Summary
In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane alkaloid levels were the highest in the root (0.9 mg g−1 on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR) and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large yields of tropane alkaloids.
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Benfey, P. N.; Chau, N. H. The cauliflower mosaic 35S promoter: combinational regulation of transcription in plants. Science 250:959–966; 1990.
Bourgaud, F.; Gravot, A.; Milesi, E.; Gontier, E. Production of plant secondary metabolietes: a historical perspective. Plant Sci. 161:839–851; 2001.
Canel, C.; Lopes, Cardoso M. I.; Whitmer, S.; van-der Fits, L.; Pasquali, G.; van-der Heijden, R.; Hoge, J. H. C.; Verpoorte, R. Effects of overexpression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta 205:414–419; 1998.
Cusido, R.; Palazon, J.; Pinol, M. T.; Bonfill, M.; Marales, C. Datura metel: in vitro propagation of tropane alkaloids. Planta Med. 65:144–148; 1999.
Dixon, R. A. Natural products and plant disease resistance. Nature 411:843–847; 2001.
Ebel, J. Biochemical analysis of the induction process. Annu. Rev. Phytopathol. 24:235–264; 1986.
Giri, A.; Narasu, M. L. Recent trends and applications. Biotechnol. Adv. 18:1–22; 2000.
Hashimoto, T.; Hayashi, A.; Amano, Y.; Kohno, J.; Iwanari, H.; Usuda, S.; Yamada, Y. Hyoscyamine 6β-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root. J. Biol. Chem. 266:4648–4653; 1991.
Hashimoto, T.; Matsuda, J.; Yamada, Y. Two-step expoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6β-hydroxylase. FEBS Lett. 329:35–39; 1993.
Hashimoto, T.; Shoji, T.; Mihara, T.; Ogari, H. Tamaki, Y.; Yamada Y. Intraspecific variability of the tandem repeats of putrescine N-methyltransferase. Plant Mol. Biol. 37:25–37; 1998.
Hashimoto, T.; Yamada, Y. Alkaloids biogenesis: molecular aspects. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45:257–285; 1994.
Hibi, N.; Fujita, T.; Hatano, M.; Hashimoto, T.; Yamada, Y. Putrescine N-methyltransferase in cultured roots of Hyoscyamus albus. Plant Physiol. 100:826–835; 1992.
Jung, H. Y.; Kang, M. J.; Kang, Y. M.; Yun, D. J., Bahk, J. D.; Chung, Y. G.; Choi, M. S. Production of tropane alkaloids by optimum culture conditions and XAD resin, on hairy root cultures from Scopolia parviflora Nak. Kor. J. Biotechnol. Bioengng 17:525–530; 2002.
Kang, Y. M.; Min, J. Y.; Kim, W. J.; Kim, Y. D.; Lee, B. H.; Choi, M. S. Growth pattern and content of tropane alkaloids of metabolic engineered Scopolia parviflora hairy root lines. Korean J. Med. Crop. Sci. 12:123–128; 2004a.
Kang, Y. M.; Min, J. Y.; Moon, S. H.; Karigar, C. S.; Prasad, D. T.; Lee, C. H.; Choi, M. S. Rapid in vitro adventitious shoot propagation of Scopolia parviflora through rhizome cultures for enhanced production of tropane alkaloids. Plant Cell Rep. 23:128–133; 2004b.
Laemmli, U. K. Cleaage of structural protein during the assembly of the lead of bacteriophage T4. Nature 227:680–685; 1970.
Mitsuhara, I.; Ugaki, M.; Hirochika, H.; Ohshima, M.; Murakami T.; Gotoh, Y.; Katayose, Y.; Nakamura, S.; Honkura, R., Nishimiya, S.; Ueno, K.; Mochizuki, A.; Tanimoto, H.; Tsugawa, H.; Otsuki, Y.; Ohashi, Y. Efficient promoter cassettes for enhanced expression of foregign genes in cotyledonous and monoctyledonous plants. Plant Cell Physiol. 37:49–59; 1996.
Moyano, E.; Fornale, S.; Palazon, J.; Cusido, R. M.; Bagni, N.; Pinol, M. T. Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene. Phytochemistry 59:697–702; 2002.
Oksman, C. K. M.; Arro, R. Regulation of tropane alkaloid metabolism in plants and plant cell cultures. In: Verpoorte, R.; Alfermann, A. W., eds. Metabolic engineering of plant secondary metabolism. Dordrecht: Kluwer Academic Publisher; 2000:254–281.
Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manual. New York: Cold Springer Harbor Laboratory Press; 1998.
Sato, F.; Hashimoto, T.; Hachiya, A.; Tamura, K.; Choi, K. B.; Morishige, T.; Fujimoto, H.; Yamada, Y. Metabolic engineering of plant alkaloid biosynthesis. Proc. Natl Acad. Sci. USA 98:367–372; 2001.
Suzuki, K.; Yamada, Y.; Hashimoto, T. Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiol. 40:298–297; 1999.
Waller, G. R.; Nowacki, E. K. Alkaloid biology and metabolism in plants. New York: Kluwer Academic Publishers/Plenum; 1978.
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Lee, OS., Kang, YM., Jung, HY. et al. Enhanced production of tropane alkaloids in Scopolia parviflora by introducing the PMT (putrescine N-methyltransferase) gene. In Vitro Cell.Dev.Biol.-Plant 41, 167–172 (2005). https://doi.org/10.1079/IVP2004621
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DOI: https://doi.org/10.1079/IVP2004621