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Pennisetum glaucum Na+/H+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea

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Abstract

Soil salinity is one of the major abiotic stresses affecting plant productivity. We report on the isolation and characterization of a different isoform of vacuolar Na+/H+ antiporter from Pennisetum glaucum (PgNHX1) that conferred high level of salinity tolerance when over expressed in Brassica juncea. PgNHX1 cDNA has an ORF of 1,413 bp and a 3′ and 5′ UTR of 350 and 166 bp, respectively. In contrast to AtNHX1 and OsNHX1, which have nine transmembrane-spanning domains, PgNHX1 has five transmembrane domains. Real time quantitative RT-PCR analysis revealed up-regulation of PgNHX1 transcript following NaCl and ABA treatments. The 3.3 kb PgNHX1 genomic clone has 12 introns spanning the entire ORF. Confocal imaging of the GFP-PgNHX1 fusion construct showed vacuolar localization in yeast cells. The fungal inhibitor, brefeldin A, inhibited this localization. PgNHX1 functionally complemented yeast mutants defective in endogenous NHX and such transformed yeast could tolerate up to 70 mM NaCl. Transgenic B. juncea plants overexpressing PgNHX1 survive, set flowers and normal seeds in presence of up to 300 mM NaCl. The transgenic plants accumulated more Na+ in the leaves than in seeds. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants for growing in high-saline areas.

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References

  • 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  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Barkla BJ, Blumwald E (1991) Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris. Proc Natl Acad Sci USA 88:11177–11181

    Article  PubMed  CAS  Google Scholar 

  • Blumwald E (2000) Sodium transport and salt tolerance in plants. Curr Opin Cell Biol 12:431–434

    Article  PubMed  CAS  Google Scholar 

  • Boyer JS (1982) Plant productivity and environment. Science 218:443–448

    Article  PubMed  Google Scholar 

  • Brown JW (1986) A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acid Res 14:9549–9559

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Dibrov P, Fliegel L (1998) Comparative molecular analysis of Na+/H+ exchangers: a unified model for Na+/H+ antiport? FEBS Lett 424:1–5

    Article  PubMed  CAS  Google Scholar 

  • Fan L, Zheng S, Wang X (1997) Antisense suppression of phospholipase Dα retards abscisic acid and ethylene-promoted senescence of post harvest Arabidopsis leaves. Plant Cell 9:2183–2196

    Article  PubMed  CAS  Google Scholar 

  • FAO (2005) Global network on integrated soil management for sustainable use of salt—affected soils. FAO Land and Plant Nutrition Management Service, Rome, Italy. http://www.fao.org/ag/agl/agll/spush

  • Finkelstein RR, Gampala SS, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell 14:15–45

    Google Scholar 

  • Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:307–319

    Article  PubMed  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

    PubMed  CAS  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 45:149–159

    Google Scholar 

  • Fukuda A, Yazaki Y, Ishikawa T, Koike S, Tanaka Y (1998) Na+/H+ antiporter in tonoplast vesicles from rice roots. Plant Cell Physiol 39:196–201

    CAS  Google Scholar 

  • 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 USA 96:1480–1485

    Article  PubMed  CAS  Google Scholar 

  • Greenway H, Munns R (1980) Mechanism of salt tolerance in non halophytes. Annu Rev Plant Physiol 31:149–190

    Article  CAS  Google Scholar 

  • Hamada A, Shona 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  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Hawes C (2005) Cell biology of the plant golgi apparatus. New Phytol 165:29–44

    Article  PubMed  Google Scholar 

  • Lu SY, Jing YX, Shen SH, Zhao HY, Ma LO, Zhou XJ, Li QRYF (2005) Antiporter gene from Hordum brevisubulatum (Trin.) Link and its overexpression in transgenic tobacco. J Integr Plant Biol 47:343–349

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

    Article  CAS  Google Scholar 

  • Mimura T, Kura-Hotta M, Tsujimura T, Ohnishi M, Miura M, Okazaki Y, Mimura M, Maeshima M, Washitani-Nemoto S (2003) Rapid increase of vacuolar volume in response to salt stress. Planta 216:397–402

    PubMed  CAS  Google Scholar 

  • Munns R (1993) Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant Cell Environ 16:15–24

    Article  CAS  Google Scholar 

  • Munns R (2005) Genes and salt tolerance: bringing them together. New Phytol 167:645–663

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:472–497

    Article  Google Scholar 

  • Nass R, Cunningham KW, Rao R (1997) Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. J Biol Chem 272:26145–26152

    Article  PubMed  CAS  Google Scholar 

  • Nebenfuhr A, Ritzenthaler C, Robinson DG (2002) Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol 130:1102–1108

    Article  PubMed  CAS  Google Scholar 

  • Ohnishi M, Fukuda-Tanaka S, Hoshino A, Takada J, Inagaki Y, Iida S (2005) Characterization of a novel Na+/H+ antiporter gene InNHX2 and comparison of InNHX2 with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory. Plant Cell Physiol 46:259–267

    Article  PubMed  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  PubMed  CAS  Google Scholar 

  • Pental D, Pradhan AK (1993) Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium mediated genetic transformation. Plant Cell Rep 12:462–467

