Molecular Biology Reports

, Volume 41, Issue 3, pp 1669–1682 | Cite as

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


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 residues. 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 functional 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.


Drought Leptochloa fusca Na+/H+ antiporter Salt tolerance Tobacco 



The authors are also very thankful to Higher Education Commission (HEC) of Pakistan for providing PhD scholarship to Mr. Muhammad Rauf for conducting his PhD studies at NIBGE and his six months fellowship at Agricultural Biotechnology Laboratories University of Connecticut, USA.


  1. 1.
    Wang W, Vinocur B, Shoseyov O, Altman A Biotechnology of plant osmotic stress tolerance physiological and molecular considerations. In: IV international symposium on in vitro culture and horticultural breeding 560 Science publications, Adelaide, pp 285–292Google Scholar
  2. 2.
    Sayar R, Khemira H, Bensalem M, Kameli A (2005) Drought tolerance evaluation tests for durum wheat (Triticum durum Desf.), Poster in international conference on integrated approaches to sustain and Improve plant production under drought stress. Inter Drought-II:P3-61Google Scholar
  3. 3.
    Sayar R, Khemira H, Kameli A, Mosbahi M (2008) Physiological tests as predictive appreciation for drought tolerance in durum wheat (Triticum durum Desf.). Agron Res 6(1):79–90Google Scholar
  4. 4.
    Brini F, Hanin M, Mezghani I, Berkowitz GA, Masmoudi K (2007) Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt-and drought-stress tolerance in Arabidopsis thaliana plants. J Exp Bot 58(2):301–308PubMedCrossRefGoogle Scholar
  5. 5.
    Wang J, Zuo K, Wu W, Song J, Sun X, Lin J, Li X, Tang K (2003) Molecular cloning and characterization of a new Na+/H+ antiporter gene from Brassica napus. Mt DNA 14(5):351–358Google Scholar
  6. 6.
    Taiz L (1992) The plant vacuole. J Exp Biol 172(1):113–122PubMedGoogle Scholar
  7. 7.
    Rodríguez-Rosales MP, Gálvez FJ, Huertas R, Aranda MN, Baghour M, Cagnac O, Venema K (2009) Plant NHX cation/proton antiporters. Plant Sign Behav 4(4):265–276CrossRefGoogle Scholar
  8. 8.
    Zhu JK (2003) Regulation of ion homeostasis under salt stress. Cur Opin Plant Biol 6(5):441–445CrossRefGoogle Scholar
  9. 9.
    Hamada A, Shono M, Xia T, Ohta M, Hayashi Y, Tanaka A, Hayakawa T (2001) Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini. Plant Mol Biol 46(1):35–42PubMedCrossRefGoogle Scholar
  10. 10.
    Li J, Jiang G, Huang P, Ma J, Zhang F (2007) Overexpression of the Na+/H+ antiporter gene from Suaeda salsa confers cold and salt tolerance to transgenic Arabidopsis thaliana. Plant Cell Tiss Org Cult 90(1):41–48CrossRefGoogle Scholar
  11. 11.
    Guan B, Hu Y, Zeng Y, Wang Y, Zhang F (2011) Molecular characterization and functional analysis of a vacuolar Na+/H+ antiporter gene (HcNHX1) from Halostachys caspica. Mol Biol Rep 38(3):1889–1899PubMedCrossRefGoogle Scholar
  12. 12.
    Jha A, Joshi M, Yadav NS, Agarwal PK, Jha B (2011) Cloning and characterization of the Salicorni abrachiata Na+/H+ antiporter gene SbNHX1 and its expression by abiotic stress. Mol Biol Rep 38(3):1965–1973PubMedCrossRefGoogle Scholar
  13. 13.
    Liu L, Zeng Y, Pan X, Zhang F (2012) Isolation, molecular characterization, and functional analysis of the vacuolar Na+/H+ antiporter genes from the halophyte Karelinia caspica. Mol Biol Rep 39(6):1–10PubMedCrossRefGoogle Scholar
  14. 14.
    Yao M, Zeng Y, Liu L, Huang Y, Zhao E, Zhang F (2012) Overexpression of the halophyte Kalidium foliatum H+-pyrophosphatase gene confers salt and drought tolerance in Arabidopsis thaliana. Mol Biol Rep 39(8):7989–7996PubMedCrossRefGoogle Scholar
  15. 15.
    Liu Q, Zhang Q, Burton RA, Shirley NJ, Atwell BJ (2010) Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice. Plant Mol Biol 72(1):47–60PubMedCrossRefGoogle Scholar
