Evolution of withanolide biosynthesis in nightshades

Abstract Withanolides are a diverse class of defensive metabolites produced by multiple clades within the nightshade family (Solanaceae). To evaluate whether withanolide biosynthesis originated independently across these lineages, we investigated the evolutionary history of a withanolide biosynthetic gene cluster (WBC) that encodes enzymes required for withanolide production. We analyzed genomes, transcriptomes, and metabolomes from dozens of Solanaceae species to assess whether the genomic presence or expression of WBC genes accounts for the distribution of withanolides among clades. Our results indicate that the WBC was likely present in the common ancestor of Solanaceae and was independently lost in several non‐producing lineages. Moreover, many WBC genes are expressed exclusively in withanolide‐producing species and show phylogenetically widespread correlations between transcript abundance and metabolite levels. These findings suggest that regulatory changes, followed by genetic degradation of the WBC, have driven repeated losses of withanolide biosynthesis during Solanaceae evolution. To explore the regulatory basis of these shifts, we inferred gene regulatory networks and found evidence that withanolide biosynthesis may be suppressed by transcriptional programs associated with steroidal glycoalkaloid production. Notably, we also identified two transcription factors that appear to promote or maintain WBC gene expression in specific lineages. These regulatory genes provide valuable tools for investigating the evolution, ecological function, and potential applications of withanolides.