The enzymatic cyclization of nerolidyl diphosphate by δ-cadinene synthase from cotton stele tissue infected with Verticillium dahliae

Soluble preparations of cotton stele tissue infected with Verticillium dahliae containing delta-cadinene synthase convert (1-RS)-[1-H-2]-E,E-farnesyl diphosphate to [5-H-2]- and [11-H-2]-delta-cadinene and convert [4,4,13,13,13-H-2(5)]-nerolidyl diphosphate to [8,8,15,15,15-H-2(5)]-delta-cadinene. These data imply that nerolidyl diphosphate is an intermediate in the enzymatic cyclization of the natural substrate E,E-farnesyl diphosphate to delta-cadinene by delta-cadinene synthase and involves the conversion of E,E-farnesyl diphosphate to nerolidyl diphosphate followed by cyclization to cis-germacradienyl cation, a 1,3-hydride shift, a second cyclization to a cadinanyl cation and deprotonation to delta-cadinene. Kinetic analyses of induced delta-cadinene synthase mRNA, delta-cadinene synthase activity and formation of sesquiterpenoid phytoalexins in, cotton stele tissue infected with Verticillium dahliae show that 12 hr after fungal inoculation the delta-cadinene synthase mRNA was at a maximum level. The tissue injected with H2O in place of fungal inoculation showed no detectable delta-cadinene synthase mRNA or delta-cadinene synthase activity after 12 to 96 hr, After 12 hr, 54% of the delta-cadinene synthase activity had developed, but no phytoalexins were detected, the midpoint in the formation of the phytoalexins was 48 hr. These data, together with the enzyme analyses, support the conclusion that Verticillium dahliae initiates a signal in the stele tissue that results in an increased steady-state revel of delta-cadinene synthase mRNA and an increased activity of delta-cadinene synthase which functions in the conversion of E,E-farnesyl diphosphate --> nerolidyl diphosphate --> delta-cadinene that is metabolically converted to desoxyhemigossypol, desoxyhemigossypol-6-methyl ether, hemigossypol and hemigossypol-6-methyl ether. (C) 1998 Elsevier Science Ltd. All rights reserved.