Functional & Integrative Genomics

, Volume 19, Issue 4, pp 541–554 | Cite as

Expressing class I wheat NHX (TaNHX2) gene in eggplant (Solanum melongena L.) improves plant performance under saline condition

  • Rajesh YarraEmail author
  • P. B. Kirti
Original Article


Brinjal or eggplant (Solanum melongena L.) is an important solanaceous edible crop, and salt stress adversely affects its growth, development, and overall productivity. To cope with excess salinity, vacuolar Na+/H+ antiporters provide the best mechanism for ionic homeostasis in plants under salt stress. We generated transgenic eggplants by introducing wheat TaNHX2 gene that encodes a vacuolar Na+/H+ antiporter in to the eggplant genome via Agrobacterium-mediated transformation using pBin438 vector that harbors double35S:TaNHX2 to confer salinity tolerance. Polymerase chain reaction and southern hybridization confirmed the presence and integration of TaNHX2 gene in T1 transgenic plants. Southern positive transgenic eggplants showed varied levels of TaNHX2 transcripts as evident by RT-PCR and qRT-PCR. Stress-inducible expression of TaNHX2 significantly improved growth performance and Na+ and K+ contents from leaf and roots tissues of T2 transgenic eggplants under salt stress, compared to non-transformed plants. Furthermore, T2 transgenic eggplants displayed the stable leaf relative water content and chlorophyll content, proline accumulation, improved photosynthetic efficiency, transpiration rate, and stomatal conductivity than the non-transformed plants under salinity stress (200 mM NaCl). Data showed that the T2 transgenic lines revealed that reduction in MDA content, hydrogen peroxide, and oxygen radical production associated with the significant increase of antioxidant enzyme activity in transgenic eggplants than non-transformed plants under salt stress (200 mM NaCl). This study suggested that the TaNHX2 gene plays an important regulatory role in conferring salinity tolerance of transgenic eggplant and thus may serve as a useful candidate gene for improving salinity tolerance in other vegetable crops.


TaNHX2 Solanum melongena Salt stress Vegetables 



Reverse transcription PCR


Quantitative real-time PCR


Superoxide dismutase


Ascorbate peroxidase


Guaiacol peroxidase


Glutathione reductase





Authors acknowledge the Head, Department of Plant Sciences for access to the research facilities provided by DST-FIST, DBT-CREBB, and UGC-SAP to the Department of Plant Sciences, University of Hyderabad. The authors are thankful to Prof. Shouyi Chen and Prof. Jinsong Zhang, Institute of Genetics and Developmental Biology, CAS, Beijing for generous offer of the plasmid used in this study. We thank the anonymous reviewers for their valuable comments in improving the manuscript.

Author contributions

RY and PBK conceived the experiment. RY performed the experiment. RY and PBK analyzed the data. RY and PBK wrote the manuscript. All authors approved the final version of the manuscript.

Funding information

The Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India provided fund and fellowship under Young Scientist Scheme (SB/FT/LS-445/2012).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10142_2019_656_MOESM1_ESM.docx (234 kb)
ESM 1 (DOCX 234 kb)


