Identification of genes in Thuja plicata foliar terpenoid defenses.

Thuja plicata (western redcedar) is a long-lived conifer species whose foliage is rarely affected by disease or insect pests, but can be severely damaged by ungulate browsing. Deterrence to browsing correlates with high foliar levels of terpenoids, in particular the monoterpenoid a-thujone. Here, we set out to identify genes whose products may be involved in the production of a-thujone and other terpenoids in this species. First, we generated a foliar transcriptome database from which to draw candidate genes. Second, we mapped the storage of thujones and other terpenoids to foliar glands. Third, we used global expression profiling to identify more than 600 genes that are expressed at high levels in foliage with glands, but can either not be detected or are expressed at low levels in a natural variant lacking foliar glands. Fourth, we used in situ RNA hybridization to map the expression of a putative monoterpene synthase to the epithelium of glands and used enzyme assays with recombinant protein of the same gene to show that it produces sabinene, the monoterpene precursor of a-thujone. Finally, we identified candidate genes with predicted enzymatic functions for the conversion of sabinene to a-thujone. Taken together, this approach generated both general resources and detailed functional characterization in the identification of genes of foliar terpenoid biosynthesis in T. plicata.