Skip to main content
SpringerLink
Log in

Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

  • Published:
Planta Aims and scope Submit manuscript

Abstract

Certain higher plants synthesize and accumulate glycine betaine, a compound with osmoprotectant properties. Biosynthesis of glycine betaine proceeds via the pathway choline → betaine aldehyde → glycine betaine. Plants such as tobacco (Nicotiana tabacum L.) which do not accumulate glycine betaine lack the enzymes catalyzing both reactions. As a step towards engineering glycine betaine accumulation into a non-accumulator, spinach and sugar beet complementary-DNA sequences encoding the second enzyme of glycine-betaine synthesis (betaine aldehyde dehydrogenase, BADH, EC 1.2.1.8) were expressed in tobacco. Despite the absence of a typical transit peptide, BADH was targeted to the chloroplast in leaves of transgenic plants. Levels of extractable BADH were comparable to those in spinach and sugar beet, and the molecular weight, isoenzyme profile and K m for betaine aldehyde of the BADH enzymes from transgenic plants were the same as for native spinach or sugar beet BADH. Transgenic plants converted supplied betaine aldehyde to glycine betaine at high rates, demonstrating that they were able to transport betaine aldehyde across both the plasma membrane and the chloroplast envelope. The glycine betaine produced in this way was not further metabolized and reached concentrations similar to those in plants which accumulate glycine betaine naturally. Betaine aldehyde was toxic to non-transformed tobacco tissues whereas transgenic tissues were resistant due to detoxification of betaine aldehyde to glycine betaine. Betaine aldehyded ehydrogenase is therefore of interest as a potential selectable marker, as well as in the metabolic engineering of osmoprotectant biosynthesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BADH:

betaine aldehyde dehydrogenase

bp:

base pairs

FAB-MS:

fast atom bombardment-mass spectrometry

GAPDH:

NADP-linked glyceraldehyde-3-phosphate dehydrogenase

References

  • An, G., Ebert, P.R., Mitra, A., Ha, S.B. (1988) Binary vectors. In: Plant molecular biology manual A3, pp. 1–19, Gelvin, S.B., Schilperoort, R.A., eds. Kluwer, Dordrecht

    Google Scholar 

  • Arakawa, K., Takabe, T., Sugiyama, T., Akazawa, T. (1987) Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody. J. Biochem. 101, 1485–1488

    Google Scholar 

  • Arnon, D.I. (1949) Copper enzymes in chloroplasts. Polyphenoloxidases in Beta vulgaris. Plant Physiol. 24, 1–15

    Google Scholar 

  • Berry-Lowe, S.L., Schmidt, G.W. (1991) Chloroplast protein transport. In: Cell culture and somatic cell genetics of plants, vol. 7A: The molecular biology of plastids, pp. 257–302, Bogorad, L., Vasil, I.K., eds. Academic Press, New York

    Google Scholar 

  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254

    Google Scholar 

  • Brouquisse, R., Weigel, P., Rhodes, D., Yocum, C.F., Hanson, A.D. (1989) Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma. Plant Physiol. 90, 322–329

    Google Scholar 

  • Budar, F., Thia-Toong, L., Van Montagu, M., Hernalsteens, J.P. (1986) Agrobacterium-mediated gene transfer results mainly in transgenic plants transmitting T-DNA as a single Mendelian factor. Genetics 114, 303–313

    Google Scholar 

  • Byerrum, R.U., Sato, C.S., Ball, C.D. (1956) Utilization of betaine as a methyl group donor in tobacco. Plant Physiol. 31, 374–377

    Google Scholar 

  • Cromwell, B.T. (1955) The tobacco alkaloids. In: Modern methods of plant analysis, vol. 4, pp. 487–516, Paech, K., Tracey, M.V. eds. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Csonka, L.N., Hanson, A.D. (1991) Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Microbiol. 45, 569–606

    Google Scholar 

  • Flavell, R.B., Dart, E., Fuchs, R.L., Fraley, R.T. (1992) Selectable marker genes: safe for plants? Bio/Technology 10, 141–144

    Google Scholar 

  • Grimm, B., Ish-Shalom, D., Even, D., Glaczinski, H., Ottersbach, P., Ohad, L., Kloppstech, K. (1989) The nuclear-coded chloroplast 22-kDa heat-shock protein of Clamydomonas: evidence for translocation into the organelle without a processing step. Eur. J. Biochem. 182, 539–546

    Google Scholar 

  • Hanson, A.D., Wyse, R. (1982) Biosynthesis, translocation, and accumulation of betaine in sugar beet and its progenitors in relation to salinity. Plant Physiol. 70, 1191–1198

    Google Scholar 

  • Hedrick, J.L., Smith, A.J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155–164

    Google Scholar 

  • Kao, K.N., Michayluk, M.R. (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126, 105–110

    Google Scholar 

  • Keegstra, K., Olsen, L.J., Theg, S.M. (1989) Chloroplastic precursors and their transport across the envelope membranes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 471–501

