Biosynthesis of Cyclic Diterpene Hydrocarbons in Rice Cell Suspensions: Conversion of 9,10-syn-Labda-8(17),13-dienyl Diphosphate to 9β-Pimara-7,15-diene and Stemar-13-ene

The biosynthesis of diterpene hydrocarbons with enzyme extracts from rice cell suspension cultures was investigated to verify proposed pathways and intermediates in the production of the momilactone and oryzalexin phytoalexins. Diterpene synthase activity in cells treated with chitin to elicit the phytoalexin response was compared with the activity in untreated cells using the acyclic substrates [1-3H](E,E,E)- and [1-3H](E,Z,E)-geranylgeranyl diphosphates (GGPPs 4-OPP and 11-OPP) as well as the bicyclic substrates [15-3H]ent-copalyl and [15-3H]syn-copalyl diphosphates (CPPs, 5-OPP, and 6-OPP).ent-kaurene (7),ent-sandaracopimaradiene (8), 9βH-pimara-7,15-diene (9), and stemar-13-ene (10) were identified as major products by comparisons with authentic standards. Marked increases in diterpene synthase activities were observed with enzyme from chitin-treated cells: (E,E,E)-GGPP (∼100 fold),ent-CPP (∼3 fold), andsyn-CPP (∼60 fold). The very low conversions of (E,Z,E)-GGPP to hydrocarbon products excludes its role in the biosynthesis of 9,10-syn-diterpenes in rice cells.ent-Kaurene was the major diterpene formed froment-CPP with enzyme from unelicited cells. In contrast the enzyme from chitin-treated cells convertedent-CPP to a mixture ofent-kaurene,ent-sandaracopimaradiene, and a third unidentified diterpene. Withsyn-CPP as substrate the induced synthases afforded a mixture of 9β-pimaradiene, stemarene, and a third, unidentifiedsyn-diterpene. Overall the results are consistent with the hypothesis that rice cells respond to treatment with chitin fragments by producing new diterpene synthases not present in the untreated cells. These induced cyclases initiate phytoalexin biosynthesis by diverting (E,E,E)-GGPP into new cyclization modes that produceent-sandaracopimaradiene, stemarene, and 9β-pimaradiene, the presumed precursors to oryzalexins A–F, oryzalexin S, and momilactones A–C, respectively. The intermediate role of 9,10-syn-CPP in syn diterpene biosynthesis is verified.