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Cloning and characterization of a novel vacuolar Na+/H+ antiporter gene (VuNHX1) from drought hardy legume, cowpea for salt tolerance

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Abstract

Soil salinity is a major abiotic stress that seriously affects crop productivity worldwide. One of the mechanisms that allow plants to withstand salt stress is vacuolar sequestration of Na+, through a Na+/H+ antiporter. We isolated a new vacuolar Na+/H+ antiporter gene (VuNHX1) from a drought hardy grain legume, cowpea (Vigna unguiculata L.). The cDNA is 1,981 bp, with an open reading frame of 1,629 bp encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. VuNHX1 displays a conserved amiloride binding domain (84LFFIYLLPPI93) in third transmembrane (TM3) region. Phylogenetic and bioinformatic analysis indicated VuNHX1 belonging to Class-I clade of plant NHX exchangers with high similarity with legume Na+/H+ antiporters. To assess its role in Na+ exchange, we performed complementation studies using the salt sensitive yeast mutant strain AXT3. The results showed that VuNHX1 complemented for the loss of yeast NHX1 under NaCl, KCl and LiCl stress in the salt sensitive phenotype of the yeast strain AXT3. The expression profiles revealed significant induction of VuNHX1 in cowpea seedlings under salt, cold and dehydration stress. Both expression analysis and ion estimation under salt stress indicated the VuNHX1 expression preferentially in roots than in leaves. Overexpression of VuNHX1 in transgenic Arabidopsis conferred enhanced salt tolerance in transgenic Arabidopsis lines while the wild type plants exhibited growth retardation. This study shows that VuNHX1 is a potential gene for salt tolerance, and can be used in future for developing cisgenic salt tolerant cowpea and transgenic salt tolerant crops.

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References

  • Ali R, Brett CL, Mukherjee S, Rao R (2004) Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast. J Biol Chem 279:4498–4506

    Article  CAS  PubMed  Google Scholar 

  • Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285:1256–1258

    Article  CAS  PubMed  Google Scholar 

  • Apse MP, Sottosanto JB, Blumwald E (2003) Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter. Plant J 36:229–239

    Article  CAS  PubMed  Google Scholar 

  • Baltierra Q, Castillo M, Gamboa MC et al (2012) Molecular characterization of a novel Na+/H+ antiporter cDNA from Eucalyptus globules. Biochem Biophys Res Commun 430:535–540

    Article  PubMed  Google Scholar 

  • Bao-Yan AN, Yan L, Jia-Rui LI et al (2008) Expression of a vacuolar Na+/H+ Antiporter gene of alfalfa enhances salinity tolerance in transgenic Arabidopsis. Acta Agron Sin 34:557–564

    Google Scholar 

  • Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207

    Article  CAS  Google Scholar 

  • Brett CL, Donowitz M, Rao R (2005a) The evolutionary origins of eukaryotic sodium/proton exchangers. Am J Physiol Cell Physiol 288:223–239

    Article  Google Scholar 

  • Brett CL, Tukaye DN, Mukherjee S, Rao R (2005b) The yeast endosomal Na+(K+)/H+ exchanger Nhx1 regulates cellular ph to control vesicle trafficking. Mol Biol Cell 16:1396–1405

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Brini F, Gaxiola RA, Berkowitz GA, Masmoudi K (2005) Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump. Plant Physiol Biochem 43:347–354

    Article  CAS  PubMed  Google Scholar 

  • Chauhan S, Forstoefel N, Ran Y, Quigley F, Nelson DE, Bohnert HJ (2000) Na+/myo-inositol symporters and Na+/H+-antiport in Mesembryanthemum crsytallinum. Plant J 24:511–522

    Article  CAS  PubMed  Google Scholar 

  • Chen LH, Zhang B, Xu ZQ (2008) Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum). Transgenic Res 17:121–132

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Darley CP, van Wuytswinkel OCM, van der Woude K, Mager WH, de Boer AH (2000) Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers. Biochem J 351:241–249

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Duzdemir O, Unlukara A, Kurunc A (2009) Response of cowpea (Vigna unguiculata) to salinity and irrigation regimes. N Z J Crop Hort Sci 37:271–280

    Article  CAS  Google Scholar 

  • Fukuda A, Nakamura A, Tanaka Y (1999) Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochim Biophys Acta 1446:149–155

