Independent evolutionary origin for insect deterrent cucurbitacins

The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Iberis spp. are Brassicaceae contain unique feeding inhibitors, cucurbitacins, that are free from insect Pieris rapae and Phyllotreta nemorum which specialized in Brassicaceae. Using de-novo assembled transcriptomics from Iberis amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase. In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyse the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the unlinked genomic position of the cucurbitacin biosynthetic genes in I. amara contrasts with the highly organized gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This investigation thus provides new insight into the mechanism of recurrent defensive chemical evolution and diversification.