    Article  CAS  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  PubMed  CAS  Google Scholar 

  • Reddy MK, Nair S, Tewari KK (1998) Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea. Plant Mol Biol 37:773–784

    Article  PubMed  CAS  Google Scholar 

  • Ren ZH, Gao JP, Li LG, Cai XL, Huang W, Chao DY, Zhu MZ, Wang ZY, Luan S, Lin HX (2005) A rice quantitative trait locus for salt tolerance encodes a sodium transporter. Nat Genet 37:1141–1146

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Sahi C, Agarwal M, Reddy MK, Sopory SK, Grover A (2003) Isolation and expression analysis of salt stress—associated ESTs from contrasting rice cultivars using a PCR—based subtraction method. Theor Appl Genet 106:620–628

    PubMed  CAS  Google Scholar 

  • Sanan-Mishra N, Pham X-H, Sopory SK, Tuteja N (2005) Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc Natl Acad Sci USA 102:509–514

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Shi H, Ishitani M, Kim C, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc Natl Acad Sci USA 97:6896–6901

    Article  PubMed  CAS  Google Scholar 

  • Shi H, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21:81–85

    Article  PubMed  CAS  Google Scholar 

  • Singla-Pareek SL, Reddy MK, Sopory SK (2003) Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc Natl Acad Sci USA 100:14672–14677

    Article  PubMed  CAS  Google Scholar 

  • Tyagi W, Singla-Pareek SL, Nair S, Reddy MK, Sopory SK (2006) A novel isoform of ATPase c subunit from pearl millet that is differentially regulated in response to salinity and calcium. Plant Cell Rep 25:156–163

    Article  PubMed  CAS  Google Scholar 

  • Vernon LP (1969) Spectrophotometric determination of chlorophylls and phaeophytins in plant extracts. Anal Chem 32:1144–1150

    Article  Google Scholar 

  • Volkov V, Wang B, Dominy PJ, Fricke W, Amtmann A (2004) Thellungiella halophila, a salt tolerant relative of Arabidopsis thaliana, possesses effective mechanisms to discriminate between potassium and sodium. Plant Cell Environ 27:1–14

    Article  CAS  Google Scholar 

  • Wakabayashi S, Pang T, Su X, Shigekawaa M (2000) A novel topology model of the human Na+/H+ exchanger isoform 1. J Biol Chem 275:7942–7949

    Article  PubMed  CAS  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 5:351–358

    Google Scholar 

  • Wells KM, Rao R (2001) The yeast Na+/H+ exchanger Nhx1 is an N-linked glycoprotein. Topological implications. J Biol Chem 276:3401–3407

    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  PubMed  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  PubMed  CAS  Google Scholar 

  • Xiao-Yan Y, Ai-Fang Y, Ke-Wei Z, Ju-Ren Z (2004) Production and analysis of transgenic maize with improved salt tolerance by the introduction of AtNHX1. Gene Acta Bot Sin 46:854–861

    Google Scholar 

  • Xue Z-Y, Zhi D-Y, Xue G-P, Zhao Y-X, Xia G-M (2004) Enhanced salt tolerance of transgenic wheat (Triticum aestivum L.) expressing a vacuolar Na+/H+ antiporter gene with improved grain yield in saline soils in the field and a reduced level of leaf Na+. Plant Sci 167:849–859

    Article  CAS  Google Scholar 

  • Yamaguchi T, Aharon GS, Sottosanto JB, Blumwald E (2005) Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca2+- and pH-dependent manner. Proc Natl Acad Sci USA 102:16107–16112

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Yokoi S, Quintero FJ, Cubero B, Ruiz MT, Bressan RA, Hasegawa PM, Pardo JM (2002) Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response. Plant J 30:529–539

    Article  PubMed  CAS  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  PubMed  CAS  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  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Zorb C, 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 162:55–66

    Article  PubMed  Google Scholar 

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Acknowledgments

We thank Prof. J. M. Pardo, and Dr. F. J. Quintero, Madrid, Spain, for providing the yeast mutant strains, Prof. V. Rajamani and Dr. J. K. Tripathi, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, for their help in ion estimation work, Dr. Shahid Jameel for providing the confocal facility (Wellcome grant) and Ms. Charu for the technical help. This work is supported by grants from DST (Indo-Russia) and DST (Young scientist project to SLS-P) projects. DR was supported by a fellowship from UGC.

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  1. Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Divya Rajagopal, Pradeep Agarwal, Wricha Tyagi, Sneh L. Singla-Pareek, M. K. Reddy & S. K. Sopory

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  1. Divya Rajagopal
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  2. Pradeep Agarwal
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  3. Wricha Tyagi
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  4. Sneh L. Singla-Pareek
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  5. M. K. Reddy
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  6. S. K. Sopory
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Correspondence to S. K. Sopory.

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Rajagopal, D., Agarwal, P., Tyagi, W. et al. Pennisetum glaucum Na+/H+ antiporter confers high level of salinity tolerance in transgenic Brassica juncea . Mol Breeding 19, 137–151 (2007). https://doi.org/10.1007/s11032-006-9052-z

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  • DOI: https://doi.org/10.1007/s11032-006-9052-z

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