  1. Abbas W, Ashraf M, Akram NA (2010) Alleviation of salt-induced adverse effects in eggplant (Solanum melongena L.) by glycinebetaine and sugarbeet extracts. Sci Hortic 125:188–195CrossRefGoogle Scholar
  2. Akinci IE, Akinci S, Yilmaz K, Dikici H (2004) Response of eggplant varieties (Solanum melongena) to salinity in germination and seedling stages. New Zealand J Crop and Hort Sci 32:193–200CrossRefGoogle Scholar
  3. Almeida DM, Oliveira MM, Saibo NJM (2017) Regulation of Na+ and K+ homeostasis in plants: towards improved salt tolerance in crop plants. Genet Mol Biol 40:326–345CrossRefGoogle Scholar
  4. Armengaud P, Thiery L, Buhot N, Grenier-De March G, Savoure A (2004) Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol Plant 120:442–450CrossRefGoogle Scholar
  5. Ashraf M, Akram NA, Al-Qurainy F, Foolad MR (2011) Drought tolerance: roles of organic osmolytes, growth regulators and mineral nutrients. Adv Agron 111:24996Google Scholar
  6. AVRDC (2006) Proceedings of the 2006 APSA-AVRDC workshop. AVRDC-The world vegetable center, Shanhua, Tainan, Taiwan. AVRDC Publication, p 06–677Google Scholar
  7. Bassil E, Blumwald E (2014) The ins and outs of intracellular ion homeostasis: NHX-type cation/H+ transporters. Curr Opin Plant Biol 22:1–6CrossRefGoogle Scholar
  8. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  9. Bhaskaran S, Savithramma DL (2011) Co-expression of Pennisetum glaucum vacuolar Na+/H+ antiporter and Arabidopsis H+-pyrophosphatase enhances salt tolerance in transgenic tomato. J Exp Bot 62:5561–5570CrossRefGoogle Scholar
  10. Blumwald E (2000) Sodium transport and salt tolerance in plants. Curr Opin Cell Biol 12:431–434CrossRefGoogle Scholar
  11. Bresler E, McNeal BL, Carter DL (1982) Saline and sodic soils. Springer-Verlag, BerlinCrossRefGoogle Scholar
  12. 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 43:347–354CrossRefGoogle Scholar
  13. Brini F, Hanin M, Mezghani I, Berkowitz GA, Masmoudi K (2007) Overexpression of wheat Na+/H+ antiporter TNHX1 and Hþ-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants. J Exp Bot 58:301–308CrossRefGoogle Scholar
  14. Bulle M, Yarra R, Abbagani S (2016) Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene. Mol Breed 36:36. CrossRefGoogle Scholar
  15. Cao D, Hou W, Liu W, Yao WW, Wu C, Liu X, Han T (2011) Overexpression of TaNHX2 enhances salt tolerance of ‘composite’ and whole transgenic soybean plants. Plant Cell Tissue Organ Cult 107:541–552CrossRefGoogle Scholar
  16. Chance B, Maehly AC (1955) Assay of catalase and peroxidases. Methods Enzymol 2:764–775CrossRefGoogle Scholar
  17. Chen GX, Asada K (1989) Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol 30(7):987–998Google Scholar
  18. Chen H, An R, Tang JH, Cui XH, Hao FS, Chen J, Wang XC (2007) Over-expression of a vacuolar Na+/H+antiporter gene improves salt tolerance in an upland rice. Mol Breed 19:215–225CrossRefGoogle Scholar
  19. Dadkhah AR, Grrifiths H (2006) The effect of salinity on growth, inorganic ions and dry matter partitioning in sugar beet cultivars. J Agric Sci Technol 8:199–210Google Scholar
  20. Daunay M (2008) Eggplant. In: Vegetables II, Prohens J, Nuez F (eds) Handbook of plant breeding. Springer, New York, pp 163–220Google Scholar
  21. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Report 1:19–21CrossRefGoogle Scholar
  22. Doganlar S, Frary A, Daunay MC, Lester RN, Tanksley SD (2002) A comparative genetic linkage map of eggplant (Solanum melongena) and its implications for genome evolution in the solanaceae. Genetics 161:1697–1711Google Scholar
