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Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections


Salinity and fungal diseases are the two significant constraints limiting soybean productivity. In order to address these problems, we have transformed soybean cv. Pusa 16 via somatic embryogenesis with salinity induced and apoplastically secreted pathogenesis-related tobacco osmotin (Tbosm) gene using Agrobacterium-mediated genetic transformation. Integration of Tbosm in randomly selected five GUS assay-positive independently transformed soybean plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Reverse transcriptase-PCR (RT-PCR) and Western blotting confirmed that the Tbosm was expressed in three of the five transformed soybean plants. Further the Western blotting revealed that the truncated osmotin protein accumulated more in apoplastic fluid. The transformed (T1) soybean plants survived up to 200 mM NaCl, whereas non-transformed (NT) plants could withstand till 100 mM and perished at 150 mM NaCl. The biochemical analysis revealed the T1 soybean plants accumulated higher amount of proline, chlorophyll, APX, CAT, SOD, DHAR, MDHAR, and RWC than NT plants. Leaf gas exchange measurements revealed that T1 soybean plants maintained higher net photosynthetic rate, CO2 assimilation, and stomatal conductance than NT plants. The three T1 soybean plants expressing the osmotin gene also showed resistance against three important fungal pathogens of soybean—Microsphaera diffusa, Septoria glycines and Phakopsora pachyrhizi. The T1 soybean plants produced 32–35 soybean pods/plant containing 10.3–12.0 g of seeds at 200 mM NaCl, whereas NT plant produced 28.6 soybean pods containing 9.6 g of seeds at 100 mM NaCl. The present investigation clearly shows that expression of Tbosm enhances salinity tolerance and fungal disease resistance in transformed soybean plants.

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  1. Abdin MZ, Kiran U, Alam A (2011) Analysis of osmotin, a PR protein as metabolic modulator in plants. Bioinformation 5(8):336–340

  2. Able GH, MacKenzie AJ (1964) Salt tolerance of soybean varieties (Glycine max L. Merrill) during germination and later growth. Crop Sci 4:157–161

  3. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:21–126

  4. Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–1274

  5. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24(1):1–15

  6. Barthakur S, Babu V, Bansal KC (2001) Overexpression of osmotin induces proline accumulation and confers tolerance to osmotic stress in transgenic tobacco. J Plant Biochem Biotechnol 10:31–37

  7. Bartoli CG, Gomez F, Martinez DE, Guiamet JJ (2004) Mitochondria are the main target for oxidative damage in leaves of wheat (Triticum aestivum L.). J Exp Bot 55:1663–1669

  8. Bates LS, Waldren RP, Teare JD (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207

  9. Birt DF, Hendrich S, Anthony M, Alkel DL (2004) Soybeans and the prevention of chronic human disease. Soybeans: Improvement, production and uses. In: Specht J, BOerma R (eds), 3rd edn. American society of Agronomy, Madison, WI. pp 1047–1117

  10. Chang RZ, Chen YW, Shao GH, Wan CW (1994) Effect of salt stress on agronomic characters and chemical quality of seeds in soybean. Soybean Sci 13:101–105

  11. Checker VG, Chhibbar AK, Khurana P (2011) Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress. Transgenic Res. doi:10.1007/s11248-011-9577-8

  12. 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 30:987–998

  13. Chen N, Liu Y, Liu X, Chai J, Hu Z, Guo G, Liu H (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 27:321–333

  14. Chen S, Cui X, Chen Y, Gu C, Miao H, Gao H, Chen F, Liu Z, Guan Z, Fang W (2011) CgDREBa transgenic Chrysanthemum confers drought and salinity tolerance. Environ Exp Bot 74:255–260

  15. Das M, Chauhan H, Chhibbar A, Rizwanul Haq QM, Khurana P (2011) High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus indica cv. K2, by constitutive and inducible expression of tobacco osmotin. Transgenic Res 20:231–246

