Cellular and Molecular Life Sciences

, Volume 75, Issue 6, pp 1133–1144 | Cite as

Structural variations in wheat HKT1;5 underpin differences in Na+ transport capacity

  • Bo Xu
  • Shane Waters
  • Caitlin S. Byrt
  • Darren Plett
  • Stephen D. Tyerman
  • Mark Tester
  • Rana Munns
  • Maria Hrmova
  • Matthew Gilliham
Original Article


An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na+) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na+-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K m) for the Na+ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D471/a gap and D474/G473 that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na+ exclusion in wheat that leads to an improved salinity tolerance in the field.


Gatekeeper cells Salt exclusion Ion transport Structure–function Einkorn Bread Salt tolerance Xenopus Mutagenesis Yeast High-affinity K+ transporter 


Author contributions

BX, MH, DP, and MG conceived the project out of work initiated by RM and MT. BX performed all experiments except the structural modelling and predictions (SW) and the cloning and original characterisation of TmHKT1;5-AK118E/L339P/Y379M (CSB). SDT advised on electrophysiology and analysis. MG, MH, and DP supervised the work. BX, SW, CSB, MH, and MG wrote the paper. All authors provided comment.

Compliance with ethical standards


This work was supported by the Grains Research and Development Corporation (UA00145, M.G.), the University of Adelaide Australian Postgraduate Award and the CJ Everald postgraduate scholarship (S.W.), and the Australian Research Council through the following schemes: Discovery (DP120100900, M.H.), Centre of Excellence (CE140100008, M.G., R.M., S.D.T), Future Fellowship (FT130100709, M.G.), and DECRA (DE150100837, C.S.B.).

Supplementary material

18_2017_2716_MOESM1_ESM.pdf (2 mb)
Supplementary material 1 (PDF 2024 kb)


