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Heterotrophic cell suspension cultures were initiated from hypocotyls of young Ammi majus L. seedlings. When these cultures were propagated continuously in the dark, they produced only traces of the coumarin umbelliferone. Upon addition of fungal cell wall fractions, i.e. a skieroglucan or an elicitor from either Phytophthora megasperma f. sp. glycinea or Alternaria carthami , the cells excreted large amounts of umbelliferone in addition to isopimpinellin, ( S )-marmesin, ( R )-ammirin, umbelliferone-[3′-methyl-buta-1 t .3-dien-1′-yl]-ether and umbelliferone-[3′-hydroxymethyl- 1 t .-buten-1′-yl]-ether. The last two compounds are new compounds and appear to be derived from 7- O -prenylumbelliferone, a coumarin that has been identified in from other plants. The Phytophthora -elicitor was the most effective inducer of coumarin accumulation. When cultures which had been elicited for 3 hr were pulsed with l -[U- 14 C]phenylalanine for 7 hr, all the coumarins and an additional, still unidentified compound in the extracts were labelled efficiently. Our results suggest that A. majus cells are particularly suitable for biosynthetic studies on various coumarins as well as for regulatory studies of the inhibition of coumarin phytoalexin accumulation.

Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

Publisher Summary This chapter discusses the induced accumulation of coumarins, the regulation of their biosynthesis, and their potential physiological function. Cultured plant cells do not accumulate secondary metabolites as readily as their parent plants. However, as coumarins are produced in large quantities in various plants belonging to families Ructacae, Umbelliferae, and Solanaceae, cell cultures derived from these plants either lack coumarins, or their coumarin content is low. Moreover, the amount of coumarin produced in cell cultures may vary from one transfer of cells to another. These differences cannot be attributed to genetic incompetence of the cultured cells, but result rather from abnormal gene expression. Coumarins isolated from cell cultures grown on standard growth media are referred as constitutive coumarins. The level of secondary metabolities in cultured cells may further be increased by several means.

Microsomes prepared from cultured Ammi majus cells that had been challenged for 14 h with an elicitor derived from the cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) converted psoralen to bergaptol (5-hydroxypsoralen) in the presence of NADPH and oxygen. The enzymatic activity was characterized as an inducible cytochrome-P-450-dependent monooxygenase associated with the endoplasmic reticulum. All of the steps involved in bergapten (5-methoxypsoralen) biosynthesis in Ammi majus have now been demonstrated in vitro. The results suggest that bergaptol and not hydroxymarmesin in the precursor of bergapten.

Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P450-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.