  16. Delgado MJ, Ligero F, Liuch C (1994) Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plant. Soil Biol Biochem 26:371–376

  17. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini preparation: Version II. Plant Mol Biol Rep 1:19–21

  18. Doulis AG, Debian N, Kingston-Smith AH, Foyer CH (1997) Differential localization of antioxidants in maize leaves. Plant Physiol 114:1031–1037

  19. Elsheikh EAE, Wood M (1995) Nodulation and N2 fixation by soybean inoculated with salt-tolerant rhizobia or salt-sensitive bradyrhizobia in saline soil. Soil Biol Biochem 27:657–661

  20. FAO (2008)

  21. FAOSTAT (2009) Agricultural data. Available on

  22. Flower DJ, Ludlow MM (1986) Contribution of osmotic adjustment to the dehydration tolerance of water-stressed pigeon pea [Cajanus cajan (L.) Mill sp.] leaves. Plant Cell Environ 9:33–40

  23. Foyer CH, Descourvieres P, Kunert KJ (1994) Protection against oxygen radicals: an important defense mechanism studied in transgenic plants. Plant Cell Environ 17:507–523

  24. Fryer MJ, Andrews JR, Oxborough K, Blowere DA, Baker NR (1998) Relationship between CO2 assimilation, photosynthetic electron transport and active O2 metabolism in leaves of maize in the field during periods of low temperature. Plant Physiol 116:571–580

  25. Fu XZ, Khan EU, Hu SS, Fan QJ, Liu JH (2011) Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.). Environ Exp Bot 74:106–113

  26. Gao SQ, Chen M, Xia LQ, Xiu HJ, Xu ZS, Li LC, Zhao CP, Chen XG, Ma YZ (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 28:301–311

  27. Goel D, Singh AK, Yadav V, Babbar SB, Bansal KC (2010) Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.). Protoplasma 245:133–141

  28. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

  29. Houot V, Etienne P, Petitot AS, Barbier S, Blein JP, Suty L (2001) Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner. J Exp Bot 52:1721–1730

  30. Hu L, Lu H, Liu Q, Chen X, Jiang X (2005) Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol. Tree Physiol 25:1273–1281

  31. Husaini AM, Abdin MZ (2008) Development of transgenic strawberry (Fragaria × ananassa Dutch.) plants tolerant to salt stress. Plant Sci 174:446–455

  32. Jefferson RA, Kavanagh TA, Bevan NW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

  33. Larosa PC, Chen Z, Nelson DE, Singh NK, Hasegawa PM, Bressan RA (1992) Osmotin gene expression is post-transcriptionally regulated. Plant Physiol 100:409–415

  34. Lauchli A (1984) Salt exclusion: an adaptation of legume crops and pastures under saline conditions. In: Staples RC, Toeniessen GH (eds) Salinity tolerance in plants: strategies for crop improvement. Wiley, New York, pp 171–187

  35. Light GG, Mahan JR, Roxas VP, Allen RD (2005) Transgenic cotton (Gossypium hirsutum L.) seedlings expressing a tobacco glutathione S-transferase fail to provide improved stress tolerance. Planta 222:346–354

  36. Liu D, Raghothama KG, Hasegawa PM, Bressan RA (1994) Osmotin overexpression in potato delays development of disease symptoms. Proc Natl Acad Sci USA 91:1888–1892

  37. Liu D, Rhodes D, D’Urzo MP, Xu Y, Narasimhan ML, Hasegawa PM, Bressan RA, Abad L (1996) In vivo and in vitro activity of truncated osmotin that is secreted into the extracellular matrix. Plant Sci 121:123–131

  38. Malatrasi M, Close TJ, Marmiroli N (2002) Identification and mapping of a putative stress response regulator gene in barley. Plant Mol Biol 50:143–152

