Biosynthesis of cyclic diterpene hydrocarbons in rice cell suspensions: conversion of 9,10-syn-labda-8(17),13-dienyl diphosphate to 9beta-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-sanda, racopimaradiene (8), 9 beta H-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 (approximately 100 fold), ent-CPP (approximately 3 fold), and syn-CPP (approximately 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 from ent-CPP with enzyme from unelicited cells. In contrast the enzyme from chitin-treated cells converted ent-CPP to a mixture of ent-kaurene, ent-sandaracopimaradiene, and a third unidentified diterpene. With syn-CPP as substrate the induced syntheses afforded a mixture of 9 beta-pimaradiene, stemarene, and a third, unidentified syn-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 produce ent-sandaracopimaradiene, stemarene, and 9 beta-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.
Mohan
R S
RS
Department of Chemistry, University of Illinois, Urbana 61801, USA.
Yee
N K
NK
Coates
R M
RM
Ren
Y Y
YY
Stamenkovic
P
P
Mendez
I
I
West
C A
CA
D001711:Biotransformation; D002686:Chitin / Q000494:pharmacology; D004224:Diterpenes / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D010755:Organophosphates / Q000378:metabolism*; D012275:Oryza / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D012717:Sesquiterpenes; D013379:Substrate Specificity; D013729:Terpenes; D014166:Transferases / Q000378:metabolism*; D014316:Tritium; D000094162:Phytoalexins
D004224:Diterpenes; D010755:Organophosphates; D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; C099623:stemar-13-ene; D014316:Tritium; D002686:Chitin; C099622:pimara-7,15-diene; C090962:copalyl pyrophosphate; D014166:Transferases; D000094162:Phytoalexins