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Euphytica

, Volume 146, Issue 1–2, pp 165–176 | Cite as

Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses

  • André M. Almeida
  • Enrique Villalobos
  • Susana S. Araújo
  • Barbara Leyman
  • Patrick Van Dijck
  • Luís Alfaro-Cardoso
  • Pedro S. Fevereiro
  • José M. Torné
  • Dulce M. Santos
Article

Summary

Trehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 C and 35C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops.

Key words

abiotic stress tolerance model plant trehalose tobacco 

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References

  1. Avonce, N., B. Leyman, O. Mascorro-Gallardo, P. van Dijck, J.M. Thevelein & G. Iturriaga, 2004. The Arabidopsis Trehalose-6-phosphate synthase AtTPS1 gene is a regulator of glucose, abscisic acid and stress signaling. Plant Physiol 136: 3649-3659.CrossRefPubMedGoogle Scholar
  2. Bajaj, S., J. Targolli, L.F. Liu, T.H. David Ho & R. Wu, 1999. Transgenic approaches to increase dehydration-stress tolerance in plants. Mol Breed 5: 493–503.CrossRefGoogle Scholar
  3. Blazquez, M.A., E. Santos, C.L. Flores, J.M. Martinez-Zapater, J. Salinas & C. Gancedo, 1998. Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase. Plant J 13: 685–689.CrossRefPubMedGoogle Scholar
  4. Chang, S., J. Poryear & J. Cairney, 1993. A simple and efficient method for RNA isolation from pine tree. Plant Mol Biol Rep 11: 113–116.Google Scholar
  5. D'Halluin, K., M. de Block, J. Denecke, J. Janssens, J. Leemans, A. Reymaerts & J. Botterman, 1995. The bar gene as selectable and screenable marker in plant engineering: Recombinant DNA methodology II, pp. 157–168. Academic Press, New York.Google Scholar
  6. Dellaporta, S., J. Wood & J. Hicks, 1983. A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1: 19–21.Google Scholar
  7. Elbein, A.D., 1974. The metabolism of α,α-trehalose. Adv Carbohydr Chem Biochem 30: 227–256.PubMedCrossRefGoogle Scholar
  8. Garg, A.K., J.K. Kim, A.P. Ranwala, Y.D. Choi, L.V. Kochian & R.J. Wu, 2002. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Nat Acad Sci USA 99: 15898–15903.CrossRefPubMedGoogle Scholar
  9. Goddijn, O.J. & K. van Dun, 1999. Trehalose metabolism in plants. Trends in Plant Science 8: 315–319.Google Scholar
  10. Goddijn, O.J., T.C. Verwoerd, E. Moogd, R.W. Krutwagen, P.T. de Graaf, J. Poels, K. van Dun, A.S. Ponstein, B. Damm & J. Pen, 1997. Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant physiol 113: 181–190.CrossRefPubMedGoogle Scholar
  11. Hellens, R.P., E.A. Edwards, N.R. Leyland, S. Bean & P.M. Mullineaux, 2000. pGreen, versatile and flexible binary T1 vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819–832.CrossRefPubMedGoogle Scholar
  12. Holmström, K.O., E. Mäntylä, B. Wellin, A. Mandal & E.T. Palva, 1996. Drought tolerance in tobacco. Nature 379: 683–684.Google Scholar
  13. Horsh, R.B., J.F. Fry, N.L. Hoffmann, D. Eichholtz, S.G. Rogers & R.T. Fraley, 1985. Transferring genes into plants. Science 227: 1229–1231.Google Scholar
  14. Ingram, J. & D. Bartels, 1996. The molecular basis of dehydration tolerance in plants. Ann Rev Plant Physiol Plant Mol Biol 47: 377–403.CrossRefGoogle Scholar
  15. Iturriaga, G., D.F. Gaff & R. Zentella, 2000. New desiccation tolerant plants, including a grass in the central highlands of Mexico, accumulate trehalose. Aust J Bot 48: 153–158.CrossRefGoogle Scholar
  16. Jang, I.C., S.J. Oh, J.S. Seo, W.B. Choi, S.Y. Song, C.H. Kim, Y.S. Kim, H.S. Seo, Y.D. Choi, B.H. Nahm & J.K. Kim, 2003. Expression of a bifunctional fusion of the Escherichia coli genes for Trehalose-6-phosphate synthase and Trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth. Plant Physiol 131: 516–524.CrossRefPubMedGoogle Scholar
