Abstract
Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.
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Abbreviations
- cDNA:
-
complementary DNA
- TLC:
-
thin-layer chromatography
References
Boyd, D., Beckwith, J. (1990) The role of charged amino acids in the localization of secreted and membrane proteins. Cell 62, 1031–1033
Carte, B.K., DeBrosse, C., Eggleston, D., Hemling, M., Mentzer, M., Poehland, B., Troupe, N., Westley, J.W., Hetch, S.M. (1990) Isolation and characterization of a presumed biosynthetic precursor of camptothecin from extracts of Camptotheca acuminata. Tetrahedron 46, 2747–2760
Cordell, G.A. (1974) The biosynthesis of indole alkaloids. Lloydia 37, 219–298
Eilert, U., De Luca, V., Constabel, F., Kurz, W.G.W. (1987) Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus. Arch. Biochem. Biophys. 254, 491–497
Farnsworth, N.R., Blomster, R.N., Damratoski, D., Meer, W.A., Cammarato, L.V. (1964) Studies on Catharanthus alkaloids. VI. Evaluation by means of thin-layer chromatography and ceric ammonium sulfate spray reagent. Lloydia 27, 302–307
Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G., Fraley, R.T. (1985) A simple and general method for transferring genes into plants. Science 227, 1229–1231
Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. (1987) GUS fusions: β-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907
Kutchan, T.M. (1989) Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli. FEBS Lett. 257, 127–130
Kutchan, T.M., Hampp, N., Lottspeich, F., Beyreuther, K., Zenk, M.H. (1988) The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli. FEBS Lett. 237, 40–44
Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, Nyston, N.Y., USA
Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10035–10038
Matzke, A.J.M., Matzke, M.A. (1986) A set of novel Ti plasmid derived vectors for the production of transgenic plants. Plant Mol. Biol. 7, 357–365
McKnight, T.D., Roessner, C.A., Devagupta, R., Scott, A.I., Nessler, C.L. (1990) Nucleotide sequence of a cDNA encoding the vacuolar protein strictosidine synthase from Catharanthus roseus. Nucleic Acids Res. 18, 4939
Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497
Schoner, R.G., Ellis, L.F., Schoner, B.E. (1985) Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. BioTechnology 3, 151–154
Songstad, D.D., De Luca, V., Brisson, N., Kurz, W.G.W., Nessler, C.L. (1990) High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase. Plant Physiol. 94, 1410–1413
Stöckigt, J., Zenk, M.H. (1977) Isovincoside (strictosidine), the key intermediate in the enzymatic formation of indole alkaloids. FEBS Lett. 79, 233–237
VandenBosch, K.A. (1986) Light and electron microscopic visualization of uricase by immunogold labeling of sections of resin-embedded soybean nodules. J. Microsc. 143, 187–197
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We thank Dr. C.A. Roessner for providing the E. coli strain expressing strictosidine synthase, Dr. J. Balsevich for providing alkaloid standards, and Dr. L. Cloney for assisting with antibody preparation. This work was supported by a National Institutes of Health Biomedical Research Support Grant to T.D.M and by a grant from the US Department of Agriculture, Competitive Research Grants Office (90-37262-5375) to C.L.N.
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McKnight, T.D., Bergey, D.R., Burnett, R.J. et al. Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants. Planta 185, 148–152 (1991). https://doi.org/10.1007/BF00194055
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DOI: https://doi.org/10.1007/BF00194055