Characterization of diterpene synthase genes in Brachypodium distachyon, a monocotyledonous model plant, provides evolutionary insight into their multiple homologs in cereals.

This article focuses on the identification and characterization of diterpene synthase genes in *Brachypodium distachyon* (Bd), a monocotyledonous model grass species, to provide evolutionary insights into their homologs in cereal crops. The Bd genome contains a single ent-copalyl diphosphate synthase gene (BdCPS) and four tandemly arrayed ent-kaurene synthase-like genes (BdKS1, BdKSL2, BdKSL3, and BdKSL4), a simpler set compared to cereals such as rice, maize, and wheat. Functional analyses confirmed BdCPS and BdKS1 are involved in gibberellin biosynthesis, while BdKSL2 and BdKSL3 likely participate in specialized diterpenoid metabolism; notably, KS activity was restored in BdKSL2 through targeted amino acid substitutions. The study highlights gene duplication and neofunctionalization events shaping diterpene synthase diversity in grasses and suggests Bd has fewer labdane-related diterpene synthase genes than cereals, offering a model for understanding the evolution of these enzymes in Poaceae.