Whole-genome duplication drives biosynthetic gene cluster fragmentation and regulatory rewiring of monoterpene indole alkaloid metabolism in Strychnos

Whole-genome duplications (WGDs) shape plant genomes by creating redundancy, which then leads to distinct architectural changes through processes like fractionation, gene loss, and rearrangement. The manner in which specialized metabolic pathways maintain functional integration after such extensive restructuring is not entirely clear, especially for biosynthetic gene clusters (BGCs) that typically cluster genes involved in specialized metabolism but can be disrupted by post-duplication events.

In this study, we introduce chromosome-level genomes for Loganiaceae, including closely spaced telomere-to-telomere assemblies of Strychnos ignatii and S. pubescens, as well as a draft genome of the extinct S. ridleyi. Following a WGD, the two surviving Strychnos species have diverged in their evolutionary paths and metabolite profiles: S. ignatii witnesses an expansion of monoterpenoid and monoterpene indole alkaloid (MIA) gene families and dominance of strychnine-type MIA, while S. pubescens demonstrates heightened transposable element activity linked to DOF-linked regulatory rewiring and a broader chemical profile featuring sesquiterpenoids and triterpenoids.

Remarkably, both species maintain active strychnine biosynthesis despite fragmentation of a crucially conserved alkaloid BGC in MIA-producing Gentianales, indicating that pathway function can persist even when ancestral BGC architecture is disrupted. By comparing metabolomic and transcriptomic pathway data, we identify norfluorocurarine oxidase (NO) as a key divergence point contributing to strychnine accumulation.

Our analysis of promoters, yeast one-hybrid assays, and electrophoretic mobility shift assays supports a model where S. ignatii retains the traditional jasmonate-responsive MYB, MYC2/bHLH, and AP2/ERF cis-regulatory module at NO, whereas S. pubescens exhibits a diminished capacity to recruit these activators and instead displays a DOF-associated regulatory architecture.

Overall, our findings reveal that WGD can separate physical cluster organization from pathway function, enabling specialized metabolic pathways to remain active while undergoing divergent chemical evolution through lineage-specific expansions in coding space and transposable-element-driven cis-regulatory rewiring.