Isolation and Characterization of Volatile Terpene Synthases in Cotton

In cotton, gossypol and related sesquiterpene aldehydes, which function as phytoalexins in direct defense reactions against pathogens and herbivores, are derived from the sesquiterpene (+)-delta-cadinene. However, cotton plants also produce and release a blend of volatile terpenes. By gas chromatography–mass spectrum (GC-MS) analysis, we detected 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12. By EST data mining combined with RACE, full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. In vitro assays of the recombinant proteins revealed that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were widely expressed in the cotton plant, whereas GhTPS2 was expressed only in leaves, and expressions of them can be induced by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA). Moreover, in the G. raimondii genome, totally 69 putative terpene synthases were identified and classified into 3 large groups of monoterpene synthases, sesquiterpene synthases and diterpene metabolism related. Some of them, including (+)-δ-cadinene synthase gene family members, are organized as clusters and showed distinct and biased expression patterns.