Subfunctionalization and epigenetic regulation of a biosynthetic gene cluster in <i>Solanaceae</i>

Biosynthetic gene clusters (BGCs) are sets of often heterologous genes that are genetically and functionally linked. Among eukaryotes, BGCs are most common in plants and fungi and ensure the coexpression of the different enzymes coordinating the biosynthesis of specialized metabolites. Here, we report the identification of a withanolide BGC in <i>Physalis grisea</i> (ground-cherry), a member of the nightshade family (<i>Solanaceae</i>). A combination of transcriptomic, epigenomic, and metabolic analyses revealed that, following a duplication event, this BGC evolved two tissue-specifically expressed subclusters, containing several pairs of paralogs that contribute to related but distinct biochemical processes; this subfunctionalization is tightly associated with epigenetic features and the local chromatin environment. The two subclusters appear strictly isolated from each other at the structural chromatin level, each forming a highly self-interacting chromatin domain with tissue-dependent levels of condensation. This correlates with gene expression in either above- or below-ground tissue, thus spatially separating the production of different withanolide compounds. By comparative phylogenomics, we show that the withanolide BGC most likely evolved before the diversification of the <i>Solanaceae</i> family and underwent lineage-specific diversifications and losses. The tissue-specific subfunctionalization is common to species of the <i>Physalideae</i> tribe but distinct from other, independent duplication events outside of this clade. In sum, our study reports on an instance of an epigenetically modulated subfunctionalization within a BGC and sheds light on the biosynthesis of withanolides, a highly diverse group of steroidal triterpenoids important in plant defense and amenable to pharmaceutical applications due to their anti-inflammatory, antibiotic, and anticancer properties.