  1. 1.
    Schachtman DP, Schroeder JI (1994) Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants. Nature 370:655–658CrossRefPubMedGoogle Scholar
  2. 2.
    Epstein E (1966) Dual pattern of ion absorption by plant cells and by plants. Nature 212:1324–1327CrossRefGoogle Scholar
  3. 3.
    Platten JD, Cotsaftis O, Berthomieu P, Bohnert H, Davenport RJ, Fairbairn DJ, Horie T, Leigh RA, Lin H-X, Luan S (2006) Nomenclature for HKT transporters, key determinants of plant salinity tolerance. Trends Plant Sci 11(8):372–374CrossRefPubMedGoogle Scholar
  4. 4.
    Rubio F, Gassmann W, Schroeder JI (1995) Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance. Science 270(5242):1660–1663CrossRefPubMedGoogle Scholar
  5. 5.
    Gierth M, Mäser P (2007) Potassium transporters in plants–involvement in K+ acquisition, redistribution and homeostasis. FEBS Lett 581(12):2348–2356CrossRefPubMedGoogle Scholar
  6. 6.
    Horie T, Hauser F, Schroeder JI (2009) HKT transporter-mediated salinity resistance mechanisms in Arabidopsis and monocot crop plants. Trends Plant Sci 14(12):660–668CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Laurie S, Feeney KA, Maathuis FJ, Heard PJ, Brown SJ, Leigh RA (2002) A role for HKT1 in sodium uptake by wheat roots. Plant J 32(2):139–149CrossRefPubMedGoogle Scholar
  8. 8.
    James RA, Davenport RJ, Munns R (2006) Physiological characterization of two genes for Na+ exclusion in durum wheat, Nax1 and Nax2. Plant Physiol 142(4):1537–1547CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Läuchli A, James RA, Huang CX, McClluy M, Munns R (2008) Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion. Plant Cell Environ 31(11):1565–1574CrossRefPubMedGoogle Scholar
  10. 10.
    Sentenac H, Bonneaud N (1992) Cloning and expression in yeast of a plant potassium ion transport system. Science 256(5057):663–665CrossRefPubMedGoogle Scholar
  11. 11.
    Corratgé-Faillie C, Jabnoune M, Zimmermann S, Véry A-A, Fizames C, Sentenac H (2010) Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family. Cell Mol Life Sci 67(15):2511–2532CrossRefPubMedGoogle Scholar
  12. 12.
    Huang CS, Pedersen BP, Stokes DL (2017) Crystal structure of the potassium-importing KdpFABC membrane complex. Nature 546(7660):681–685CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mäser P, Hosoo Y, Goshima S, Horie T, Eckelman B, Yamada K, Yoshida K, Bakker EP, Shinmyo A, Oiki S (2002) Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants. Proc Natl Acad Sci USA 99(9):6428–6433CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cotsaftis O, Plett D, Shirley N, Tester M, Hrmova M (2012) A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing. PLoS One 7(7):e39865CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Waters S, Gilliham M, Hrmova M (2013) Plant high-affinity potassium (HKT) transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity. Int J Mol Sci 14(4):7660–7680CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Singh A, Bhushan B, Gaikwad K, Yadav OP, Kumar S, Rai RD (2015) Induced defence responses of contrasting bread wheat genotypes under differential salt stress imposition. Indian J Biochem Biophys 52(1):75–85PubMedGoogle Scholar
  17. 17.
    Asins MJ, Villalta I, Aly MM, Olias R, Álvarez De Morales P, Huertas R, Li J, Jaime-Pérez N, Haro R, Raga V (2013) Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis. Plant Cell Environ 36(6):1171–1191CrossRefPubMedGoogle Scholar
  18. 18.
    Uozumi N, Kim EJ, Rubio F, Yamaguchi T, Muto S, Tsuboi A, Bakker EP, Nakamura T, Schroeder JI (2000) The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant Physiol 122(4):1249–1260CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    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(10):1141–1146CrossRefPubMedGoogle Scholar
  20. 20.
    Negrão S, Cecília Almadanim M, Pires IS, Abreu IA, Maroco J, Courtois B, Gregorio GB, McNally KL, Margarida Oliveira M (2013) New allelic variants found in key rice salt-tolerance genes: an association study. Plant Biotech J 11(1):87–100CrossRefGoogle Scholar
  21. 21.
    Ariyarathna HCK, Ul-Haq T, Colmer TD, Francki MG (2014) Characterization of the multigene family TaHKT 2;1 in bread wheat and the role of gene members in plant Na+ and K+ status. BMC Plant Biol 14(1):1CrossRefGoogle Scholar
