Amino Acids

, Volume 30, Issue 2, pp 185–194 | Cite as

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

  • I. Matityahu
  • L. Kachan
  • I. Bar Ilan
  • R. Amir
Article

Summary.

The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO2, the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.

Keywords: Cysteine biosynthesis – Glutathione – O-Acetylhomoserine sulfhydrylase – O-Acetyl(thiol)lyase – Oxidative stress – Transgenic tobacco plants 

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References

  1. Antoniewski, J, Robichon-Szulmajster, H 1973Biosynthesis of methionine and its control in wild type and regulatory mutants of Saccharomyces cerevisiae.Biochimie55529539PubMedGoogle Scholar
  2. Becker, D, Kemper, E, Schell, J, Masterson, R 1992New plant binary vectors with selectable markers located proximal to the left T-DNA border.Plant Mol Biol2011951197PubMedGoogle Scholar
  3. Blaszczyk, A, Brodzik, R, Sirko, A 1999Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serine acetyltransferase.Plant J20237243CrossRefPubMedGoogle Scholar
  4. Buchner, P, Takahashi, H, Hawkesford, MJ 2004Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport.J Exp Botany5517651773CrossRefGoogle Scholar
  5. Cherest, H, Thomas, D, Surdin-Kerjan, Y 1993Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulphuration pathway which has been built up by enzyme recruitment.J Bacteriol17553665374PubMedGoogle Scholar
  6. D’Andrea, R, Surdin-Kerjan, Y, Pure, G, Cherest, H 1987Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene.Mol Gen Genet207165170PubMedGoogle Scholar
  7. Dominguez-Solis, JR, Gutierrez-Alcala, G, Vega, JM, Romero, LC, Gotor, C 2001The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance.J Biol Chem27631474CrossRefGoogle Scholar
  8. Droux, M, Ruffet, ML, Douce, R, Job, D 1998Interactions between serine acetyltransferase and O-acetylserine(thiol)lyase in higher plants–structural and kinetic properties of the free and bound enzymes.Eur J Biochem255235245CrossRefPubMedGoogle Scholar
  9. Gamrasni, D, Matityahu, I, Amir, R 2005Aggregates 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.Mol Breeding156574CrossRefGoogle Scholar
  10. Hacham, Y, Gofna, U, Amir, R 2003In vivo analysis of various substrates utilized by cystathionine γ-synthase and O-acetylhomoserine sulfhydrylase in methionine biosynthesis.Mol Biol Evol2015131520PubMedGoogle Scholar
  11. Harms, K, von Ballmoos, P, Brunold, C, Hofgen, R, Hesse, H 2000Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione.Plant J22335343CrossRefPubMedGoogle Scholar
  12. Hell, R, Jost, R, Berkowitz, O, Wirtz, M 2002Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant Arabidopsis thaliana.Amino Acids22245257CrossRefPubMedGoogle Scholar
  13. Hesse, H, Nikiforova, V, Gakiere, B, Hoefgen, R 2004Molecular analysis and control of cysteine biosynthesis: integration of nitrogen and sulphur metabolism.J Exp Botany5512831292Google Scholar
  14. Hofgen, R, Kreft, O, Willmitzer, L, Hesse, H 2001Manipulation of thiol contents in plants.Amino Acids20291299PubMedGoogle Scholar
  15. Horsch, R, Fry, B, Hoffmann, NL, Eichholtz, D, Rogers, SG, Fraley, RT 1985A simple and general method for transferring genes into plants.Science22712291231Google Scholar
  16. Kawashima, CG, Noji, M, Nakamura, M, Ogra, Y, Suzuki, KT, Saito, K 2004Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase.Biotechnol Lett26153157CrossRefPubMedGoogle Scholar
  17. Laemmli, UK 1970Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature227680685CrossRefPubMedGoogle Scholar