  16. 16.
    Malik KA, Aslam Z, Naqvi M (1986) Kallar grass: a plant for saline land. Nuclear Institute for Agriculture and Biology Faisalabad, PakistanGoogle Scholar
  17. 17.
    Abdullah M, Akram M, Khan A, Qureshi R (1990) Internal water resources management by plants under various root environment stresses with special reference to kallar grass Leptochloa fusca. In: Davidsont NJ (ed) In: National seminar on water resource development and its management in arid areas. ESA Bulletin, columbia, pp 6–8Google Scholar
  18. 18.
    Panahi B, Ahmadi FS, Mehrjerdi MZ, Moshtaghi N (2013) Molecular cloning and the expression of the Na+/H+ antiporter in the monocot halophyte Leptochloa fusca (L.) Kunth. NJAS-Wageningen J Life Sci 65:87–93CrossRefGoogle Scholar
  19. 19.
    Spyropoulos IC, Liakopoulos TD, Bagos PG, Hamodrakas SJ (2004) TMRPres2D: high quality visual representation of transmembrane protein models. Bioinformatics 20(17):3258–3260PubMedCrossRefGoogle Scholar
  20. 20.
    Hofmann K (1993) TMbase-A database of membrane spanning protein segments. Biol Chem Hoppe-Seyler 374:166Google Scholar
  21. 21.
    Lambert C, Léonard N, De Bolle X, Depiereux E (2002) ESyPred3D: prediction of proteins 3D structures. Bioinformatics 18(9):1250–1256PubMedCrossRefGoogle Scholar
  22. 22.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Computat Chem 25(13):1605–1612CrossRefGoogle Scholar
  23. 23.
    Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E, Pupko T, Ben-Tal N (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucl Acids Res 33(2):299–302CrossRefGoogle Scholar
  24. 24.
    Schonfeld MA, Johnson RC, Carver BF, Mornhinweg DW (1988) Water relations in winter wheat as drought resistance indicators. Crop Sci 28(3):526–531CrossRefGoogle Scholar
  25. 25.
    Premachandra G, Saneoka H, Ogata S (1990) Cell membrane stability, an indicator of drought tolerance as affected by applied nitrogen in soybean. J Agr Sci 115:63–66CrossRefGoogle Scholar
  26. 26.
    Sairam R (1994) Effect of moisture-stress on physiological activities of two contrasting wheat genotypes. Ind Exp Biol 32:594Google Scholar
  27. 27.
    Steel R, Torrie J, Dickey D (1997) Principles and procedures of statistics: a biometrical approach. WCB/McGraw-Hill, BostonGoogle Scholar
  28. 28.
    Khan M, Duke N (2001) Halophytes—a resource for the future. Wetlands Ecol Manage 9:455–456CrossRefGoogle Scholar
  29. 29.
    Khan MS (2011) Role of sodium and hydrogen (Na+/H+) antiporters in salt tolerance of plants: present and future challenges. Afr J Biotechnol 10(63):13693–13704Google Scholar
  30. 30.
    Lan T, Duan Y, Wang B, Zhou Y, Wu W (2011) Molecular cloning and functional characterization of a Na+/H+ antiporter gene from halophyte Spartina anglica. Turk J Agric For 35:535–543Google Scholar
  31. 31.
    Ma XL, Zhang Q, Shi HZ, Zhu JK, Zhao YX, Ma CL, Zhang H (2004) Molecular cloning and different expression of a vacuolar Na+/H+ antiporter gene in Suaeda salsa under salt stress. Biol Plant 48(2):219–225CrossRefGoogle Scholar
  32. 32.
    Wu C-A, Yang G-D, Meng Q-W, Zheng C–C (2004) The cotton GhNHX1 gene encoding a novel putative tonoplast Na+/H+ antiporter plays an important role in salt stress. Plant Cell Physiol 45(5):600–607PubMedCrossRefGoogle Scholar
  33. 33.
    Wu C, Gao X, Kong X, Zhao Y, Zhang H (2009) Molecular cloning and functional analysis of a Na+/H+ antiporter gene ThNHX1 from a halophytic plant Thellungiella halophila. Plant Mol Biol Rep 27(1):1–12CrossRefGoogle Scholar
  34. 34.
    Wu GQ, Xi JJ, Wang Q, Bao AK, Ma Q, Zhang JL, Wang SM (2011) The ZxNHX gene encoding tonoplast Na+/H+ antiporter from the xerophyte Zygophyllum xanthoxylum plays important roles in response to salt and drought. J Plant Physiol 168(8):758–767PubMedCrossRefGoogle Scholar
  35. 35.
    Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci 96(4):1480–1485PubMedCrossRefGoogle Scholar
