Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops. Keywords Drought  Leptochloa fusca  Na ? /H ? antiporter  Salt tolerance  Tobacco Introduction Plants constantly face threat from diverse biotic and abiotic factors. Among these, salinity and drought are emerging threats that affect various critical physiological, biochem- ical and molecular processes require for plant growth and productivity [1]. Several physiological characters have been identified and reported to contribute in plant growth under salt and osmotic stress [2, 3]. Salt stress affects the plant growth by impairing metabolism and photosynthetic activity by causing an osmotic imbalance as a result of high concentration of Na ? in the cytosol. Plant can adapt under salt stress through osmotic adjustment which is character- ized by cellular ion homeostasis, intercellular uptake of Na ? and Cl - and vacuolar sequestration of toxic ions from cytosol [4]. Molecular mechanisms involved in abiotic stress tolerance are characterized by activation and regu- lation of stress related genes. Such genes are reported to be involved in signaling, transcriptional control, membrane and protein protection, ion homeostasis and scavenging of toxic ions and free radicals [5]. Vacuolar compartmental- ization of toxic ions from cytoplasm facilitates functions of specific ions ratios as signal determinants and is largely achieved by H ? -ATPase, located on plasma membrane or ATPase and H ? -pyrophosphatase, located on tonoplast. These pumps create a H ? electrochemical potential across membranes and provide driving force for secondary active transport by various transporters [6]. Two types of M. Rauf  K. Shahzad  M. Ahmad  I. Habib  S. Mansoor  N. A. Saeed (&) Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan e-mail: nasaeedpk@yahoo.com M. Rauf  K. Shahzad Pakistan Institute for Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan R. Ali  G. A. Berkowitz Agricultural Biotechnology Laboratories, Department of Plant Science, University of Connecticut, Mansfield, CT, USA 123 Mol Biol Rep DOI 10.1007/s11033-013-3015-3