    Google Scholar 

  • Ladyman, J.A.R., Hitz, W.D., Hanson, A.D. (1980) Translocation and metabolism of glycine betaine. Planta 150, 191–196

    Google Scholar 

  • LeRudulier, D., Strom, A.R., Dandekar, A.M., Smith, L.T., Valentine, R.C. (1984) Molecular biology of osmoregulation. Science 224, 1064–1068

    Google Scholar 

  • Lilley, R.M.., Fitzgerald, M.P., Rienits, K.G., Walker, D.A. (1975) Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol. 75, 1–10

    Google Scholar 

  • Luck, H. (1965) Catalase. In: Methods of enzymatic analysis, pp. 886–888, Bergmeyer, H.U. ed. Academic Press, New York

    Google Scholar 

  • McCue, K.F., Hanson, A.D. (1990) Drought and salt tolerance: towards understanding and application. Trends Biotechnol. 8, 358–362

    Google Scholar 

  • McCue, K.F., Hanson, A.D. (1992) Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression. Plant Mol. Biol. 18, 1–11

    Google Scholar 

  • Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497

    Google Scholar 

  • Puissant, C., Houdebine, L.-M. (1990) An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. BioTechniques 8, 148–149

    Google Scholar 

  • Racker, E. (1950) Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids. Biochim. Biophys. Acta 4, 211–214

    Google Scholar 

  • Rathinasabapathi, B., King, J. (1992) An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts. Plant Cell Tissue Organ Culture 29, 207–214

    Google Scholar 

  • Rhodes, D., Hanson, A.D. (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 357–384

    Google Scholar 

  • Rhodes, D., Rich, P.J., Myers, A.C., Reuter, C.C., Jamieson, G.C. (1987) Determination of betaines by fast atom bombardment mass spectrometry: identification of glycine betaine deficient genotypes of Zea mays. Plant Physiol. 84, 781–788

    Google Scholar 

  • Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, New York

    Google Scholar 

  • Somero, G.N. (1986) Protons, osmolytes, and fitness of internal milieu for protein function. Am. J. Physiol. 251, R197-R213

    Google Scholar 

  • Tarczynski, M.C., Jensen, R.G., Bohnert, H.J. (1993) Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 259, 508–510

    Google Scholar 

  • Weigel, P., Weretilnyk, E.A., Hanson, A.D. (1986) Betaine aldehyde oxidation by spinach chloroplasts. Plant Physiol. 82, 753–759

    Google Scholar 

  • Weretilnyk, E.A., Hanson, A.D. (1988) Betaine aldehyde dehydrogenase polymorphism in spinach: genetic and biochemical characterization. Biochem. Genet. 26, 143–151

    Google Scholar 

  • Weretilnyk, E.A., Hanson, A.D. (1989) Betaine aldehyde dehydrogenase from spinach leaves: purification, in vitro translation of the mRNA, and regulation by salinity. Arch. Biochem. Biophys. 271, 56–63

    Google Scholar 

  • Weretilnyk, E.A., Hanson, A.D. (1990) Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. Proc. Natl. Acad. Sci. USA 87, 2745–2749

    Google Scholar 

  • Weretilnyk, E.A., Bednarek, S., McCue, K.F., Rhodes, D., Hanson, A.D. (1989) Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons. Planta 178, 342–352

    Google Scholar 

  • Wolosiuk, R.A., Buchanan, B.B. (1976) Studies on the regulation of chloroplast NADP-linked glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem. 251, 6456–6461

    Google Scholar 

  • Wyn Jones, R.G. (1984) Phytochemical aspects of osmotic adaptation. Recent Adv. Phytochem. 18, 55–78

    Google Scholar 

  • Wyn Jones, G.R., Rippin, A.J., Storey, R. (1973) Metabolism of choline in the rhizosphere and its possible influence on plant growth. Pestic. Sci. 4, 375–383

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut de Recherche en Biologie Végétale de l'Université de Montréal, 4101 Est Rue Sherbrooke, H1X 2B2, Montréal, Québec, Canada

    Bala Rathinasabapathi & Andrew D. Hanson

  2. USDA-ARS, 800 Buchanan St., 94710, Albany, CA, USA

    Kent F. McCue

  3. Biochemistry Department, Michigan State University, 48824, East Lansing, MI, USA

    Douglas A. Gage

Authors
  1. Bala Rathinasabapathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kent F. McCue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas A. Gage
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew D. Hanson
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

We thank Dr. G. An for the gift of the vector pGA643 and Mr. Sylvain Lebeurier for help in maintaining plants. This work was supported, in part, by grants from the Natural Sciences and Engineering Research Council of Canada, the Rockefeller Foundation, and the U.S. Department of Agriculture, and by gifts from CIBAGEIGY Biotechnology.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rathinasabapathi, B., McCue, K.F., Gage, D.A. et al. Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance. Planta 193, 155–162 (1994). https://doi.org/10.1007/BF00192524

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00192524

Key words

Search

Navigation