  36. 36.
    Darley CP, Van Wuytswinkel O, Van Der Woude K, Mager W, De Boer A (2000) Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers. Biochem J 351(Pt 1):241PubMedCrossRefGoogle Scholar
  37. 37.
    Kagami T, Suzuki M (2005) Molecular and functional analysis of a vacuolar Na+/H+ antiporter gene of Rosa hybrida. Gen Genet Sys 80(2):121–128CrossRefGoogle Scholar
  38. 38.
    Sato Y, Sakaguchi M (2005) Topogenic properties of transmembrane segments of Arabidopsis thaliana NHX1 reveal a common topology model of the Na+/H+ exchanger family. J Biochem 138(4):425–431PubMedCrossRefGoogle Scholar
  39. 39.
    Ye CY, Zhang HC, Chen JH, Xia XL, Yin WL (2009) Molecular characterization of putative vacuolar NHX1 type Na+/H+ exchanger genes from the salt resistant tree Populus euphratica. Physiol Plant 137(2):166–174PubMedCrossRefGoogle Scholar
  40. 40.
    Xing J, Wang B, Jia K, Wan S, Meng J, Guo F, Li X (2011) Isolation of Arachis hypogaea Na+/H+ antiporter and its expression analysis under salt stress. Afr J Biotechnol 10(65):14302–14310Google Scholar
  41. 41.
    Visarada K, Saikishore N, Kuriakose S, Rani VS, Royer M, Rao S, Seetharama N (2008) A simple model for selection and rapid advancement of transgenic progeny in sorghum. Plant Biotechnol Rep 2(1):47–58CrossRefGoogle Scholar
  42. 42.
    Vengadesan G, Anand RP, Selvaraj N, Perl-Treves R, Ganapathi A (2005) Transfer and expression of nptII and bar genes in cucumber (Cucumis satavus L.). In Vitro Cell Develop Biol Plant 41(1):17–21CrossRefGoogle Scholar
  43. 43.
    Soliman MH, Omar HS, El-Awady MA, Al-Assal S, El-Din AAYG (2009) Transformation and expression of Na+/H+ antiporter vacuolar (AtNHX1) gene in tobacco plants under salt stress. Arab J Biotechnol 12:99–108Google Scholar
  44. 44.
    Wei Q, Hu P, Kuai B (2011) Ectopic expression of an Ammopiptanthus mongolicus H+-pyrophosphatase gene enhances drought and salt tolerance in Arabidopsis. Plant Cell Tiss Org Cult 110(3):359–369CrossRefGoogle Scholar
  45. 45.
    He C, Yan J, Shen G, Fu L, Holaday AS, Auld D, Blumwald E, Zhang H (2005) Expression of an Arabidopsis vacuolar sodium/proton antiporter gene in cotton improves photosynthetic performance under salt conditions and increases fiber yield in the field. Plant Cell Physiol 46(11):1848–1854PubMedCrossRefGoogle Scholar
  46. 46.
    Banjara M, Zhu L, Shen G, Payton P, Zhang H (2012) Expression of an Arabidopsis sodium/proton antiporter gene (AtNHX1) in peanut to improve salt tolerance. Plant Biotechnol Rep 6(1):59–67CrossRefGoogle Scholar
  47. 47.
    Jiang C, Zheng Q, Liu Z, Xu W, Liu L, Zhao G, Long X (2012) Overexpression of Arabidopsis thaliana Na+/H+ antiporter gene enhanced salt resistance in transgenic poplar (Populus euramericana ‘Neva’). Trees 26(3):685–694CrossRefGoogle Scholar
  48. 48.
    Bassil E, Tajima H, Liang Y, Ohto M, Ushijima KRN, Esumi T, Coku A, Belmonte M, Blumwald E (2011) The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction. Plant Cell Environ 23(9):3482–3497CrossRefGoogle Scholar
  49. 49.
    Xu K, Hong P, Luo L, Xia T (2009) Overexpression of AtNHX1, a vacuolar Na+/H+ antiporter from Arabidopsis thaliana, in Petunia hybrida enhances salt and drought tolerance. J Plant Biol 52(5):453–461CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Muhammad Rauf
    • 1
    • 2
  • Khurram Shahzad
    • 1
    • 2
  • Rashid Ali
    • 3
  • Moddassir Ahmad
    • 1
  • Imran Habib
    • 1
  • Shahid Mansoor
    • 1
  • Gerald A. Berkowitz
    • 3
  • Nasir A. Saeed
    • 1
  1. 1.Agricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  2. 2.Pakistan Institute for Engineering and Applied Sciences (PIEAS)IslamabadPakistan
  3. 3.Agricultural Biotechnology Laboratories, Department of Plant ScienceUniversity of ConnecticutMansfieldUSA

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