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Molecular Breeding

, Volume 15, Issue 1, pp 65–74 | Cite as

Aggregates formed as a result of the expression of yeast Met2 gene in transgenic tobacco plants, stimulate the production of stress-protective metabolites and increased the plants tolerance to heat stress

  • Dan Gamrasni
  • Ifat Matityahu
  • Rachel Amir
Article

Abstract

Methionine biosynthesis has taken different evolutionary pathways in bacteria, fungi and plants. To gain insight into these differences and to search for new ways of manipulating methionine biosynthesis in plants, the yeast (Saccharomyces cerevisiae) Met2 gene and the bacteria (Leptospira meyeri) MetX gene, both encoding homoserine O-acetyltransferase, were expressed in tobacco plants. We found protein aggregates in extracts of these transgenic plants, whose levels were much higher in plants grown at 35 °C than at 25 °C. It appears that the yeast and the bacterial proteins are heat labile and tend to change their intracellular conformation. These conformational changes of the transgenic proteins were more prominent at high temperature and most probably triggered aggregation of the yeast and the bacterial proteins. Moreover, plants expressing the yeast gene that grew at 35 °C over-accumulated stress-associated metabolites, such as phenolic compounds, including tannins, as well as the amino acid arginine. In addition, the transgenic plants expressing high levels of the foreign genes show growth retardation, which further suggests that, these plants suffer from internal stress. The changes in protein conformation and the consequent triggering of stress response may account for the ability of these transgenic plants to tolerate more extreme heat stress (60 °C) than the wild-type plants.

Keywords

Arginine Heat stress Homoserine O-acetyltransferase Methionine biosynthesis Phenols Protein aggregation 