  23. Elstner EF, Heupel A (1976) Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase. Anal Biochem 70:616–620CrossRefGoogle Scholar
  24. Fan W, Deng G, Wang H, Zhang H, Zhang P (2015) Elevated compartmentalization of Na+ into vacuoles improves salt and cold stress tolerance in sweet potato (Ipomoea batatas). Physiol Plant 154:560–571CrossRefGoogle Scholar
  25. FAO (2002) Working with local institutions to support sustainable livelihoods. Food and Agriculture Organization, RomeGoogle Scholar
  26. Fukuda A, Nakamura A, Tanaka Y (1999) Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochim Biophys Acta 1446:149–155CrossRefGoogle Scholar
  27. Gantasala NP, Papolu PK, Thakur PK, Kamaraju D, Sreevathsa R, Rao U (2013) Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L). BMC Res Notes 6:312CrossRefGoogle Scholar
  28. Gaxiola RA, Rao R, Sherman A, Grisafi F, Alper SL, Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNHX1 and AVP1, can function in cation detoxification in yeast. Proc Natl Acad Sci U S A 96:1480–1485CrossRefGoogle Scholar
  29. Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001) Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci U S A 98:11444–11449CrossRefGoogle Scholar
  30. Gaxiola RA, Palmgren MG, Schumacher K (2007) Plant proton pumps. FEBS Lett 581:2204–2214CrossRefGoogle Scholar
  31. Gouiaa S, Khoudi H, Leidi EO, Pardo JM, Masmoudi K (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(1):137–155CrossRefGoogle Scholar
  32. Haripriya D, Selvan N, Jeyakumar N, Periasamy R, Marimuthu J, Irudayaraj V (2010) The effect of extracts of Selaginella involvens and Selaginella inaequalifolia leaves on poultry pathogens. Asian Pac J Trop Med 3:67881Google Scholar
  33. Hasegawa PM (2013) Sodium (Na+) homeostasis and salt tolerance of plants. Environ Exp Bot 92:19–31CrossRefGoogle Scholar
  34. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198CrossRefGoogle Scholar
  35. Hiscox JD, Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57:1332–1334CrossRefGoogle Scholar
  36. Jaleel CA, Riadh K, Gopi R, Manivannan P, Ines J, Al-Juburi HJ et al (2009) Antioxidant defense response: physiological plasticity in higher plants under abiotic constrains. Acta Physiol Plant 31:427–436CrossRefGoogle Scholar
  37. Jiang X, Leidi EO, Pardo JM (2010) How do vacuolar NHX exchangers function in plant salt tolerance? Plant Signal Behav 55:792–795CrossRefGoogle Scholar
  38. Koike M, Sugimoto M, Aiuchi D, Nagao H, Shinya R, Tani M, Kuramochi K (2007) Reclassification of Japanese isolate of Verticillium lecanii to Lecanicillium spp. Jpn J Appl Entomol Zool 51:234–237CrossRefGoogle Scholar
  39. Kumar SK, Sivanesan I, Murugesan K, Jeong BR, Hwang SJ et al (2014) Enhancing salt tolerance in eggplant by introduction of foreign halotolerance gene, HAL1isolated from yeast. Hortic Environ Biotechnol 55:222–229. CrossRefGoogle Scholar
  40. Kumar S, Kalita A, Srivastava R, Sahoo L (2017) Co-expression of Arabidopsis NHX1 and bar improves the tolerance to salinity, oxidative stress, and herbicide in transgenic mungbean. Front Plant Sci 8:1896CrossRefGoogle Scholar
  41. Leidi EO, Barragan V, Rubio L, El-Hamdaoui A, Ruiz MT, Cubero B, Fernandez JA, Bressan RA, Hasegawa PM, Quintero FJ, Pardo JM (2010) The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato. Plant J 61:495–450CrossRefGoogle Scholar
  42. Li J, Jiang G, Huang P, Ma J, Zhang F (2007) Overexpression of the Na+/H+ antiporter gene from Suaeda salsa confers cold and salt tolerance to transgenic Arabidopsis thaliana. Plant Cell Tissue Organ Cult 90:41–48CrossRefGoogle Scholar