  22. 22.
    Mishra S, Singh B, Panda K, Singh BP, Singh N, Misra P, Rai V, Singh NK (2016) Association of SNP haplotypes of HKT family genes with salt tolerance in indian wild rice germplasm. Rice 9(1):1CrossRefGoogle Scholar
  23. 23.
    Kumar S, Beena A, Awana M, Singh A (2017) Physiological, biochemical, epigenetic and molecular analyses of wheat (Triticum aestivum) genotypes with contrasting salt tolerance. Front Plant Sci 8:1151CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Diatloff E, Kumar R, Schachtman DP (1998) Site directed mutagenesis reduces the Na+ affinity of HKT1, an Na+ energized high affinity K+ transporter. FEBS Lett 432(1–2):31–36CrossRefPubMedGoogle Scholar
  25. 25.
    Böhm J, Scherzer S, Shabala S, Krol E, Neher E, Mueller T, Hedrich R (2016) Venus flytrap HKT1-type channel provides for prey sodium uptake into carnivorous plant without conflicting with electrical excitability. Mol Plant 9(3):428–436CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Almeida P, Katschnig D, de Boer AH (2013) HKT transporters-state of the art. Int J Mol Sci 14(10):20359–20385CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Almeida P, de Boer G-J, de Boer AH (2014) Differences in shoot Na+ accumulation between two tomato species are due to differences in ion affinity of HKT1;2. J Plant Physiol 171(6):438–447CrossRefPubMedGoogle Scholar
  28. 28.
    Ali A, Raddatz N, Aman R, Kim S, Park HC, Jan M, Baek D, Khan IU, Oh D-H, Lee SY (2016) A single amino acid substitution in the sodium transporter HKT1 associated with plant salt tolerance. Plant Physiol 171:2112–2126CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Cao Y, Jin X, Huang H, Derebe MG, Levin EJ, Kabaleeswaran V, Pan Y, Punta M, Love J, Weng J (2011) Crystal structure of a potassium ion transporter, TrkH. Nature 471(7338):336–340CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Mian A, Oomen RJ, Isayenkov S, Sentenac H, Maathuis FJ, Véry AA (2011) Over-expression of an Na+- and K+-permeable HKT transporter in barley improves salt tolerance. Plant J 68(3):468–479CrossRefPubMedGoogle Scholar
  31. 31.
    Jabnoune M, Espeout S, Mieulet D, Fizames C, Verdeil J-L, Conéjéro G, Rodríguez-Navarro A, Sentenac H, Guiderdoni E, Abdelly C (2009) Diversity in expression patterns and functional properties in the rice HKT transporter family. Plant Physiol 150(4):1955–1971CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Amar SB, Brini F, Sentenac H, Masmoudi K, Véry A-A (2014) Functional characterization in Xenopus oocytes of Na+ transport systems from durum wheat reveals diversity among two HKT1;4 transporters. J Exp Bot 65(1):213–222CrossRefPubMedGoogle Scholar
  33. 33.
    Munns R, James RA, Xu B, Athman A, Conn SJ, Jordans C, Byrt CS, Hare RA, Tyerman SD, Tester M, Plett D, Gilliham M (2012) Wheat grain yield on saline soils is improved by an ancestral Na+ transporter gene. Nat Biotech 30(4):360–364CrossRefGoogle Scholar
  34. 34.
    Byrt CS, Xu B, Krishnan M, Lightfoot DJ, Athman A, Jacobs AK, Watson-Haigh NS, Plett D, Munns R, Tester M (2014) The Na+ transporter, TaHKT1;5-D, limits shoot Na+ accumulation in bread wheat. Plant J 80(3):516–526CrossRefPubMedGoogle Scholar
  35. 35.
    Tounsi S, Amar SB, Masmoudi K, Sentenac H, Brini F, Véry A-A (2016) Characterization of two HKT1;4 transporters from Triticum monococcum to elucidate the determinants of the wheat salt tolerance Nax1 QTL. Plant Cell Physiol 57(10):2047–2057CrossRefPubMedGoogle Scholar
  36. 36.
    Baxter I, Brazelton JN, Yu D, Huang YS, Lahner B, Yakubova E, Li Y, Bergelson J, Borevitz JO, Nordborg M (2010) A coastal cline in sodium accumulation in Arabidopsis thaliana is driven by natural variation of the sodium transporter AtHKT1; 1. PLoS Genet 6(11):e1001193CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Møller IS, Gilliham M, Jha D, Mayo GM, Roy SJ, Coates JC, Haseloff J, Tester M (2009) Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis. Plant Cell 21(7):2163–2178CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Plett D, Safwat G, Gilliham M, Møller IS, Roy S, Shirley N, Jacobs A, Johnson A, Tester M (2010) Improved salinity tolerance of rice through cell type-specific expression of AtHKT1; 1. PLoS One 5(9):e12571CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Munns R, Gilliham M (2015) Salinity tolerance of crops–what is the cost? New Phytol 208(3):668–673CrossRefPubMedGoogle Scholar
