Advertisement

Planta

, Volume 233, Issue 1, pp 175–188 | Cite as

Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes

  • Atsunori FukudaEmail author
  • Atsuko Nakamura
  • Naho Hara
  • Seiichi Toki
  • Yoshiyuki Tanaka
Original paper

Abstract

We previously cloned a vacuolar Na+/H+ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na+, Li+, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K+ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na+ and K+ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.

Keywords

Gene expression Na+/H+ antiporter gene Oryza Salt tolerance 

Abbreviations

ABA

Abscisic acid

ABRE

ABA-responsive element

bZIP

Basic-domain leucine zipper

EST

Expressed sequence tag

GUS

β-Glucuronidase

kb

Kilobase

4-MU

4-Methylumbelliferone

4-MUG

4-Methylumbelliferyl-β-d-glucuronide

PAC

P1-derived artificial chromosome

RACE

Rapid amplification of cDNA ends

RGRP

Rice Genome Research Program

Notes

Acknowledgments

We thank Dr. Rajini Rao (Johns Hopkins University, USA) for kindly providing the yeast strains, K601 and R100. We also thank Drs. I. Mitsuhara and Y. Ohashi for kindly providing the vectors, pTN2 and pE2113–GUS. The cDNA clones including OsNHX2 and OsNHX5 and PAC clones including OsNHX1 and OsNHX5 were gifts from the Rice Genome Research Program. We thank Drs. Y. Nagamura and M. Yano for technical advice. We also thank C. Tsuiki, K. Toyoshima, T. Kataoka, and S. Li for technical assistance. This work was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Development of Innovative Transgenic Plants no.2113 and Rice Genome Project MP-2126).

Supplementary material

425_2010_1289_MOESM1_ESM.pdf (142 kb)
Supplementary material 1 (PDF 141 kb)
425_2010_1289_MOESM2_ESM.pdf (65 kb)
Supplementary material 2 (PDF 64 kb)
425_2010_1289_MOESM3_ESM.pdf (90 kb)
Supplementary material 3 (PDF 90 kb)

