Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L.)

  • Kondeti Subramanyam
  • K. V. Sailaja
  • Koona Subramanyam
  • D. Muralidhara Rao
  • K. LakshmideviEmail author
Original Paper


Plants, when exposed to abiotic or biotic stress, produce several pathogenesis-related proteins to counteract the effects of stress. Osmotin is one of the important pathogenesis-related proteins induced during several stress conditions. We have developed improved salt stress tolerant transgenic chilli pepper plants (Capsicum annum L. var. Aiswarya 2103) by ectopic expression of the Nicotiana tabaccum osmotin gene using Agrobacterium tumefaciens EHA105 as a vector. Four-week-old chilli pepper leaves were used as an explant and A. tumefaciens EHA105 harboring pBINASCOSM plasmid that contains osmotin gene under the control of CaMV 35S promoter and npt II as a selectable marker was used in co-cultivation. Transgene integration and expression were analyzed using molecular, immunochemical, and biochemical assays. PCR and Southern blot analysis confirmed that osmotin gene has been successfully integrated into the genome of chilli pepper plants. The osmotin gene was stably segregated and expressed in T2 generation transgenic chilli pepper plants, and it was confirmed by Western blot analysis. Biochemical assays of these putative transgenic plants revealed enhanced levels of chlorophyll, proline, glycinebetaine, APX, SOD, DHAR, MDHAR, GR, and relative water content. Yield potential of the putative transgenic chilli pepper plants was evaluated under salinity stress conditions in a green house. The putative transgenic chilli pepper plants overexpressing the osmotin gene were morphologically similar to wild-type plants and produced 3.32 kg chilli pepper fruits per plant at 300 mM NaCl concentration.


Capsicum annum L. Agrobacterium tumefaciens EHA105 Osmotin gene Binary vector CaMV 35S promoter Western blotting 


CaMV 35S

Cauliflower mosaic virus 35S promoter


Ascorbate peroxidase


Superoxide dismutase


Dehydro ascorbate reductase


Monodehydroascorbate reductase


Glutathione reductase


Murashige and Skoog medium




Indole-3-acetic acid


Gibberellic acid


Indole-3-butyric acid


Silver nitrate



We thank Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India for providing financial support to carry out the present work. The authors are grateful to Prof. M.V. Rajam, Department of Genetics, Delhi University—South campus, India for the critical correction and evaluation of this manuscript.


