BIOSYNTHESIS OF CYCLIC DITERPENE HYDROCARBONS IN RICE CELL-SUSPENSIONS - CONVERSION OF 9,10-SYN-LABDA-8(17),13-DIENYL DIPHOSPHATE TO 9-BETA-PIMARA-7,15-DIENE AND STEMAR-13-ENE

The biosynthesis of diterpene hydrocarbons with enzyme extracts from r ice cell suspension cultures was investigated to verify proposed pathw ays and intermediates in the production of the momilactone and oryzale xin phytoalexins. Diterpene synthase activity in cells treated with ch itin to elicit the phytoalexin response was compared with the activity in untreated cells using the acyclic substrates [1-H-3](E,E,E)- and [ 1-H-3](E,Z,E)-geranylgeranyl diphosphates (GGPPs 4-OPP and 11-OPP) as well as the bicyclic substrates [15-H-3]ent-copalyl and [15-H-3]syn-co palyl diphosphates (CPPs, 5-OPP, and 6-OPP). ent-kaurene (7), ent-sand aracopimaradiene (8), 9 beta H-pimara-7,15-diene (9), and stemar-13-en e (10) were identified as major products by comparisons with authentic standards. Marked increases in diterpene synthase activities were obs erved with enzyme from chitin-treated cells: (E,E,E)-GGPP (similar to 100 fold), ent-CPP (similar to 3 fold), and syn-CPP (similar to 60 fol d). The very low conversions of (E,Z,E)-GGPP to hydrocarbon products e xcludes its role in the biosynthesis of 9,10-syn-diterpenes in rice ce lls. ent-Kaurene was the major diterpene formed from ent-CPP with enzy me from unelicited cells. In contrast the enzyme from chitin-treated c ells converted ent-CPP to a mixture of ent-kaurene, ent-sandaracopimar adiene, and a third unidentified diterpene. With syn-CPP as substrate the induced synthases afforded a mixture of 9 beta-pimaradiene, stemar ene, and a third, unidentified syn-diterpene. Overall the results are consistent with the hypothesis that rice cells respond to treatment wi th chitin fragments by producing new diterpene synthases not present i n the untreated cells, These induced cyclases initiate phytoalexin bio synthesis by diverting (E,E,E)-GGPP into new cyclization modes that pr oduce ent-sandaracopimaradiene, stemarene, and 9 beta-pimaradiene, the presumed precursors to oryzalexins A-F, oryzalexin S, and momilactone s A-C, respectively. The intermediate role of 9,10-syn-CPP in syn dite rpene biosynthesis is verified. (C) 1996 Academic Press, Inc.