Ectopic expression of a grape vine vacuolar NHX antiporter enhances transgenic potato plant tolerance to salinity

  • Safa Charfeddine
  • Mariam Charfeddine
  • Mohsen Hanana
  • Radhia Gargouri-Bouzid
Original Article


Salinity is a crucial environmental constraint that reduces plant productivity. However, plants activate different signaling pathways to overcome the abiotic stress. The NHX (Na+/H+ exchanger) antiporter corresponds to one of the antiporters involved in response to salinity. They are known to be responsible for the vacuole compartmentation of toxic Na+. In this report, a grapevine vacuolar antiporter (VvNHX1) cDNA was introduced into potato, response of transgenic plants to salinity was evaluated under in vitro and greenhouse culture conditions. The transgenic plants showed higher growth rate than wild type (WT) after the salinity treatment suggesting an improved tolerance both in vitro and under greenhouse culture conditions. In addition, a lower oxidative stress level was observed while a higher relative water and soluble sugar content were measured in transgenic plants compared to WT plants. Furthermore, in contrast to WT plants, the transgenic plants displayed an increase of leaf ion (K+, Mg2+) content and a decline in Na+ accumulation. The increase in the antioxidant enzyme activities in transgenic plants suggests that they can overcome oxidative stress resulting from salt treatment. The measurement of the tuber yield and the weight loss of plants sprinkled with 100 mM NaCl in the greenhouse showed a low negative effect on transgenic plants (12.5 and 40%) in comparison to WT (80%).


Salinity Vacuolar Na+/H+ antiporter VvNHX1 potato 



Reactive oxygen species


Na+/H+ antiporter


Superoxide dismutase




Gluthatione peroxidase




Trichloroacetic acid


Thiobarbituric acid


Relative water content


Analysis of variance


Wild type



This work was financed by the Tunisian Ministry of High Education and Scientific Research. The authors thank to Anne-Lise Haenni from the Institute Jacques Monod (France) and Mrs Najoua Neifar english teacher from the University of Sfax-Tunisia and Miss cyrine Bouzid from ODDO BHF for improving the English throughout the manuscript.

Authors’ contributions

SC and MC carried out the experimental work. The insertion of the VvNHX1 gene into the pCAMBIA99.1 vector was generated by MH. RGB participated in the data analysis and contributed in writing and revising the manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

