Characterization Of The Late Metabolic Steps In Poison Ivy Urushiol Biosynthesis

Abstract

Urushiol is the natural product allergen responsible for the dreaded skin rash symptoms caused by poison ivy (Toxicodendron radicans). Although none of the urushiol biosynthetic enzymes or genes have been characterized, there are two alkylphenol metabolites present in poison ivy—anacardic acid and cardanol—hypothesized as urushiol metabolic intermediates. Urushiol is hypothesized to be synthesized from either of these metabolites through a hydroxylation reaction on the aromatic ring. Thus, the objective of this work is to identify poison ivy transcripts that encode for oxidative enzymes capable of converting anacardic acid or cardanol into urushiol. To identify potential candidate genes responsible for catalyzing these hydroxylations, we identified 13,087 differentially expressed poison ivy transcripts (≥ 3-fold difference with a p-value ≤ 0.05) that were also significantly correlated with differential urushiol accumulation levels (Pearson correlation p-value ≤ 0.05). These differentially expressed transcripts were subsequently filtered for enzyme families known to catalyze hydroxylation reactions on an aromatic ring. This resulted in eighteen transcripts, representing three different enzyme families, that are high priority candidates for encoding predicted enzymes responsible for catalyzing hydroxylation reactions of either anacardic acid or cardanol to produce urushiol. Additionally, coordinately expressed transcripts were grouped into "modules" with coexpression analysis. Coexpression analysis revealed a single module that was significantly, positively correlated with C15 urushiol levels (0.79, p-value=0.01) and contained sixteen poison ivy transcripts that may catalyze hydroxylation reactions of either anacardic acid or cardanol. Each candidate transcript was expressed in Nicotiana benthamiana or Saccharomyces cerevisiae, the recombinant protein purified and then assayed for hydroxylation of either anacardic acid or cardanol resulting in urushiol production. The in vitro enzyme assays are necessary to biochemically validate which transcript(s) encode a cardanol or anacardic acid hydroxylase, thereby identifying genes involved in late steps in urushiol biosynthesis. Eleven CYPs and two 2ODOs were expressed in tobacco or in yeast; however, biochemical analyses with the recombinant enzymes failed to confirm the hypothesized enzymic production of the expected urushiol metabolic products. These negative results prompted a reevaluation of the hypothesis that anacardic acid and/or cardanol are urushiol metabolic intermediates. Future work will focus on sampling younger poison ivy tissues—in the case that urushiol biosynthesis is developmentally regulated—and using scRNA-seq for analyzing transcriptomes of cell types most associated with urushiol synthesis.