  1. Aono M, Saji H, Sakamoto A, Tanaka K, Kondo N (1995) Paraquat tolerance of transgenic Nicotiana tabacum with enhanced activities of glutathione reductase and superoxide dismutase. Plant Cell Physiol 36:1687–1691PubMedGoogle Scholar
  2. Aziz A, Larher F (1998) Osmotic stress induced changes in lipid composition and peroxidation in leaf discs of Brassica napus L. J Plant Physiol 153:754–762Google Scholar
  3. Barthakur S, Babu V, Bansal KC (2001) over expression of osmotin induces proline accumulation and confers tolerance to osmotic stress in transgenic tobacco. J Plant Biochem Biotechnol 10:31–37Google Scholar
  4. Bates LS, Waldren RP, Teare JD (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  5. Benavides MP, Marconi PL, Gallego SM, Comba ME, Tomaro ML (2000) Relationship between antioxidant defense system and salt tolerance in Solanum tuberosum. Aust J Plant Physiol 27:273–278Google Scholar
  6. Bressan RA, Singh NK, Handa AK, Mount R, Clithero J, Hasegawa PM (1987) Stability of altered gene expression in cultured plant cells adapted to salt. In: Monti L, Porceddu E (eds) Drought resistance in plants, physiological and genetic aspects. Commission of the European Communities, Brussels, pp 41–57Google Scholar
  7. Brini F, Hanin M, Lumbreras V, Amara J, Khoudi H et al (2007) Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana. Plant Cell Rep 26:2017–2026PubMedCrossRefGoogle Scholar
  8. 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 30:987–998Google Scholar
  9. Chen Ni, Liu Yan, Liu Xin, Chai Juan, Zhang Hu, Guo Guangqiv, Liu Heng (2009) Enhanced tolerance to water deficit and salinity stress in transgenic Lycium barbarum L. plants ectopically expressing ATHK1, an Arabidopsis thaliana histidine kinase gene. Plant Mol Biol Rep 27:321–333CrossRefGoogle Scholar
  10. Cromwell BT, Rennie SD (1953) The estimation and distribution of glycinebetaine (Betaine) in Beta vulgaris L. and other plants. Biochem J 55(1):189–192Google Scholar
  11. Della Porta SL, Wood J, Hicks JB (1983) A plant DNA mini preparation: verson II. Plant Mol Biol Rep 1:19–21CrossRefGoogle Scholar
  12. Dhindsa RS, Matowe W (1981) Drought tolerance in two mosses correlated with enzymatic defense against lipid peroxidation. J Exp Bot 22:79–91CrossRefGoogle Scholar
  13. Doulis AG, Debian N, Kingston-Smith AH, Foyer CH (1997) Differential localization of antioxidants in maize leaves. Plant Physiol 114:1031–1037PubMedGoogle Scholar
  14. Fan L, Zhen S, Wang X (1997) Antisense suppression of phospholipase D retards abscisic acid and ethylene promoted senescence of postharvest Arabidopsis leaves. Plant Cell 9:2183–2196PubMedCrossRefGoogle Scholar
  15. Flowers TJ, Yeo AR (1986) Ion relations of plants under drought and salinity. Aust J Plant Physiol 13:75–91CrossRefGoogle Scholar
  16. Flowers TJ, Yeo AR (1995) Breeding for salinity resistance in crop plants-Where next? Aust J Plant Physiol 22:875–884CrossRefGoogle Scholar
  17. Floyd RA, Nagy ZS (1984) Formation of long lived hydroxyl free radical adducts of proline and hydroxyl-proline in a Fenton reaction. Biochim Biophys Acta 790:94–97PubMedCrossRefGoogle Scholar
  18. Foyer CH, Halliwell B (1976) The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133:21–25CrossRefGoogle Scholar
  19. Foyer C, Lelandais M, Galap C, Kunert KJ (1991) Effect of elevated cytosolic glutathione reductase activity on the cellular glutathione pool and photosynthesis in leaves under normal and stress conditions. Plant Physiol 97:863–872PubMedCrossRefGoogle Scholar
  20. Foyer CH, Souriau N, Perret S, Lelandais M, Kunert KJ, Pruvost C (1995) Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance in photo inhibition in poplar trees. Plant Physiol 109:1047–1057PubMedCrossRefGoogle Scholar
  21. Gao S-Q, Chen M, Xia L-Q, Xiu H-J, Xu Z-S, Li L-C, Zhao C-P, Cheng X-G, Ma Y-Z (2009) A cotton (Gossypium hirsutum) DRE-binding transcription factor gene, GhDREB, confers enhanced tolerance to drought, high salt, and freezing stresses in transgenic wheat. Plant Cell Rep 28:301–311PubMedCrossRefGoogle Scholar
  22. Giannopolitis CN, Ries SK (1977) Superoxide dismutases. I. Occurrence in higher planta. Plant Physiol 59:309–314PubMedCrossRefGoogle Scholar
  23. Gilmour SJ, Zarka DG, Stockinger EJ (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcription activators as an early step in cold-induced cor gene expression. Plant J 16:433–442PubMedCrossRefGoogle Scholar
  24. Hiscox JD, Israelstam GF (1979) A method for extraction of Chlorophyll from leaf tissue without maceration. Can J Bot 59:463–469Google Scholar
  25. Hong ZL, Lakkineni K, Zhang ZM, Verma DPS (2000) Removal of feedback inhibition of DELTA1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136PubMedCrossRefGoogle Scholar
