Biosynthesis of Cardiac Glycosides in Wallflowers (Erysimum, Brassicaceae)

Accumulation of cardiac glycosides, small molecule inhibitors of essential Na⁺/K⁺ ATPases in animal cells, evolved independently as a defensive trait in at least twelve plant families. Although they are lethal at higher concentrations, plant-derived cardiac glycosides have a long history of use in both traditional and modern medicine as a treatment for congestive heart failure and other diseases. However, despite extensive research on the ecological functions and medical applications of cardiac glycosides, the complete biosynthesis pathway has not been identified in any plant species. Within the Brassicaceae family, the Erysimum genus is unique in its accumulation of cardiac glycosides. To provide new insight into cardiac glycoside biosynthesis, we sequenced the genome of Erysimum cheiranthoides (wormseed wallflower) and the foliar transcriptomes of 47 additional Erysimum species. Metabolite profiling detected almost 100 different cardiac glycosides, with up to fifty in some individual Erysimum species. Comparison of the cardiac glycoside content and a transcriptome-based Erysimum phylogeny provided evidence for rapid gain and loss of enzyme functions. There appears to be no tradeoff between the production of glucosinolates, characteristic defensive metabolites of plants in the Brassicaceae, and cardiac glycosides in Erysimum. We identified candidate genes for cardiac glycoside biosynthesis in E. cheiranthoides with a combination of ethylmethanesulfonate mutagenesis, co-expression analysis, and genetic mapping of natural variation in cardiac glycoside content. Both Agrobacterium-mediated transient expression and virus-induced gene expression silencing were used to confirm the in vivo function of the identified E. cheiranthoides genes.