UBC Theses and Dissertations
Thujone biosynthesis in western redcedar (Thuja plicata) Kshatriya, Kristina
Western redcedar (WRC; Thuja plicata) is a highly valued conifer species native to the west coast of North America. However, herbivore feeding, specifically browsing by deer and elk, is a major problem for WRC reforestation. Browsing deterrence correlates with high levels of foliar monoterpenes, particularly α- and β-thujone, which accumulate in resin glands in WRC foliage and seeds. Thujone is proposed to derive from geranyl diphosphate via a four-step biosynthetic pathway. The first two enzymes of the pathway have recently been identified: a monoterpene synthase converts geranyl diphosphate into (+)-sabinene, which is oxidized into (+)-trans-sabinol through a cytochrome P450. It is hypothesized that (+)-trans-sabinol is further oxidized to (+)-sabinone and then reduced to α- and β-thujone. In this study, I further investigated thujone biosynthesis in WRC. First, I monitored thujone pathway intermediates and resin storage structures throughout the growing season. Both foliage and seed cones had a temporal shift from sabinene to α-thujone as the predominant monoterpene. This shift in monoterpene predominance was accompanied by the deterioration of epithelial cells surrounding oleoresin storage structures. Second, I selected candidate genes for the third step of the thujone pathway, proposed to be an alcohol dehydrogenase (ADH) catalyzing the oxidation of (+)-trans-sabinol to (+)-sabinone. Using transcriptomic analysis of wild type WRC foliage as well as a naturally occurring WRC variant that does not produce monoterpenes, I identified 21 putative ADHs. Third, I tested candidate ADH gene functions through in vitro assays with recombinant protein that was heterologously expressed in E. coli. Three ADHs (ADH5, ADH10, and ADH17) were found to have activity, producing β-thujone rather than the expected product of (+)-sabinone. Optimization in protein expression and activity assay conditions led to ADH5 producing both β-thujone and (+)-sabinone as major products. Understanding the genes involved in thujone biosynthesis will provide valuable information for WRC tree breeding. Discovering mechanisms of resistance in WRC will support efforts to produce seedlings that are more resistant to ungulate browsing and have the desired resistance attributes for reforestation.
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