Cardiac glycosides protect wormseed wallflower () against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model.We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field.CYP87A126 initiates cardiac glycoside biosynthesis via sterol side chain cleavage, and CYP716A418 has a role in cardiac glycoside hydroxylation. In CYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores.These results begin elucidation of the cardiac glycoside biosynthetic pathway and demonstrate that cardiac glycoside production allows to escape from some, but not all, specialist herbivores.