  40. 40.
    Ismail AM, Horie T (2017) Genomics, physiology, and molecular breeding approaches for improving salt tolerance. Annu Rev Plant Biol 68:405–434CrossRefPubMedGoogle Scholar
  41. 41.
    Byrt CS, Platten JD, Spielmeyer W, James RA, Lagudah ES, Dennis ES, Tester M, Munns R (2007) HKT1; 5-like cation transporters linked to Na+ exclusion loci in wheat, Nax2 and Kna1. Plant Physiol 143(4):1918–1928CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Vieira-Pires RS, Szollosi A, Morais-Cabral JH (2013) The structure of the KtrAB potassium transporter. Nature 496(7445):323–328CrossRefPubMedGoogle Scholar
  43. 43.
    Fairbairn DJ, Liu W, Schachtman DP, Gomez-Gallego S, Day SR, Teasdale RD (2000) Characterisation of two distinct HKT1-like potassium transporters from Eucalyptus camaldulensis. Plant Mol Biol 43(4):515–525CrossRefPubMedGoogle Scholar
  44. 44.
    Liu W, Fairbairn DJ, Reid RJ, Schachtman DP (2001) Characterization of two HKT1 homologues from Eucalyptus camaldulensis that display intrinsic osmosensing capability. Plant Physiol 127(1):283–294CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Rodríguez-Navarro A, Ramos J (1984) Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159(3):940–945PubMedPubMedCentralGoogle Scholar
  46. 46.
    Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26(2):283–291CrossRefGoogle Scholar
  47. 47.
    Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17(4):355–362CrossRefPubMedGoogle Scholar
  48. 48.
    Gille C, Birgit W, Gille A (2013) Sequence alignment visualization in HTML5 without Java. Bioinformatics 30(1):121–122CrossRefPubMedGoogle Scholar
  49. 49.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    My Shen, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Sci 15(11):2507–2524CrossRefGoogle Scholar
  51. 51.
    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. Nucleic Acids Res 33(suppl 2):W299–W302CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Celniker G, Nimrod G, Ashkenazy H, Glaser F, Martz E, Mayrose I, Pupko T, Ben-Tal N (2013) ConSurf: using evolutionary data to raise testable hypotheses about protein function. Isr J Chem 53(3–4):199–206CrossRefGoogle Scholar
  53. 53.
    James RA, Blake C, Byrt CS, Munns R (2011) Major genes for Na+ exclusion, Nax1 and Nax2 (wheat HKT1;4 and HKT1;5), decrease Na+ accumulation in bread wheat leaves under saline and waterlogged conditions. J Exp Bot 62(8):2939–2947CrossRefPubMedGoogle Scholar
  54. 54.
    Henderson SW, Gilliham M (2015) The “Gatekeeper” concept: cell-type specific molecular mechanisms of plant adaptation to abiotic stress. In: Laitinen RAE (ed) Molecular mechanisms in plant adaptation. Wiley, New Jersey, pp 83–115Google Scholar
  55. 55.
    Byrt CS (2008) PhD thesis: Genes for sodium exclusion in wheat. University of Adelaide, AdelaideGoogle Scholar
  56. 56.
    Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4(3):363–371CrossRefPubMedGoogle Scholar
  57. 57.
    Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinform 9(1):1CrossRefGoogle Scholar
  58. 58.
    Wu S, Zhang Y (2007) LOMETS: a local meta-threading-server for protein structure prediction. Nucleic Acids Res 35(10):3375–3382CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Pei J, Kim BH, Grishin NV (2008) PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res 36(7):2295–2300CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Sali A, Blundell T (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234(3):779–815CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Australian Research Council Centre of Excellence in Plant Energy Biology, Waite Research PrecinctUniversity of AdelaideGlen OsmondAustralia
  2. 2.School of Agriculture, Food and Wine, and Waite Research Institute, Waite Research PrecinctUniversity of AdelaideGlen OsmondAustralia
  3. 3.Division of Biological and Environmental Sciences and Engineering, Center for Desert AgricultureKing Abdullah University of Science and TechnologyThuwalKingdom of Saudi Arabia
  4. 4.School of Agriculture and Environment, and ARC Centre of Excellence in Plant Energy BiologyUniversity of Western AustraliaCrawleyAustralia

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