Molecular genetic, biochemical & anatomical characterization of monoterpenoid synthesis in western redcedar (Thuja plicata).

Western redcedar (Thuja plicata) is an economically and culturally important tree species to British Columbia (BC). Wood from T. plicata is highly valued for being light weight, dimensionally stable and resistant to rot. However, T. plicata silviculture is not without problems. Reforestation with T. plicata is expensive due to extensive browsing of seedlings by ungulates. Also, woody stems of mature trees can be susceptible to heartwood rotting fungi. Resistance to ungulate browsing positively correlates with the overall abundance of monoterpenoids in foliage, notably the oxygenated monoterpenoid α-thujone, while heartwood rot resistance is relative to the amount of tropolones, a class of compounds derived from monoterpenes, in the wood. To better understand factors that affect monoterpenoid content in young T. plicata foliage, leaves were examined for ontogenetic changes in morphology, anatomy and monoterpenoid content during the first year of growth. The two morphologically different leaf types, needles and scales, possess different monoterpenoid storage structures and very different chemical contents, punctuated by the absence of α-thujone in needles. To determine the genetic basis for the production of foliar monoterpenoids, a transcriptome database was generated from which to draw candidate genes. Complementary DNA profiling was used to determine the differential gene expression between a natural variant lacking foliar monoterpenoids and wild type tree. This analysis yielded more than 600 genes with low or undetectable expression in foliage of the variant, and up to 10000-fold higher transcript abundance in foliage with resin glands. Amongst the candidate genes was a single monoterpene synthase gene that produced sabinene, the likely monoterpene precursor to α-thujone, as its major product in vitro. A second, more comprehensive, transcriptome was generated to identify additional monoterpene synthases from other tissues. Eleven terpene synthase gene fragments were identified, one of which was found to produce terpinolene, the likely precursor of the tropolone β-thujaplicin. The transcriptome datasets together with the characterization of monoterpene synthases represents a major first step into understanding the genetics of T. plicata terpenoid based defenses.