Abstract
The AbH6H gene for hyoscyamine 6β-hydroxylase (H6H), which converts hyoscyamine to scopolamine, was isolated from Atropa belladonna. This plant also possesses a related sequence, AbψH6H, which appears to be a non-functional pseudo-gene. AbH6H RNA was detected in cultured root, native root and anther, but not in stem, leaf, pistil, petal, and sepal tissues. In situ hybridization, immunohistochemistry and promoter::GUS transgene analysis showed that AbH6H is expressed specifically in root pericycle cells, and in tapetum and pollen mother cells. A 671 bp 5′-upstream region from AbH6H was sufficient for pericycle-specific expression in hairy roots of A. belladonna and Hyoscyamus niger, which both produce scopolamine, but cell-specific regulation was severely compromised in tobacco hairy roots, which do not produce scopolamine.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bajaj, Y.P.S. and Simola, L.K. 1991. Atropa belladonna L.: in vitro culture, regeneration of plants, cytopreservation, and the production of tropane alkaloids. In: Biotechnology in Agriculture and Forestry 15. Springer-Verlag, Berlin/Heidelberg, pp. 1-23.
Beeckman, T. and Engler, G. 1994. An easy technique for the clearing of histochemically stained plant tissue. Plant Mol. Biol. Rep. 12: 37-42.
Burbulis, I.E., Lacobucci, M. and Shirley, B.W. 1996. A null mutation in the first enzyme of flavonoid biosynthesis does not affect male fertility in arabidopsis. Plant Cell 8: 1013-1025.
Detzel, A. and Wink, M. 1993. Attraction, deterrence or intoxication of bees (Apis mellifera) by plant allelochemicals. Chemoecology 4: 8-18.
Duck, N.B. 1994. RNA in situ hybridization in plants. In: S.B. Gelvin and R.A. Schilperoort <nt>(Eds.)</nt>, Plant Molecular Biology Manual, 2nd ed, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. G1/1-G1/13.
Evans, W.C., 1979. Tropane alkaloids in the Solanaceae. The biology and taxonomy of the Solanaceae, Linnean Society Symposium Series, vol. 7, Academic Press, London, pp. 241-254.
Eyal, Y., Curie, C. and McCormick, S. 1995. Pollen specificity elements reside in 30 bp of the proximal promoters of two pollen-expressed genes. Plant Cell 7: 373-384.
Green, P.J., Yong, M.H., Cuozzo, M., Kano-Murakami, Y., Silverstein, P. and Chua, N.H. 1988. Binding site requirements for pea nuclear protein factor GT-1 correlate with sequences required for light-dependent transcriptional activation of the rbcS-3A gene. EMBO J 20: 4035-4044.
Hanley, B.A. and Schuler, M.A. 1988. Plant intron sequences: evidence for distinct groups of intron. Nucl. Acids Res. 16: 7159-7176.
Harborne, J.B. and Khan, M.B. 1993. Variations in the alkaloidal and phenolic profiles in the genus Atropa (Solanaceae). Bot. J. Linnean Soc. 111: 47-53.
Hartmann, T., Witte, L., Oprach, F. and Toppel, G. 1986. Reinvestigation of the alkaloid composition of Atropa belladonna plants, root cultures, and cell suspension cultures. Planta Med. 52: 390-395.
Hashimoto, T. and Yamada, Y. 1986. Hyoscyamine 6_-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures. Plant Physiol. 81: 619-625.
Hashimoto, T., Yukimune, Y. and Yamada, Y. 1986. Tropane alkaloid production in Hyoscyamus root cultures. J. Plant Physiol. 124: 61-75.
Hashimoto, T., Hayashi, A., Amano, Y., Kohno, J. and Yamada, Y. 1991. Hyoscyamine 6_-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root. J. Biol. Chem. 266: 4648-4653.
Hashimoto, T., Matsuda, J. and Yamada, Y. 1993. Two step epoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6_-hydroxylase. FEBS Lett. 329: 35-39.
