Taxus cell culture may be an alternative source of paclitaxel and related taxane production. Significantly increased amounts of paclitaxel and baccatin III were observed in cultured cells of Taxus species after exposure to methyl jasmonate. Among the three species of Taxus tested, Taxus media showed the highest paclitaxel content while Taxus baccata showed the highest baccatin III content when 100 microM of methyl jasmonate was added to the culture media. Furthermore, the activities of methyl jasmonate and related substances for inducing paclitaxel production were compared in cell suspension cultures of T. media. Methyl jasmonate and its free acid showed the strongest promoting activity. Reduction of the keto group at the C-3 position greatly reduced this activity. cis-Jasmone, which does not have a carboxyl group at the C-1 position, had almost no activity. These results suggest that these two regions of methyl jasmonate are important for promoting the production of paclitaxel and related taxanes in Taxus cell cultures.

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[1,4-(14)C]Putrescine, administered to cultured roots of Hyoscyamus albus L., was converted to spermidine and spermine as well as to conjugated forms. A substantial fraction of its radioactivity (approx. 6%) was, however, incorporated into N-methylputrescine within 4 h, followed by an increase in radioactive tropane alkaloids (mostly hyoscyamine). Incubation of the roots with DL-[5-(14)C]ornithine and L-[2,3-(3)H]arginine separately resulted in rapid incorporation of each label into free putrescine and N-methylputrescine, followed by incorporation into hyoscyamine. During active alkaloid biosynthesis in H. albus roots, scarcely any radioactivity was recovered in δ-N-methylornithine. When the roots had been treated with 5 mM DL-α-difluoromethylornithine for 1 d prior to the administration of labeled ornithine, the total uptake of radioactivity into the root cells was not reduced, even though ornithine decarboxylase was specifically inactivated and the incorporation of the label to putrescine, N-methylputrescine, hyoscyamine and scopolamine markedly reduced. These and other results (Hashimoto et al., 1989, Planta 178, 123-130) are strong indications that tropane alkaloids in H. albus root cultures are synthesized from both ornithine and arginine by way of putrescine.

The activity of arginine decarboxylase (EC 4.1.1.19) in cultured roots of Hyoscyamus albus L., which produce considerable amounts of tropane alkaloids, was twice that of ornithine decarboxylase (EC 4.1.1.17), both activities being highest during active root growth, whereas arginase (EC 3.5.3.1) activity was negligible. Actively growing roots had putrescine conjugates as their major polyamines, and spermidine was the most abundant free polyamine. Putrescine N-methyltransferase (PMT; EC 2.1.1.53) activity was high, the peak occurring on the sixth day of culture when root growth became slower. Thereafter, the free N-methylputrescine content of the roots increased and was followed by an increase in alkaloid content (mostly hyoscyamine). The amounts of arginine and, especially, of ornithine were low. No N-methylornithine was detected. The PMT activity was present only in root, shoot and cell-suspension cultures of plants that synthesized tropane alkaloids or nicotine; no enzyme activities that methylate ornithine at the δ-amino group or that decarboxylate δ-N-methylornithine were detected in any of the cultures tested. Our data indicate that tropane alkaloids in H. albus roots are synthesized by way of the symmetrical putrescine, i.e. a pathway different from that proposed by E. Leete (1962, J. Am. Chem. Soc. 84, 55) according to which these alkaloids are synthesized by way of asymmetrical δ-N-methylornithine.

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