    Article  CAS  PubMed  Google Scholar 

  • Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A, Hirochika H, Tanaka Y (2004) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice. Plant Cell Physiol 45:146–159

    Article  CAS  PubMed  Google Scholar 

  • Gattiker A, Gasteiger E, Bairoch A (2002) ScanPROSITE: a reference implementation of a PROSITE scanning tool. Appl Bioinformatics 1:107–108

    CAS  PubMed  Google Scholar 

  • Geourjon C, Deleage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11:681–684

    CAS  PubMed  Google Scholar 

  • Gietz D, St. Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gouiaa S, Khoudi H, Leidi EO et al (2012) Expression of wheat Na+/H+ antiporter TNHXS1 and H+-pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance. Plant Mol Biol 79:137–155

    Article  CAS  PubMed  Google Scholar 

  • Hall AE (2012) Phenotyping cowpeas for adaptation to drought. Front Physiol 3:00155

    Article  Google Scholar 

  • 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 46:35–42

    Article  CAS  PubMed  Google Scholar 

  • Hanana M, Cagnac O, Yamaguchi T, Hamdi S, Ghorbel A, Blumwald E (2007) A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening. Plant Cell Physiol 48:804–811

    Article  CAS  PubMed  Google Scholar 

  • Hasegawa PM (2013) Sodium (Na+) homeostasis and salt tolerance of plants. Environ Exp Bot 92:19–31

    Article  CAS  Google Scholar 

  • Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Bio 51:463–499

    Article  CAS  Google Scholar 

  • He CX, Yan JQ, Shen GX, Fu LH, 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 46:1848–1854

    Article  CAS  PubMed  Google Scholar 

  • Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetic and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

    Article  CAS  PubMed  Google Scholar 

  • Hernandez A, Jiang X, Cubero B, Nieto PM, Bressan RA, Hasegawa PM, Pardo JM (2009) Mutants of the Arabidopsis thaliana cation/H+ antiporter AtNHX1 conferring increased salt tolerance in yeast: the endosome/prevacuolar compartment is a target for salt toxicity. J Biol Chem 284:14276–14285

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hofmann K, Stoffel W (1993) A database of membrane spanning proteins segments. Biol Chem 374:166

    Google Scholar 

  • Jha A, Joshi M, Yadav NS, Agarwal PK, Jha B (2011) Cloning and characterization of the Salicornia brachiata Na+/H+ antiporter gene SbNHX1 and its expression by abiotic stress. Mol Biol Rep 38:1965–1973

    Article  CAS  PubMed  Google Scholar 

  • Kagami T, Suzuki M (2005) Molecular and functional analysis of a vacuolar Na+/H+ antiporter gene of Rosa hybrida. Genes Genet Syst 80:121–128

    Article  CAS  PubMed  Google Scholar 

  • Kinclova-Zimmermannova O, Flegelova H, Sychrova H (2004) Rice Na+/H+-antiporter Nhx1 partially complements the alkali-metal-cation sensitivity of yeast strains lacking three sodium transporters. Folia Microbiol 49:519–525

    Article  CAS  Google Scholar 

  • Kronzucker HJ, Britto DT (2011) Sodium transport in plants: a critical review. New Phytol 189:54–81

    Article  CAS  PubMed  Google Scholar 

  • Lee BH, Zhu JK (2010) Phenotypic analysis of Arabidopsis mutants: germination rate under salt/hormone-induced stress. Cold Spring Harb Protoc 2010:pdb-prot4969

  • Li WYF, Wong FL, Tsai SN, Phang TH et al (2006) Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. Plant Cell Environ 29:1122–1137

    Article  CAS  PubMed  Google Scholar 

  • Lichtenthaler HK (1987) Chlorophyll fluorescence signatures of leaves during the autumnal chlorophyll breakdown. J Plant Physiol 131:101–110

    Article  CAS  Google Scholar 

  • 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 Suada salsa under salt stress. Biol Plant 48:219–225

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murillo-Amador B, Troyo-Dieguez E, Garcia-Hernandez JL et al (2006) Effect of NaCl salinity in the genotypic variation of cowpea (Vigna unguiculata) during early vegetative growth. Sci Hortic 108:423–431