Higo, K., Ugawa, Y., Iwamoto, M. and Higo, H. 1998. PLACE: a database of plant cis-acting regulatory DNA elements. Nucl. Acids Res. 26: 358-359.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusion: _-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907.
Joshi, C.P. 1987. An inspection of the domain between putative TATAbox and translation start site in 79 plant genes. Nucl. Acids Res. 15: 6643-6653.
Kanegae, T., Kajiya, H., Amano, Y., Hashimoto, T. and Yamada, Y. 1994. Species-dependent expression of the hyoscyamine 6_-hydroxylase gene in the pericycle. Plant Physiol. 105: 483-490.
Luckner, M. 1990. Long distance transport of secondary products in the producer organism. In: M. Luckner <nt>(Ed.)</nt>, Secondary Metabolism in Microorganisms, Plants, and Animals, 3rd ed., VEB Gustav Fischer Verlag, Jena, p. 39.
Mathis, N.L. and Hinchee, M.A.W. 1994. Agrobacterium inoculation techniques for plant tissues. In: S.B. Gelvin and R.A. Schilperoort (Eds.), Plant Molecular Biology Manual, 2nd ed., Kluwer Academic Publishers, Dordrecht, Netherlands, pp. B6/1-B6/9.
Matsuda, J., Okabe, S., Hashimoto, T. and Yamada, Y. 1991. Molecular cloning of hyoscyamine 6_-hydroxylase, a 2-oxoglutarate dependent dioxygenase, from cultured roots of Hyoscyamus niger. J. Biol. Chem. 266: 9460-9464.
Meyerowiz, E.M. 1987. In situ hybridization to RNA in plant tissue. Plant Mol. Biol. Rep. 5: 242-250.
Mo, Y., Nagel, C. and Taylor, L.P. 1992. Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen. Proc. Natl. Acad. Sci. USA 89: 7213-7217.
Murry, M.G. and Thompson, W.F. 1980. Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res. 8: 4321-4325.
Nagel, R., Elliott, A., Masel, A., Birch, R.G. and Manners, J.M. 1990. Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol. Lett. 67: 325-328.
Puente, P., Wei, N. and Deng, X.W. 1996. Combinatorial interplay of promoter elements constitutes the minimal determinants for light and developmental control of gene expression in Arabidopsis. EMBO J. 15: 3732-3743.
Romero, I., Fuertes, A., Benito, M.J., Malpica, J.M., Leyva, A. and Paz-Arez, J. 1998. More than 80 R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant J. 14: 273-284.
Saito, K., Yamazaki, M., Anzai, H., Yoneyama, K. and Murakoshi, I. 1992. Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait. Plant Cell. Physiol. 11: 219-224.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Suzuki, K., Yamada, Y. and Hashimoto, T. Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiol., in press.
Villain, P., Mache, R. and Zhou, D.X. 1996. The mechanism of GT element-mediated cell type-specific transcriptional control. J. Biol. Chem. 271: 32593-32598.
Wink, M. 1998. A short history of alkaloids. In: M.F. Roberts and M. Wink <nt>(Eds.)</nt>, Alkaloids, Plenum Press, New York, pp. 11-44.
Ylstra, B., Busscher, J., Franken, J., Hollman, P.C.H., Mol, J.N.M. and van Tunen, A.J. 1994. Flavonol and fertilization in Petunia hybrida: localization and mode of action during pollen tube growth. Plant Cell 6: 201-212.
Yun, D.J., Hashimoto, T. and Yamada, Y. 1992. Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition. Proc. Natl. Acad. Sci. USA 89: 11799-11803.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Suzuki, Ki., Yun, DJ., Chen, XY. et al. An Atropa belladonna hyoscyamine 6β-hydroxylase gene is differentially expressed in the root pericycle and anthers. Plant Mol Biol 40, 141–152 (1999). https://doi.org/10.1023/A:1026465518112
Issue Date:
DOI: https://doi.org/10.1023/A:1026465518112