  17. Leyman, B., P. van Dijck & J.M. Thevelein, 2001. An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana. Trends in Plant Science 6: 510–513.CrossRefPubMedGoogle Scholar
  18. Murashige, T. & F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497.Google Scholar
  19. Nuccio, M.J., D. Rhodes, S.D. McNeil & A.D. Hanson, 1999. Metabolic engineering of plants for osmotic stress resistance. Curr Opin Plant Biol 2: 128–134.CrossRefPubMedGoogle Scholar
  20. Pellny, T.K., O. Ghannoum, J.P. Conroy, H. Schueppman, S. Smeekens, J. Androlojc, K.P. Krause, O. Goddijn & M. Paul, 2004. Genetic modification of photosynthesis with E. coli genes for trehalose synthesis. Plant Biotechnol J 2: 71–82.Google Scholar
  21. Penna, S., 2003. Building stress tolerance through over-producing trehalose in transgenic plants. Trends Plant Sci 8: 355-357.CrossRefPubMedGoogle Scholar
  22. Pilon-Smits, E., N. Terry, T. Sears, H. Kim, A. Zayed, S. Hwang, K. van Dun, E. Voogd, T.C. Verwoerd, R.H. Krutwagen & O.J. Goddijn, 1998. Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. J Plant Physiol 152: 525–532.Google Scholar
  23. Romero, C., J.M. Bellés, J.L. Vayá, R. Serrano & F.A. Culiañez-Maciá, 1997. Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: Pleiotropic phenotypes include drought tolerance. Planta 201: 293–297.CrossRefPubMedGoogle Scholar
  24. Sambrook, J., E.F. Fritsch & T. Maniatis, 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbour Laboratory Press, Woodbury, NY.Google Scholar
  25. Scott, P., 1999. Resurrection plants and the secrets of eternal leaf. Ann Bot 85: 159–166.Google Scholar
  26. Siedow, J.N., 2001. Feeding ten billion people, three views. Plant Physiol 126: 20–22.CrossRefPubMedGoogle Scholar
  27. Sharma, H.C., J.H. Crouch, K.K. Sharma, N. Seetharama & C.T. Hash, 2002. Applications of biotechnology for crop improvement: Prospects and constraints. Plant Sci 163: 381–395.CrossRefGoogle Scholar
  28. Tobwin, H., T. Stachelin & J. Gordon, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat Acad Sci USA 76: 4350–4354.Google Scholar
  29. Veluthambi, K., S. Mahadevan & R. Maheshwari, 1981. Trehalose toxicity in Cuscuta reflexa – correlation with low trehalase activity. Plant Physiol 68: 1369–1374.PubMedCrossRefGoogle Scholar
  30. Willadino, L., T. Câmara, N. Boget, M.A. Santos & J.M. Torné, 1996. Polyamine and free amino acid variations in NaCl treated embryogenic maize callus from sensitive and resistant cultivars. J. Plant Physiol 149: 178–185.Google Scholar
  31. Wingler, A., 2002. The function of trehalose biosynthesis in plants. Phyochemistry 60: 437–440.Google Scholar
  32. Yeo, E.T., H.B. Kwon, S.E. Han, J.T. Lee, J.C. Ryu & M.O. Byun, 2000. Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Sacharomyces cerevisae. Mol Cells 10: 263–268.PubMedGoogle Scholar
  33. Zhu, J.K., 2001. Plant salt tolerance. Trends Plant Sci 6: 66–71.PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • André M. Almeida
    • 1
  • Enrique Villalobos
    • 2
  • Susana S. Araújo
    • 1
  • Barbara Leyman
    • 3
  • Patrick Van Dijck
    • 3
  • Luís Alfaro-Cardoso
    • 4
  • Pedro S. Fevereiro
    • 1
    • 5
  • José M. Torné
    • 2
  • Dulce M. Santos
    • 6
  1. 1.Laboratório de Biotecnologia de Células VegetaisInstituto de Tecnologia Química e Biológica (ITQB)OeirasPortugal
  2. 2.Instituto de Biologia Molecular de BarcelonaCSICBarcelonaSpain
  3. 3.Laboratorium voor moleculaire celbiologieKatholieke Universiteit Leuven and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB)Leuven-HeverleeBelgium
  4. 4.Instituto de Investigação Cientifica e TropicalLisboaPortugal
  5. 5.Departamento de Biologia VegetalFaculdade de Ciências da Universidade de LisboaLisboaPortugal
  6. 6.Laboratório de Biologia Funcional de PlantasInstituto de Biologia Molecular e Celular (IBMC)PortoPortugal

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