    Article  CAS  Google Scholar 

  • Ohta M, Hayashi Y, Nakashima A, Hamada A, Tanaka A, Nakamura T, Hayakawa T (2002) Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice. FEBS Lett 532:279–282

    Article  CAS  PubMed  Google Scholar 

  • Qiao WH, Zhao XY, Li W et al (2007) Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants. Plant Cell Rep 26:1663–1672

    Article  CAS  PubMed  Google Scholar 

  • Qingxia Z, Xuefeng X, Wang Y, Tianzhong L, Jin K, Zhenhai H (2009) Isolation and preliminary function analysis of a Na+/H+ antiporter gene from Malus zumi. Afr J Biotechnol 8:4774–4781

    Google Scholar 

  • Quintero FJ, Blatt MR, Pardo JM (2000) Functional conservation between yeast and plant endosomal Na+/H+ antiporters. FEBS Lett 471:224–228

    Article  CAS  PubMed  Google Scholar 

  • Rajagopal D, Agarwal P, Tyagi W et al (2007) Pennisetum glaucum Na+/H+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea. Mol Breed 19:137–151

    Article  CAS  Google Scholar 

  • Reguera M, Bassil E, Blumwald E (2014) Intracellular NHX-type cation/H+ antiporters in plants. Mol Plant 7:261–263

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez-Navarro A, Ramos J (1984) Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159:940–945

    CAS  PubMed Central  PubMed  Google Scholar 

  • Shi H, Zhu JK (2002) Regulation of expression of the vacuolar Na+/H+ antiporter gene AtNHX1 by salt stress and abscisic acid. Plant Mol Biol 50:543–550

    Article  CAS  PubMed  Google Scholar 

  • Singh BB (2005) Cowpea [Vigna unguiculata (L.) Walp. In: Singh RJ, Jauhar PP (eds) Genetic resources, chromosomal engineering and crop improvement, vol 1. CRC Press, Boca Raton, pp 117–162

    Chapter  Google Scholar 

  • Sottosanto JB, Gelli A, Blumwald E (2004) DNA array analyses of Arabidopsis thaliana lacking a vacuolar Na+/H+ antiporter: impact of AtNHX1 on gene expression. Plant J 40:752–771

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  PubMed  Google Scholar 

  • Tang R, Li C, Xu K et al (2010) Isolation, functional characterization, and expression pattern of a vacuolar Na+/H+ antiporter gene TrNHX1 from Trifolium repens L. Plant Mol Biol Rep 28:102–111

    Article  CAS  Google Scholar 

  • Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot 91:503–527

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgin DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Timko MP, Ehlers JD, Roberts PA (2007) Cowpea. In: Kole (ed) Genome mapping and molecular breeding in plants, vol 3, pulses, sugar and tuber crops. Springer, Berlin pp 49–67

  • Venema K, Belver A, Marin-Manzano MC, Rodgriguez-Rosales MP, Donaire JP (2003) A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants. J Biol Chem 278:22453–22459

    Article  CAS  PubMed  Google Scholar 

  • 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. DNA Seq 14:351–358

    CAS  PubMed  Google Scholar 

  • Wang S, Zhang YD, Perez PG et al (2011) Isolation and characterization of a vacuolar Na+/H+ antiporter gene from Cucumis melo L. Afr J Biotechnol 10:1752–1759

    CAS  Google Scholar 

  • Wu CA, Yang GD, Meng QW, Zheng CC (2004) The cotton GhNHX1 gene encoding a novel putative tonoplast Na+/H+ antiporter plays an important role in salt stress. Plant Cell Physiol 45:600–607

    Article  CAS  PubMed  Google Scholar 

  • Wu YY, Chen QJ, Chen M, Chen J, Wang XC (2005) Salt-tolerant transgenic perennial ryegrass (Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na+/H+ antiporter gene. Plant Sci 169:65–73

    Article  CAS  Google Scholar 

  • Wu C, Gao X, Kong X et al (2009) Molecular cloning and functional analysis of a Na+/H+ antiporter gene ThNHX1 from a halophytic plant Thellungiella halophila. Plant Mol Biol Rep 27:1–12