  43. Li W, Wang D, Jin T, Chang Q, Yin D, Xu S, Liu B, Liu L (2011) The vacuolar Na+/H+ antiporter gene SsNHX1 from the halophyte Salsola soda confers salt tolerance in transgenic alfalfa (Medicago sativa L.). Plant Mol Biol Report 29:278–290CrossRefGoogle Scholar
  44. Li N, Wang X, Ma B, Du C, Zheng L, Wang Y (2017) Expression of a Na+/H+ antiporter RtNHX1 from a recretohalophyte Reaumuria trigyna improved salt tolerance of transgenic Arabidopsis thaliana. J Plant Physiol 218:109–120CrossRefGoogle Scholar
  45. Liang W, Ma X, Wan P, Liu L (2018) Plant salt-tolerance mechanism: a review. Biochem Biophys Res Commun 495:286–291CrossRefGoogle Scholar
  46. Liu J, Zhu JK (1997) Proline accumulation and salt–stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114:591–596CrossRefGoogle Scholar
  47. Lu W, Guo C, Li X, Duan W, Ma C, Zhao M, Gu J, Du X, Liu Z, Xiao K (2014) Overexpression of TaNHX3, a vacuolar Na+/H+ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes. Plant Physiol Biochem 76:17–28CrossRefGoogle Scholar
  48. McCubbin T, Bassil E, Zhang S, Blumwald E (2014) Vacuolar Na+/H+ NHX-type antiporters are required for cellular K+ homeostasis, microtubule organization and directional root growth. Plants 3:409–426CrossRefGoogle Scholar
  49. Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33:453–467CrossRefGoogle Scholar
  50. Mozafariyan M, Bayat KSAE, Bakhtiari S (2013) The effects of different sodium chloride concentrations on the growth and photosynthesis parameters of tomato (Lycopersicum esculentum cv. Foria). Int J Agri Crop Sci 6(4):203–207Google Scholar
  51. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays of tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  52. Prabhavathi VR, Rajam MV (2007) Polyamine accumulation in transgenic eggplant enhances tolerance to multiple abiotic stresses and fungal resistance. Plant Biotechnol 24:273–282CrossRefGoogle Scholar
  53. Prabhavathi V, Yadav JS, Kumar PA, Rajam MV (2002) Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene. Mol Breed 9:137–147CrossRefGoogle Scholar
  54. Prasad SM, Parihar P, Singh VP (2014) Effect of salt stress on nutritional value of vegetables. Biochem Pharmacol 3:e160. CrossRefGoogle Scholar
  55. Sagisaka S (1976) The occurrence of peroxide in a perennial plant, Populus gelrica. Plant Physiol 57:308–309CrossRefGoogle Scholar
  56. Sahoo DB, Kumar S, Mishra S, Kobayashi Y, Panda SK, Sahoo L (2016) Enhanced salinity tolerance in transgenic mungbean overexpressing Arabidopsis antiporter (NHX1) gene. Mol Breed 36:144. CrossRefGoogle Scholar
  57. Shahbaz M, Ashraf M, Al-Qurainy F, Harris PJC (2012) Salt tolerance in selected vegetable crops. Crit Rev Plant Sci 31(4):303–320. CrossRefGoogle Scholar
  58. Shaheen S, Naseer S, Ashraf M, Akram NA (2013) Salt stress affects water relations, photosynthesis, and oxidative defense mechanisms in Solanum melongena L. J Plant Interact 8:85–96CrossRefGoogle Scholar
  59. Shalhevet J, Heuer B, Meiri A (1983) Irrigation interval as a factor in the salt tolerance of eggplant. Irrig Sci 4:83–93CrossRefGoogle Scholar
  60. Shi H, Lee BH, Wu SJ et al (2002) Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21:81–85CrossRefGoogle Scholar
  61. Sixto H, Aranda I, Grau JM (2006) Assessment of salt tolerance in Populus alba clones using chlorophyll fluorescence. Photosynthetica 44:169–173CrossRefGoogle Scholar