  39. Mariashibu TS, Subramanyam K, Arun M, Mayavan S, Rajesh M, Theboral J, Manickavasagam M, Ganapathi A (2012) Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars. Acta Physiol Plant. doi:10.1007/s11738-012-1046-3

  40. McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055

  41. 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 35:539–549

  42. Narasimhan ML, Damsz B, Coca MA, Ibeas JI, Yun DJ, Pardo JM, Hasegawa PM, Bressan RA (2001) A plant defense response effector induces microbial apoptosis. Mol Cell 8:921–930

  43. Narasimhan ML, Lee H, Damsz B, Singh NK, Ibeas JL, Mat sumoto TK, Woloshuk CP, Bressan RA (2003) Overexpression of a cell wall glycoprotein in Fusarium oxysporum increases virulence and resistance to a plant PR-5 protein. Plant J 36:390–400

  44. Narasimhan ML, Coca MA, Jin J, Yamauchi T, Ito Y, Kadowaki T, Kim KK, Pardo JM, Damsz B, Hasegawa PM, Yun DJ, Bressan RA (2005) Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of mammalian adiponectin receptor. Mol Cell 17:171–180

  45. Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279

  46. Noori SAS, Sokhansanj A (2008) Wheat plants containing an osmotin gene show enhanced ability to produce roots at high NaCl concentration. Russ J Plant Physiol 55:256–258

  47. Parida AK, Das AB (2005) Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf 60:324–349

  48. 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 23:625–639

  49. Pasapula V, Shen G, Kuppu S, Paez-Valencia J, Mendoza M, Hou P, Chen J, Qiu X, Zhu L, Zhang X, Auld D, Blumwald E, Zhang H, Gaxiola R, Payton P (2011) Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought-and salt tolerance and increases fiber yield in the field conditions. Plant Biotechnol J 9(1):88–99

  50. Raghothama KG, Maggio A, Narasimhan ML, Kononowicz AK, Wang GL, Durzo MP, Hasegawa PM, Bressan RA (1997) Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region. Plant Mol Biol 34:393–402

  51. Rampino P, Pataleo S, Gerardi C, Mita G, Perrotta C (2006) Drought stress response in wheat: physiological and molecular analysis of resistant and sensitive genotypes. Plant Cell Environ 29:2143–2152

  52. Roychowdhury A, Roy C, Sengupta DN (2007) Transgenic tobacco plants overexpressing the heterologous lea gene Rab 16a from rice during high salt and water deficit display enhanced tolerance to salinity stress. Plant Cell Rep 26:1839–1859

  53. 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, Australia

  54. Saravanakumar D, Vijayakumar C, Kumar N, Samiyappan R (2007) PGPR-induced defense responses in the tea plant against blister blight disease. Crop Prot 26:556–565

  55. 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 104:169–174

  56. Singh NK, Handa AK, Hasegawa PM, Bressan RA (1985) Proteins associated with adaptation of cultured tobacco cells to NaCl. Plant Physiol 79:126–137

  57. Singh NK, Bracker CA, Hasegawa PM, Handa AK, Buckle S, Hermodson MA, Pfankoch E, Regnier FE, Bressan RA (1987) Characterization of osmotin. Plant Physiol 85:529–536

  58. Singh NK, Nelson DE, Kuhn D, Hasegawa PM, Bressan RA (1989) Molecular cloning of osmotin and regulation of its expression by ABA and adaptation to low water potential. Plant Physiol 90:1096–1101

  59. Singh SK, Sharma HC, Goswami AM, Datta SP, Singh SP (2000) In vitro growth and leaf composition of grapevine cultivars as affected by sodium chloride. Biol Plantarum 43:283–286

  60. Singleton PW, Bohlool BB (1984) Effect of salinity on nodule formation by soybean. Plant Physiol 74:72–76

  61. 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 53:110–115

  62. Subramanyam K, Sailaja KV, Subramanyam K, Muralidhara Rao D, Lakshmidevi K (2011) Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L.). Plant Cell Tissue Organ Cult 105:181–192

