Conserved trans-regulatory logic and evolving cis-modules drive the diversification of inducible diterpenoid...

Diterpenoid phytoalexins are major inducible defense metabolites in rice, whose biosynthetic genes are organized into biosynthetic gene clusters (BGCs). While BGC architecture varies across the Oryzoideae, their shared inducibility is known; however, the regulatory logic and evolutionary trajectory governing this response remains poorly understood. Here, we investigate the evolution of clustered gene regulation by examining the interplay between conserved trans-acting factors and progressively diverged cis-regulatory elements. We demonstrate that diterpenoid BGC induction is governed by a stable trans-acting framework built upon rapidly evolving cis-regulatory landscapes. Comparative genomics reveals that the basic helix-loop-helix (bHLH)-type diterpenoid phytoalexin factor (DPF) is deeply conserved across the Oryzoideae and broader grass lineages. By contrast, the promoters of diterpenoid biosynthetic genes exhibit extensive sequence divergence, particularly between AA and non-AA genome lineages of Oryza, which represent distinct genome groups defined by cytogenetic and phylogenetic relationships. Despite this overall divergence, we identified discrete, gene-specific regulatory modules (evolutionary 'islands') that recur across species. These modules are enriched in canonical DPF-recognizable N-boxes and newly defined N-box-like elements (NLEs). Functional assays demonstrate that variation in the number and arrangement of these cis-elements quantitatively calibrates DPF-mediated activation, providing a mechanistic basis for species-specific differences in metabolic inducibility. Together, our findings elucidate how BGC regulatory systems diversify through a synergy of 'trans stability and cis plasticity', offering a framework for understanding the evolution of inducible specialized metabolism in plants.