Abbreviations

HAT

homoserine O-acetyltransferase

HST

homoserine O-succinyltransferase

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References

  1. Becker, D., Kemper, E., Schell, J., Masterson, R. 1992New plant binary vectors with selectable markers located proximally to the left T-DNA borderPlant Mol. Biol.2011951197Google Scholar
  2. Biran, D., Brot, N., Weissbach, H., Ron, E.Z. 1995Heat-shock dependent transcriptional activation of the metA gene of Escherichia coliJ. Bacteriol.17713741379Google Scholar
  3. Boggess, S., Stewart, C., Aspinall, D., Peleg, L. 1976Effect of water stress on proline synthesis from radioactive precursorsPlant Physiol.58398401Google Scholar
  4. Bourhy, P., Martel, A., Margarita, D., Saint-Girons, I., Belfaiza, J. 1997Homoserine O-acetyltransferaseinvolved in Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibitedJ. Bacteriol.17943964398Google Scholar
  5. Datko, A.H., Giovanelli, J., Mudd, H. 1974Homocysteine biosynthesis in green plants. Phosphohomoserine as the physiological substrate for cystathionineJ. Biol. Chem.24911391155Google Scholar
  6. Forlani, N., Martegani, E., Alberghina, L. 1991Posttranscriptional regulation of the expression of MET2 gene of Saccharomyces cerevisiaeBiochim. Biophys. Acta10894753Google Scholar
  7. Gur, E., Biran, D., Gazit, E., Ron, E.Z. 2002In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperaturesMol. Microbiol.4613911397Google Scholar
  8. Hacham, Y., Avraham, T., Amir, R. 2002The N-terminal region of Arabidopsis cystathionine gamma synthase plays an important role in methionine metabolismPlant Physiol.128454462Google Scholar
  9. Hacham, Y., Gophna, U., Amir, R. 2003In vivo analysis of various substrates utilized by cystathionine gamma synthase and O-acetylhomoserine sulfhydrylase in methionine biosynthesisMol. Biol. Evol.2015131520Google Scholar
  10. Hagerman, A., Butler, L.G. 1978Protein precipitation method for the quantitative determination of tanninsJ. Agric. Food Chem.26809812Google Scholar
  11. Hauslanden, A., Stamler, J.S. 1998Nitric oxide in plant immunityProc. Natl. Acad. Sci. USA951034510347Google Scholar
  12. Horsch, R., Fry, B., Hoffmann, N.L., Eichholtz, D., Rogers, S.G., Fraley, R.T. 1985A simple and general method for transferring genes into plantsScience22712291231Google Scholar
  13. Howard, L.R., Pandjaitan, N., Morelock, T., Gil, M.I. 2002Antioxidant capacity and phenolic content of spinach as affected by genetics and growing seasonJ. Agric. Food Chem.5058915896Google Scholar
  14. Iba, K. 2002Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature toleranceAnn. Rev. Plant Biol.53225245Google Scholar
  15. Jaenicke, R. 1995Folding and association versus misfolding and aggregation of proteinsPhilos. Trans. R. Soc. London B. Biol. Sci.34897105Google Scholar
  16. Lam, H.M., Hsieh, M.H., Coruzzi, G. 1998Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thalianaPlant J.16345353Google Scholar
  17. Langin, T., Faugeron, G., Goyon, C., Nicolas, A., Rossignol, J.L. 1986The Met2 gene of Saccharomyces cervisiae: Molecular cloning and nucleotide sequenceGene49283293Google Scholar
  18. Ma, Y., Hendershot, H.L. 2001The unfolding tale of the unfolded protein responseCell107827830Google Scholar
  19. Macnitol, P.K., Datko, A.H., Giovanelli, J., Mudd, H. 1981Homocysteine biosynthesis in green plants: physiological importance of the transsulfuration pathway in Lemna paucicostataPlant Physiol.86619625Google Scholar
  20. Nagai, S., Flavin, M. 1971Synthesis of O-acetylhomoserineMethods Enzymol.17B423424Google Scholar
  21. Okamura, H., Akio, M., Yasuko, Y., Mitsuru, N., Yoshimasa, T. 1993Antioxidant activity of tannins and flavonoids in Eucalytus rostrataPhytochemistry33557561Google Scholar
  22. Rabe, E., Lovatt, C. 1986Increased arginine biosynthesis during phosphorus deficiencyPlant Physiol.81774779Google Scholar
  23. Ravanel, S., Gakiere, B., Job, D., Douce, R. 1998Cystathionine gama-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme over-expressed in Escherichia coliBiochem. J.331639648Google Scholar
  24. Rivero, R.M., Ruiz, J.M., Garcia, P.C., Lopez-Lefebre, L.R., Sanchez, E., Romero, L. 2001Resistance to cold and heat stress: accumulation of phenolic compounds in tomato and watermelon plantsPlant Sci.160315321Google Scholar
  25. Ron, E.Z. 1975Growth rate of Enterobacteriaceae at elevated temperatures: limitation by methionineJ. Bacteriol.124243246Google Scholar
  26. Rosen, R., Biran, D., Gur, E., Becher, D., Hecker, M., Ron, E.Z. 2002Protein aggregation in Escherichia coli: role of proteasesFEMS Microbiol. Lett.207912Google Scholar
  27. Saint-Girons, I., Parsot, C., Zakin, M., Barzu, O., Cohen, G.N. 1988Methionine biosynthesis in enterobacteriaceae: biochemical, regulatory, and evolutionary aspectsCRC Crit. Rev. Biochem.23S1S42Google Scholar
  28. Salvucci, M.E., Osteryoung, K.W., Crafts-Brandner, S.J., Vierling, E. 2001Exceptional sensitivity of Rubisco activase to thermal denaturation in vitroin vivoPlant Physiol.12710531064Google Scholar
  29. Shaul, O., Galili, G. 1992Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplastsPlant J.2203209Google Scholar
  30. Swain, T., Hillis, W.E. 1959The phenolic constituents of Prunus somestica. The quantities analysis of phenolic constituentsJ. Agric. Food Chem.106368Google Scholar
  31. Tran, P.B., Miller, R.J. 1999Aggregates in neurodegenerative disease: crowds and power?Trends Neurosci.22194197Google Scholar
  32. Wickner, S., Maurizi, M.R., Gottesman, S. 1999Posttranslational quality control: folding, refolding, and degrading proteinsScience28618881893CrossRefPubMedGoogle Scholar
  33. Ye, B., Muller, H.H., Zhang, J., Gressel, J. 1997Constitutively elevated levels of putrescine and putrescine-generating enzymes correlated with oxidant stress resistance in Conyza bonariensis and wheatPlant Physiol.11514431451Google Scholar
  34. Zhu, M., Phillipson, J.D., Greengrass, P.M., Bowery, N.E., Cai, Y. 1997Plant polyphenols: biologically active compounds or non-selective binders to protein?Phytochemistry44441447Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Dan Gamrasni
    • 1
  • Ifat Matityahu
    • 1
  • Rachel Amir
    • 1
    • 2
  1. 1.Plant Science LaboratoryMigal–Galilee Technology CenterKiryat ShmonaIsrael
  2. 2.Tel-Hai Academic CollegeUpper GalileeIsrael

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