  26. Husaini AM, Abdin MZ (2008) Development of transgenic strawberry (Fragaria x ananassa Dutch.) plants tolerant to salt stress. Plant Sci 174:446–455CrossRefGoogle Scholar
  27. La Rosa PC, Chen Z, Nelson DE, Singh NK, Hasegawa PM, Bressan RA (1992) Osmotin gene expression is post transcriptionally regulated. Plant Physiol 100:409–415CrossRefGoogle Scholar
  28. Linthorst HJM (1991) Pathogenesis related proteins of plants. Crit Rev Plant Sci 10:123–150CrossRefGoogle Scholar
  29. Maas EV, Hoffman GJ (1977) Crop salt tolerance—current assessment. J Irrigation Drainage Div 103:115–134Google Scholar
  30. Miyake C, Asada K (1992) Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in the thylakoids. Plant Cell Physiol 35:539–549Google Scholar
  31. Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250PubMedCrossRefGoogle Scholar
  32. Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279CrossRefGoogle Scholar
  33. Parkhi V, Kumar V, Sunilkumar G, Campbell LAM, Singh NK, Rathore KS (2009) Expression of apoplastically secreted tobacco osmotin in cotton confers drought tolerance. Mol Breed 23:625–639CrossRefGoogle Scholar
  34. Raghothama KG, Liu D, Nelson DE, Hasegawa PM, Bressan RA (1993) Analysis of an osmotically regulated pathogenesis related osmotin gene promoter. Plant Mol Boil 23:1117–1128CrossRefGoogle Scholar
  35. Sambrook J, Fritch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, NYGoogle Scholar
  36. Sarad N, Rathore M, Singh NK, Kumar N (2004) Genetically engineered tomatoes: new vista for sustainable agriculture in high altitude regions. In: Proceedings of the Fourth International Crop Science Congress Brisbane, AustraliaGoogle Scholar
  37. Shalata A, Tal M (1998) The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiol Plant 104:169–174CrossRefGoogle Scholar
  38. Shannon MC, Grieve CM (1999) Tolerance of vegetable crops to salinity. Sci Hortic 78:5–38CrossRefGoogle Scholar
  39. Sinclair TR, Ludlow MM (1985) Who taught plants thermodynamics? The unfulfilled potential of plant water potential. Aust J Plant Physiol 12:213–217CrossRefGoogle Scholar
  40. Singh NK, Handa AK, Hasegawa PM, Bressan RA (1985) Proteins associated with adaptation of cultured tobacco cells to NaCl. Plant Physiol 79:126–137PubMedCrossRefGoogle Scholar
  41. Singh NK, Bracker CA, Hasegava PM et al (1987) Characterization of osmotin. Plant Physiol 85:529–536PubMedCrossRefGoogle Scholar
  42. Skriver K, Mundy J (1990) Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503–512PubMedCrossRefGoogle Scholar
  43. Sokhansanj S, Sadat N, Niknam V (2006) Comparison of bacterial and plant genes participating in proline biosynthesis with osmotin gene, with respect to enhancing salinity tolerance of transgenic tobacco plants, Russ. J Plant Physiol 53:110–115Google Scholar
  44. Velikova V, Yordanov J, Edreva A (2000) Oxidative stress and some antioxidant systems in acid rain treated bean plants. Protective role of exogenous polyamines. Plant Sci 151:59–66CrossRefGoogle Scholar
  45. Wang Y, Wisniewski M, Meilen R, Uratsu SL, Cui M, Dandekar A, Fuchigami L (2007) Ectopic expression of Mn-SOD in Lycopercicum esculentum leads to enhanced tolerance to salt and oxidative stress. J Appl Horti 9(1):3–8Google Scholar
  46. Wu FB, Zhang GP, Dominy P (2003) Four barley genotypes respond differently to cadmium: lipid peroxidation and activities of antioxidant capacity. Environ Exp Bot 50:67–78CrossRefGoogle Scholar
  47. Xiong L, Zhu JK (2002) Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ 25:131–139PubMedCrossRefGoogle Scholar
  48. Yamada M, Morishita H, Urano K et al (2005) Effects of free proline accumulation in petunias under drought stress. J Exp Bot 56:1975–1981PubMedCrossRefGoogle Scholar
  49. Zhang HW, Huang ZJ, Xie BY, Chen Q, Tian X, Zhang XL, Zhang HB, Lu XY, Huang DF, Huang RF (2004) The ethylene, jasmonate, abscisic acid and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box containing genes and salt tolerance in tobacco. Planta 220:262–270PubMedCrossRefGoogle Scholar
  50. Zhu B, Chen THH, Li PH (1995) Activation of two osmotin-like protein genes by abiotic stimuli and fungal pathogen in transgenic potato plants. Plant Physiol 108:929–937PubMedCrossRefGoogle Scholar
  51. Zhu B, Chen THH, Li PH (1996) Analysis of late blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein. Planta 198:70–77PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Kondeti Subramanyam
    • 1
  • K. V. Sailaja
    • 1
  • Koona Subramanyam
    • 2
  • D. Muralidhara Rao
    • 3
  • K. Lakshmidevi
    • 1
    Email author
  1. 1.Department of BiochemistrySri Krishnadevaraya UniversityAnantapurIndia
  2. 2.Department of BiotechnologySreenidhi Institute of Science and Technology (Autonomous), Jawaharlal Nehru Technological UniversityHyderabadIndia
  3. 3.Department of BiotechnologySri Krishnadevaraya UniversityAnantapurIndia

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