  63. Talamè V, Ozturk NZ, Bohnert HJ, Tuberosa R (2007) The dynamics of water loss affects the expression of drought-related genes in barley. J Exp Bot 58:229–240

  64. Tambussi EA, Bartoli CG, Beltrano J, Guiamet JJ, Araus JL (2000) Oxidative damage to thylakoid proteins in water-stressed leaves of wheat (Triticum aestivum). Physiol Plantarum 108:398–404

  65. Türkan I, Demiral T (2009) Recent developments in understanding salinity tolerance. Environ Exp Bot 67(1):2–9

  66. Turner NC (1981) Techniques and experimental approaches for the measurement of plant water stress. Plant Soil 58:339–366

  67. 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 151:59–66

  68. Veronese P, Ruiz MT, Coca MA, Hernandez-Lopez A, Lee H, Ibeas JI, Damsz B, Pardo JM, Hasegawa PM, Bressan RA, Narasimhan ML (2003) In defense against pathogens. Both plant sentinels and foot soldier need to know the enemy. Plant Physiol 131:1580–1590

  69. Vigers AJ, Wiedemann S, Roberts WK, Legrand M, Selitrennikoff CP, Fritig B (1992) Thaumatin like pathogenesis-related proteins are antifungal. Plant Sci 83:155–161

  70. Wan C, Shao G, Chen Y, Yan S (2002) Relationship between salt tolerance and chemical quality of soybean under salt stress. Chin J Oil Crop Sci 24:67–72

  71. Wang J, Zuo K, Wu W, Song J, Sun X, Lin J, Li X, Tang K (2004) Expression of a novel antiporter gene from Brassica napus resulted in enhanced salt tolerance in transgenic tobacco plants. Biol Plant 48:509–515

  72. Xu GY, Rocha PSCF, Wang ML, Xu ML, Cui YC, Li LY, Zhu YX, Xia X (2011) A novel rice calmodulin-like gene, OsMSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis. Planta 234:47–59

  73. Yoon JY, Hamayun M, Lee SK, Lee IJ (2009) Methyl jasmonate alleviated salinity stress in soybean. J Crop Sci Biotech 12(2):63–68

  74. Yue Y, Zhang M, Zhang J, Duan L, Li Z (2011) Arabidopsis LOS5/ABA3 overexpression in transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) results in enhanced drought tolerance. Plant Sci 181(4):405–411

  75. Yun DJ, Zhao Y, Pardo JM, Narasimhan ML, Damsz B, Lee H, Abad LR, D’Urzo MP, Hasegawa PM, Bressan RA (1997) Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein. Proc Natl Acad Sci USA 94:7082–7087

  76. Yun DJ, Ibeas JI, Lee H, Coca MA, Narasimhan ML, Uesono Y, Hasegawa PM, Pardo JM, Bressan RA (1998) Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility. Mol Cell 1:807–817

  77. 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 220:262–270

  78. Zhang WJ, Yang SS, Shen XY, Jin YS, Zhao HJ, Wang T (2009) The salt-tolerance gene rstB can be used as a selectable marker in plant genetic transformation. Mol Breed 23:269–277

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The authors are thankful to the Department of Biotechnology (DBT) of Ministry of Science and Technology, Government of India, for the financial support (BT/PR9622/AGR/02/464/2007) to carry out the present work. Subramanyam is grateful to Council of Scientific and Industrial Research (CSIR), Govt. of India, for the award of Senior Research Fellowship (SRF) for his Doctoral research.

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Correspondence to Andy Ganapathi.

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Subramanyam, K., Arun, M., Mariashibu, T.S. et al. Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections. Planta 236, 1909–1925 (2012).

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  • Agrobacterium tumefaciens KYRT1
  • Truncated osmotin protein
  • Salinity
  • Microsphaera diffusa
  • Septoria glycines
  • Phakopsora pachyrhizi