title: Involvement of protein kinase and G proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis.
abstract: We have previously reported that elicitor-induced benzophenanthridine alkaloid biosynthesis in suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) is mediated by a signal transduction system that involves calcium and possibly protein kinase(s). In this work, a number of exogenous agents were employed to further investigate the components of the signal transduction pathway involved in the induction of alkaloid biosynthesis by a fungal elicitor and abscisic acid (ABA). SCP-GM suspension-cells were treated with compounds that modify protein kinase activity, including phorbol esters, and 1-oleoyl-2-acetyl-rac-glycerol (OAG), a synthetic diacylglycerol analogue. Phorbol-12-myristate-13-acetate induced alkaloid accumulation by as much as 65-fold over control values, while the negative control, phorbol-13-monoacetate, had no effect. OAG also increased alkaloid production by approximately 25-fold as compared to controls. Likewise, pretreatment of the suspension-cell cultures with H-7 or staurosporine, significantly suppressed ABA- or fungal-induction of benzophenanthridine alkaloid biosynthesis. Modulators of GTP-binding protein activity were also active in this system. Treatment of the suspension-cells with cholera toxin (CHX) induced alkaloid accumulation by 25-fold, which increased to 34-fold when CHX was combined with a fungal elicitor derived from Penicillium expansum (PE), and 32-fold when CHX was combined with ABA. Treatment of SCP-GM cells with CHX also enhanced the activities of two N-methyltransferases in the benzophenanthridine biosynthetic pathway namely, tetrahydroberberine-N-methyltransferase and tetrahydrocoptisine-N-methyltransferase, by six and seven fold, respectively. Furthermore, benzophenanthridine alkaloid biosynthesis was induced by treating the suspension-cells with the G-protein activators, mastoparan, mas-7 or melittin, while the inactive homologue, mas-17, did not. Suppression of alkaloid accumulation occurred when the suspension-cells were treated with GDP beta S or pertussis toxin prior to treatment of the SCP-GM cells with either PE or ABA. The results support the hypothesis that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago 60605, USA.
authors.forename: G B
authors.initials: GB
authors.lastname: Mahady
authors.forename: C
authors.initials: C
authors.lastname: Liu
authors.forename: C W
authors.initials: CW
authors.lastname: Beecher
mesh_terms: D000040:Abscisic Acid / Q000494:pharmacology; D000470:Alkaloids / Q000096:biosynthesis*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D059828:Plant Cells; D010944:Plants / Q000201:enzymology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D000040:Abscisic Acid; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D019311:Staurosporine