  18. Liszewska F, Sirko A (2003) Analysis of transgenic tobacco lines expressing bacterial cysK gene encoding O-acetylserine(thiol)lyase A. In: Davidian J-C, Grill D, De Kok LJ, Stulen I, Hawkesford MJ, Schnung E, Rennerberg H (eds) Sulphur transport and assimilation in plants: regulation, interaction and signaling. Backhuys Publishers, Leiden, pp 269–271Google Scholar
  19. Liszewska, F, Blaszczyk, A, Sirko, A 2001Modification of non-protein thiols contents in transgenic tobacco plants producing bacterial enzymes of cysteine biosynthesis pathway.Acta Biochimica Polonica48647656PubMedGoogle Scholar
  20. Leustek, T, Saito, K 1999Sulfate transport and assimilation in plants.Plant Physiol120637644CrossRefPubMedGoogle Scholar
  21. Noji, M, Saito, K 2002Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants.Amino Acids22231243CrossRefPubMedGoogle Scholar
  22. Noji, M, Saito, M, Nakamura, M, Aono, M, Saji, H, Saito, K 2001Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants.Plant Physiol126973980CrossRefPubMedGoogle Scholar
  23. Riemenschneider, A, Riedel, K, Hoefgen, R, Papenbrock, J, Hesse, H 2005Impact of reduced O-acetylserine(thiol)lyase isoform contents on potato plant metabolism.Plant Physiol137892900CrossRefPubMedGoogle Scholar
  24. Rochat, C, Boutin, JP 1991Metabolism of phloem-borne amino acids in maternal tissues of fruit of Nodulated or nitrate-fed pea plants (Pisum sativum L.).J Exp Bot42207214Google Scholar
  25. Saito, K 2000Regulation of sulfate transport and synthesis of sulfur-containing amino acids.Curr Opin Plant Biol3188195PubMedGoogle Scholar
  26. Saito, K, Kurosawa, M, Tatsuguchi, K, Takagi, Y, Murakoshi, I 1994Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase].Plant Physiol106887895CrossRefPubMedGoogle Scholar
  27. Shaul, O, Galili, G 1992Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts.Plant J2203209CrossRefGoogle Scholar
  28. Sirko, A, Blaszczyk, A, Liszewska, F 2004Overproduction of SAT and/or OASTL in transgenic plants: a survey of effects.J Exp Botany5518811888CrossRefGoogle Scholar
  29. Thomas, D, Surdin-Kerjan, Y 1997Metabolism of sulfur amino acids in Saccharomyces cerevisiae.Microbiol Mol Biol Rev61503532PubMedGoogle Scholar
  30. Vanoni, M, Vai, M, Popolo, L, Alberghina, L 1983Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.J Bacteriol15612821291PubMedGoogle Scholar
  31. Wirtz, M, Hell, R 2003Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase.Amino Acids24195203PubMedGoogle Scholar
  32. Yamagata, S, Takeshima, K, Naiki, N 1975O-acetylserine and O-acetylhomoserine sulfhydrylase of yeast; studies with methionine auxotrophs.J Biochem (Tokyo)7710291036Google Scholar
  33. Yamagata, S, Isaji, M, Nakamura, K, Fujisaki, S, Doi, K, Bawden, S, D’Andrea, R 1994Overexpression of the Saccharomyces cerevisiae MET17/MET25 gene in Escherichia coli and comparative characterization of the product with O-acetylserine. O-acetylhomoserine sulfhydrylase of the yeast.Appl Microbiol Biotechnol429299PubMedGoogle Scholar
  34. Youssefian, S, Nakamura, M, Sano, H 1993Tobacco plants transformed with the O-acetylserine(thiol)lyase gene of wheat are resistant to toxic levels of hydrogen sulphide gas.Plant J4759769CrossRefPubMedGoogle Scholar
  35. Youssefian, S, Nakamura, M, Orudgev, E, Kondo, N 2001Increased cysteine biosynthesis capacity of transgenic tobacco overexpressing an O-acetylserine(thiol)lyase modifies plant responses to oxidative stress.Plant Physiol12610011111CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag/Wien 2005

Authors and Affiliations

  • I. Matityahu
    • 1
  • L. Kachan
    • 1
  • I. Bar Ilan
    • 1
  • R. Amir
    • 1
    • 2
  1. 1.Plant Science Laboratory, Migal – Galilee Technology CenterKiryat ShmonaIsrael
  2. 2.Tel-Hai Academic CollegeUpper GalileeIsrael

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