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  1. Alexieva V, Sergio I, Mapelli S, Karanov E (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell Environ 24:1337–1344CrossRefGoogle Scholar
  2. Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285:1256–1258CrossRefPubMedGoogle Scholar
  3. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta Vulgaris. Plant Physiol 24:1–15CrossRefPubMedPubMedCentralGoogle Scholar
  4. Barragán V, Leidi EO, Andres Z, Rubio L, De Luca A, Fernández JA, Cubero B, Pardo JM (2012) Ion exchangers NHX1 and NHX2 mediate active K uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis. Plant Cell 24:1127–1142CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Brini F, Masmoudi K (2012) Ion transporters and abiotic stress tolerance in plants. ISRN Mol Biotechnol. CrossRefGoogle Scholar
  7. 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 58:301–308CrossRefPubMedGoogle Scholar
  8. Chen HT, Chen X, Wu BY, Yuan XX, Zhang HM, Cui XY, Liu XQ (2015) Whole-genome identification and expression analysis of K+ efflux antiporter (KEA) and Na+/H+ antiporter (NHX) families under abiotic stress in soybean. J Integ Agri 14:1171–1183CrossRefGoogle Scholar
  9. Claiborne A (1985) Catalase activity. In: Greenwald RA (ed) CRC Handbook of methods in oxygen radical research. CRC Press, Boca Raton, pp 283–284Google Scholar
  10. Dellaporta SL, Woods J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21CrossRefGoogle Scholar
  11. Duan XW, Liu T, Zhang DD, Su XG, Lin HT, Jiang YM (2011) Effect of pure oxygen atmosphere on antioxidant enzyme and antioxidant activity of harvested litchi fruit during storage. Food Res Inter 44:1905–1911CrossRefGoogle Scholar
  12. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric Method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  13. Floh L, Gunzler WA (1984) Glutathione peroxidase. Methods Enzymol 105:115–121Google Scholar
  14. Gargouri-Bouzid R, Jaoua L, Ben Mansour R, Hathat Y, Ayadi M, Ellouz R (2005) PVY resistant transgenic potato plants (CV Claustar) expressing the viral coat. Protein Plant Biotechnol 7:143–148Google Scholar
  15. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930CrossRefPubMedGoogle Scholar
  16. 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 48:804–811CrossRefPubMedGoogle Scholar
  17. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463CrossRefPubMedGoogle Scholar
  18. Islam ST, Seraj ZI (2009) Vacuolar Na+/H+ Antiporter expression and salt tolerance conferred by actin1D and CaMV35S are similar in transgenic binna to a rice. Plant Tiss Cult Biotechnol 19:257–262Google Scholar
  19. Kikuchi A, Huynh HD, Endo T, Watanabe K (2015) Review of recent transgenic studies on abiotic stress tolerance and future molecular breeding in potato. Breed Sci 65:85–102CrossRefPubMedPubMedCentralGoogle Scholar
  20. Leidi EO, Barragan V, Rubio L, El-Hamdaoui A, Ruiz T, Cubero B, Fernádez JA, Bressan RA, Hasegawa PM, Quintero FJ, Pardo JM (2010) The AtNHX1 exchanger mediates K compartmentation in vacuoles of transgenic tomato. Plant J 61:495–506CrossRefPubMedGoogle Scholar
  21. Li TX, Zhang Y, Liu H, Ting WY, Li WB, Zhang HX (2010a) Stable expression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 and salt tolerance in transgenic soybean for over six generations. Chin Sci Bull 55:1127–1134CrossRefGoogle Scholar
  22. Li Y, Zhang Y, Feng F, Liang D, Cheng L, Ma F, Shi S (2010b) Overexpression of a Malus vacuolar Na+/H+ antiporter gene (MdNHX1) in apple rootstock M26 and its influence on salt tolerance. Plant Cell Tiss Org Cult 102:337–345CrossRefGoogle Scholar
  23. Liu S, Zheng L, Xue Y, Zhang Q, Wang L, Shou H (2010) Overexpression of OsVP1 and OsNHX1 increases tolerance to drought and salinity in rice. J Plant Biol 53:444–452CrossRefGoogle Scholar
  24. Maathuis FJM (2013) Na + in plants: perception, signaling and regulation of Na + fluxes. J Exp Bot 65:849–858CrossRefPubMedGoogle Scholar
  25. Mishra S, Alavilli H, Lee B, Panda SK, Sahoo L (2014a) Cloning and Functional Characterization of a Vacuolar Na+/H+ antiporter gene from Mungbean (VrNHX1) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mishra S, Behura R, Awasthi JP, Dey M, Sahoo D, Das Bhowmik SS, Panda SK, Sahoo L (2014b) Ectopic overexpression of a mungbean vacuolar Na+/H+ antiporter gene (VrNHX1) leads to increased salinity stress tolerance in transgenic Vignaun guiculata L. Walp Mol Breed 34:1345–1359CrossRefGoogle Scholar
  27. Morel G, Wetmore RH (1951) Fern Callus tissue culture. Am J Bot 38:141–143CrossRefGoogle Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  29. Rodríguez AA, Lascano HR, Bustos D, Taleisnik E (2007) Salinity-induced decrease in NADPH oxidase activity in the maize leaf blade elongation zone. J Plant Physiol 164:223–230CrossRefPubMedGoogle Scholar
  30. Rodríguez-Rosales MP, Gálvez FJ, Huertas R, Aranda MN, Baghour M, Cagnac O, Venema K (2009) Plant NHX cation/proton antiporters. Plant Signal Behav 4:265–276CrossRefPubMedPubMedCentralGoogle Scholar
  31. Saïdi MN, Gargouri-Bouzid R, Rayanni M, Drira N (2009) Optimization of RNA isolation from Brittle leaf disease affected date palm leaves and construction of a subtractive cDNA library. Mol Biotechnol 41:63–68CrossRefGoogle Scholar
  32. Tian N, Wang J, Xu ZQ (2011) Overexpression antiporter gene AtNHX1 from Arabidopsis thaliana improves the salt tolerance of kiwi fruit (Actinidia deliciosa). South Afri J Bot 77:160–169CrossRefGoogle Scholar
  33. Väänänen D, Ikonen T, Rokka VM, Kuronen P, Serimaa R, Ollilainen V (2005) Influence of incorporated wild Solanum genomes on potato properties in terms of starch nanostructure and glycoalkaloid content. J Agri Food Chem 53:5313–5325CrossRefGoogle Scholar
  34. Vaewoerd TC, Dekker BMM, Hoekema A (1989) A small scale procedure for the rapid isolation of plant RNAs. Nucleic Acid Res 17:23–62Google Scholar
  35. Wang LJ, Zhang J, Wang D, Zhang J, Cui Y, Liu Y, Yang H, Binyu (2010) Assessment of salt tolerance in transgenic potato carrying AtNHX1 gene. Crop Sci 53:2643–2651CrossRefGoogle Scholar
  36. Wu XX, Li J, Wu XD, Liu Q, Wang ZK, Liu SS, Li SN, Ma YL, Sun J, Zhao L, Li HY, Li DM, Li WB, Su AY (2016) Ectopic expression of Arabidopsis thaliana Na+(K+)/H+ antiporter gene, AtNHX5, enhances soybean salt tolerance. Genet Mol Res. CrossRefPubMedGoogle Scholar
  37. Yamaguchi T, Fukuda-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 42:451–461CrossRefPubMedGoogle Scholar
  38. Yuanchun M, Wang J, Zhong Y, Geng F, Grant RC, Cheng ZM (2015) Subfunctionalization of cation/proton antiporter 1 genes in grapevine in response to salt stress in different organs. Horti Res 2:15031CrossRefGoogle Scholar
  39. Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19:765–768CrossRefPubMedGoogle Scholar
  40. Zhang YM, Zhang HM, Liu ZH, Li HC, Guo XL, Li GL (2015) The wheat NHX antiporter gene TaNHX2 confers salt tolerance in transgenic alfalfa by increasing the retention capacity of intracellular K. Plant Mol Biotechnol 87:317–327CrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2018

Authors and Affiliations

  • Safa Charfeddine
    • 1
  • Mariam Charfeddine
    • 1
  • Mohsen Hanana
    • 2
  • Radhia Gargouri-Bouzid
    • 1
  1. 1.Laboratory of Plant improvement and Valorization of Agro-ressources, National School of Engineering of Sfax (ENIS)Sfax UniversitySfaxTunisia
  2. 2.Laboratory of Extremophilic PlantBiotechnology Center of Borj CedriaBorj CedriaTunisia

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