    Article  CAS  Google Scholar 

  • Xia T, Apse MP, Aharon GS, Blumwald E (2002) Identification and characterization of a NaCl-inducible vacuolar Na+/H+ antiporter in Beta vulgaris. Physiol Plant 116:206–212

    Article  CAS  PubMed  Google Scholar 

  • Xue ZY, Zhi DY, Xue GP, Zhang H, Zhao YX, Xia GM (2004) Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.) expressing a vacuolar Na+/H+ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na+. Plant Sci 167:849–859

    Article  CAS  Google Scholar 

  • Yamaguchi T, Fukada-Tanaka S, Inagaki Y, Saito N, Yonekura-Sakakibara K, Tanaka Y, Kusumi T, Iida S (2001) Genes encoding the vacuolar Na+/H+ exchanger and flower coloration. Plant Cell Physiol 42:451–461

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi T, Apse MP, Shi H, Blumwald E (2003) Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity. Proc Natl Acad Sci USA 100:12510–12515

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19:765–768

    Article  CAS  PubMed  Google Scholar 

  • Zhang HX, Hodson JN, Williams JP, Blumwald E (2001) Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proc Natl Acad Sci USA 98:12832–12836

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang GH, Su Q, An LJ, Wu S (2008) Charcterization and expression of a vacuolar Na+/H+ antiporter gene from the monocot halophyte Aeluropus littoralis. Plant Physiol Biochem 46:117–126

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Liu Y, Xu Y, Chapman S, Love AJ, Xia T (2012) A newly isolated Na+/H+ antiporter gene, DmNHX1, confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana. Plant Cell Tissue Organ Cult 110:189–200

    Article  CAS  Google Scholar 

  • Zhao JS, Zhi DY, Xue ZY, Liu H, Xia GM (2007) Enhanced salt tolerance of transgenic progeny of tall fescue (Festuca arundinacea) expressing a vacuolar Na+/H+ antiporter gene from Arabidopsis. J Plant Physiol 164:1377–1383

    Article  CAS  PubMed  Google Scholar 

  • Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71

    Article  CAS  PubMed  Google Scholar 

  • Zhu JK (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445

    Article  CAS  PubMed  Google Scholar 

  • Zorb C, Noll A, Noll A, Karl S, Leib K, Yan F, Schubert S (2005) Molecular characterization of Na+/H+ antiporters (ZmNHX) of maize (Zea mays L.) and their expression under salt stress. J Plant Physiol 162:55–65

    Article  PubMed  Google Scholar 

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Acknowledgments

We express our sincere thanks to Prof. Edward Blumwald and Dr. Olivier Cagnac for the yeast strains, W303 and AXT3 respectively. We also thank Dr. Luciana LoureiroPenha for providing pYES2.0 vector for yeast expression analysis, Department of Civil Engineering, IIT Guwahati for use of Flame Photometry. LS is grateful to DBT (Department of Biotechnology, Government of India) for its support through various Grants (BT/PR10818/AGR/02/591/2008 and BT/01/NE/PS/08) for legume improvement program. BhL is grateful to Rural Development Administration, Republic of Korea for its support by Next-Generation BioGreen 21 Program (PJ009104). SM is grateful to MHRD for Research Fellowship. SKP is grateful to DBT (Department of Biotechnology, Government of India) for its support.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, India

    Sagarika Mishra & Lingaraj Sahoo

  2. Department of Life Science, Sogang University, Mapo-gu, Seoul, 121-742, Korea

    Hemasundar Alavilli & Byeong-ha Lee

  3. Department of Life Science and Bioinformatics, Assam University, Silchar, 788011, India

    Sanjib Kumar Panda

  4. Faculty of Applied Biological Science, Gifu University, 1-1, Yanagido, Gifu, 501-1193, Japan

    Lingaraj Sahoo

  5. Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, 74078, USA

    Sanjib Kumar Panda

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  1. Sagarika Mishra
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  2. Hemasundar Alavilli
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  3. Byeong-ha Lee
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  4. Sanjib Kumar Panda
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Mishra, S., Alavilli, H., Lee, Bh. et al. Cloning and characterization of a novel vacuolar Na+/H+ antiporter gene (VuNHX1) from drought hardy legume, cowpea for salt tolerance. Plant Cell Tiss Organ Cult 120, 19–33 (2015). https://doi.org/10.1007/s11240-014-0572-7

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