References

  1. Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M (1997) Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9:841–857CrossRefPubMedGoogle Scholar
  2. Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis thaliana. Science 285:1256–1258CrossRefPubMedGoogle Scholar
  3. Axelsen KB, Palmgren MG (2001) Inventory of the superfamily of P-type ion pumps in Arabidopsis. Plant Physiol 126:696–706CrossRefPubMedGoogle Scholar
  4. Bowers K, Levi BP, Patel FI, Stevens TH (2000) The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast. Mol Biol Cell 11:4277–4294PubMedGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Counillon L, Franchi A, Pouysségur J (1993) A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis. Proc Natl Acad Sci USA 90:4508–4512CrossRefPubMedGoogle Scholar
  7. Fukuda A, Nakamura A, Tanaka Y (1999) Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochim Biophys Acta 1446:149–155PubMedGoogle Scholar
  8. Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A, Hirochika H, Tanaka Y (2004a) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice. Plant Cell Physiol 45:146–159CrossRefPubMedGoogle Scholar
  9. Fukuda A, Chiba K, Maeda M, Nakamura A, Maeshima M, Tanaka Y (2004b) Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley. J Exp Bot 55:585–594CrossRefPubMedGoogle Scholar
  10. Fukuoka H, Ogawa T, Mitsuhara I, Iwai T, Isuzugawa K, Nishizawa Y, Gotoh Y, Nishizawa Y, Tagiri A, Ugaki M, Ohshima M, Yano H, Murai N, Niwa Y, Hibi T, Ohashi Y (2000) Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus. Plant Cell Rep 19:815–820CrossRefGoogle Scholar
  11. Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999) The Arabidopsis thaliana transporters, AtNHX1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci USA 96:1480–1485CrossRefPubMedGoogle Scholar
  12. Gietz RD, Schiestl RH (1995) Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269Google Scholar
  13. Goff SA, Ricke D, Lan T-H, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J, Miguel T, Paszkowski U, Zhang S, Colbert M, Sun W, Chen L, Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92–100CrossRefPubMedGoogle Scholar
  14. Hamada A, Hibino T, Nakamura T, Takabe T (2001) Na+/H+ antiporter from Synechocystis species PCC 6803, homologous to SOS1, contains an aspartic residue and long C-terminal tail important for the carrier activity. Plant Physiol 125:437–446CrossRefPubMedGoogle Scholar
  15. Hobo T, Asada M, Kowyama Y, Hattori T (1999) ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Plant J 19:679–689CrossRefPubMedGoogle Scholar
  16. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301PubMedGoogle Scholar
  17. Kumagai H, Kouchi H (2003) Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Mol Plant Microbe Interact 16:663–668CrossRefPubMedGoogle Scholar
  18. Lam E, Chua N-H (1991) Tetramer of a 21-base pair synthetic element confers seed expression and transcriptional enhancement in response to water stress and abscisic acid. J Biol Chem 266:17131–17135PubMedGoogle Scholar
  19. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, Katayose Y, Nakamura S, Honkura R, Nishimiya S, Ueno K, Mochizuki A, Tanimoto H, Tsugawa H, Otsuki Y, Ohashi Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49–59PubMedGoogle Scholar
  20. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  21. Nakamura N, Tanaka S, Teko Y, Mitsui K, Kanazawa H (2005) Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. J Biol Chem 280:1561–1572CrossRefPubMedGoogle Scholar
  22. Nass R, Rao R (1998) Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast. J Biol Chem 273:21054–21060CrossRefPubMedGoogle Scholar
  23. 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 272:26145–26152CrossRefPubMedGoogle Scholar
  24. Orlowski J, Grinstein S (1997) Na+/H+ exchangers of mammalian cells. J Biol Chem 272:22373–22376CrossRefPubMedGoogle Scholar
  25. Pardo JM, Cubero B, Leidi EO, Quintero FJ (2006) Alkali cation exchangers: roles in cellular homeostasis and stress tolerance. J Exp Bot 57:1181–1199CrossRefPubMedGoogle Scholar
  26. Prior C, Potier S, Souciet J-L, Sychrova H (1996) Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae. FEBS Lett 387:89–93CrossRefPubMedGoogle Scholar
  27. Rob M, Roelfsema G, Hedrich R (2005) In the light of stomatal opening: new insights into ‘the Watergate’. New Phytol 167:665–691CrossRefGoogle Scholar
  28. Rodriguez-Rosales MP, Jiang X, Gálvez FJ, Aranda MN, Cubero B, Venema K (2008) Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization. New Phytol 179:366–377CrossRefPubMedGoogle Scholar
  29. 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 138:425–431CrossRefPubMedGoogle Scholar
  30. Shi H, Zhu J-K (2002) Regulation of expression of the vacuolar Na+/H+ antiporter gene AtNHX1 by salt stress and abscisic acid. Plant Mol Biol 50:543–550CrossRefPubMedGoogle Scholar
  31. Shi H, Ishitani M, Kim C, Zhu J-K (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc Natl Acad Sci USA 97:6896–6901CrossRefPubMedGoogle Scholar
  32. Shi H, Quintero FJ, Pardo JM, Zhu J-K (2002) The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport in plants. Plant Cell 14:465–477CrossRefPubMedGoogle Scholar
  33. Shinozaki K, Yamaguchi-Shinozaki K (1997) Gene expression and signal transduction in water-stress response. Plant Physiol 115:327–334CrossRefPubMedGoogle Scholar
  34. Song C-P, Guo Y, Qiu Q, Lambert G, Galbraith DW, Jagendorf A, Zhu J-K (2004) A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana. Proc Natl Acad Sci USA 101:10211–10216CrossRefPubMedGoogle Scholar
  35. Sze H, Padmanaban S, Cellier F, Honys D, Cheng N-H, Bock KW, Conéjéro G, Li X, Twell D, Ward JM, Hirschi KD (2004) Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development. Plant Physiol 136:2532–2547CrossRefPubMedGoogle Scholar
  36. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  37. Thompson J, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedGoogle Scholar
  38. Venema K, Belver A, Marín-Manzano MC, Rodríguez-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 278:22453–22459CrossRefPubMedGoogle Scholar
  39. Véry A-A, Sentenac H (2003) Molecular mechanisms and regulation of K+ transport in higher plants. Annu Rev Plant Biol 54:575–603CrossRefPubMedGoogle Scholar
  40. 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 100:12510–12515CrossRefPubMedGoogle Scholar
  41. Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci 10:88–94CrossRefPubMedGoogle Scholar
  42. 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 30:529–539CrossRefPubMedGoogle Scholar
  43. Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19:765–768CrossRefPubMedGoogle Scholar
  44. 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 98:12832–12836CrossRefPubMedGoogle Scholar
  45. Zhu J-K (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Atsunori Fukuda
    • 1
    Email author
  • Atsuko Nakamura
    • 2
  • Naho Hara
    • 1
  • Seiichi Toki
    • 1
  • Yoshiyuki Tanaka
    • 1
  1. 1.Division of Plant SciencesNational Institute of Agrobiological SciencesTsukubaJapan
  2. 2.Graduate School of Life and Environmental SciencesTsukuba UniversityTsukubaJapan

Personalised recommendations