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

Abstract

Soluble preparations of cotton stele tissue infected with Verticillium dahliae containing δ-cadinene synthase convert (1- RS)-[1- 2H]- E,E-farnesyl diphosphate to [5- 2H]- and [11- 2H]-δ-cadinene and convert [4,4,13,13,13- 2H 5]-nerolidyl diphosphate to [8,8,15,15,15- 2H 5]-δ-cadinene. These data imply that nerolidyl diphosphate is an intermediate in the enzymatic cyclization of the natural substrate E,E-farnesyl diphosphate to δ-cadinene by δ-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 δ-cadinene. Kinetic analyses of induced δ-cadinene synthase mRNA, δ-cadinene synthase activity and formation of sesquiterpenoid phytoalexins in cotton stele tissue infected with Verticillium dahliae show that 12 hr after fungal inoculation the δ-cadinene synthase mRNA was at a maximum level. The tissue injected with H 2O in place of fungal inoculation showed no detectable δ-cadinene synthase mRNA or δ-cadinene synthase activity after 12 to 96 hr. After 12 hr, 54% of the δ-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 level of δ-cadinene synthase mRNA and an increased activity of δ-cadinene synthase which functions in the conversion of E,E-farnesyl diphosphate → nerolidyl diphosphate → δ-cadinene that is metabolically converted to desoxyhemigossypol, desoxyhemigossypol-6-methyl ether, hemigossypol and hemigossypol-6-methyl ether.