  62. Smith IK, Vierheller TL, Thorne CA (1988) Assay of glutathione reductase in crude tissue homogenates using 5,50-dithiobis (2-nitrobenzoic acid). Anal Biochem 175:408–413CrossRefGoogle Scholar
  63. Tang R, Li C, Xu K, Du Y, Xia T (2010) Isolation, functional characterization, and expression pattern of a vacuolar Na(+)/H(+) antiporter gene TrNHX1 from Trifolium repens L. Plant Mol Biol Report 28:102–111CrossRefGoogle Scholar
  64. Unlukara A, Kurunc A, Kesmez GD, Yurtseven E, Suarez DL (2010) Effects of salinity on eggplant (Solanum melongena L.) growth and evapotranspiration. Irrig Drain 59:203–214Google Scholar
  65. Verslues PE, Batelli G, Grillo S, Agius F, Kim YS, Zhu JH, Agarwal M, Katiyar-Agarwal S, Zhu JK (2007) Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signalling in Arabidopsis thaliana. Mol Cell Biol 27:7771–7780CrossRefGoogle Scholar
  66. Wang N, Hua H, EgrinyaEneji A, Li Z, Duan L, Tian X (2012) Genotypic variations in photosynthetic and physiological adjustment to potassium deficiency in cotton (Gossypium hirsutum L.). J. Photochem Photobiol 110:1–8CrossRefGoogle Scholar
  67. Wang B, Zhai H, He S, Zhang H, Ren Z, Zhang D, Liu Q (2016) A vacuolar Na+/H+, antiporter gene, IbNHX2, enhances salt and drought tolerance in transgenic sweetpotato. Sci Hortic 201:153–166CrossRefGoogle Scholar
  68. Wei Q, Guo YJ, Cao HM, Kuai BK (2011) Cloning and characterization of an AtNHX2-like Na+/H+ antiporter gene from Ammopiptanthus mongolicus (Leguminosae) and its ectopic expression enhanced drought and salt tolerance in Arabidopsis thaliana. Plant Cell Tissue Organ Cult 105:309–316CrossRefGoogle Scholar
  69. Wu M, Chen W, Zhao Y, Feng SG, Ying QC, Liu JJ, Wang HZ (2012) Salt tolerance enhancement of transgenic rice with Na+/H+ antiporter gene driven by root specific promoter PmPgPR10. Chin J. Rice Sci 26:643–650Google Scholar
  70. 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 45:600–607CrossRefGoogle Scholar
  71. 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 116:206–212CrossRefGoogle Scholar
  72. Yadav S, Irfan M, Ahmad A, Hayat S (2011) Causes of salinity and plant manifestations to salt stress: a review. J Environ Biol 32:667–685Google Scholar
  73. Yamaguchi T, Hamamoto S, Uozumi N (2013) Sodium transport system in plant cells. Front Plant Sci 4:410CrossRefGoogle Scholar
  74. Yarra R, He SJ, Abbagani S, Ma B, Bulle M, Zhang WK (2012) Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.). Plant Cell Tissue Organ Cult 111(1):49–57CrossRefGoogle Scholar
  75. Yu JN, Huang J, Wang ZN, Zhang JS, Chen SY (2007) An Na+/H+ antiporter gene from wheat plays an important role in stress tolerance. J Biosci 32:1153–1161CrossRefGoogle Scholar
  76. Zeng Y, Li Q, Wang H, Zhang J, Du J, Feng H, Blumwald E, Yu L, Xu G (2017) Two NHX-type transporters from Helianthus tuberosus improve the tolerance of rice to salinity and nutrient deficiency stress. Plant Biotechnol J 16:310–321. CrossRefGoogle Scholar
  77. 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 potassium. Plant Mol Biol 87:317–327CrossRefGoogle Scholar
  78. Zhu JK (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445CrossRefGoogle Scholar
  79. Zhuang J, Zhang J, Hou XL, Wang F, Xiong AS (2014) Transcriptomic, proteomic, metabolomic and functional genomic approaches for the study of abiotic stress in vegetable crops. Crit Rev Plant Sci 33(2–3):225–237CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Plant Sciences, School of Life SciencesUniversity of HyderabadHyderabadIndia

Personalised recommendations