Characterization of switchgrass (Panicum virgatum L.) PvKSL1 as a levopimaradiene/abietadiene‐type diterpene synthase

The diverse class of plant diterpenoid metabolites serves important functions in mediating growth, chemical defence, and ecological adaptation. In major monocot crops, such as maize (Zea mays), rice (Oryza sativa), and barley (Hordeum vulgare), diterpenoids function as core components of biotic and abiotic stress resilience. Switchgrass (Panicum virgatum) is a perennial grass valued as a stress-resilient biofuel model crop. Previously we identified an unusually large diterpene synthase family that produces both common and species-specific diterpenoids, several of which accumulate in response to abiotic stress. Here, we report discovery and functional characterization of a previously unrecognized monofunctional class I diterpene synthase (PvKSL1) via in vivo co-expression assays with different copalyl pyrophosphate (CPP) isomers, structural and mutagenesis studies, as well as genomic and transcriptomic analyses. In particular, PvKSL1 converts ent-CPP into ent-abietadiene, ent-palustradiene, ent-levopimaradiene, and ent-neoabietadiene via a 13-hydroxy-8(14)-ent-abietene intermediate. Notably, although featuring a distinct ent-stereochemistry, this product profile is near-identical to bifunctional (+)-levopimaradiene/abietadiene synthases occurring in conifer trees. PvKSL1 has three of four active site residues previously shown to control (+)-levopimaradiene/abietadiene synthase catalytic specificity. However, mutagenesis studies suggest a distinct catalytic mechanism in PvKSL1. Genome localization of PvKSL1 distant from other diterpene synthases, and its phylogenetic distinctiveness from known abietane-forming diterpene synthases, support an independent evolution of PvKSL1 activity. Albeit at low levels, PvKSL1 gene expression predominantly in roots suggests a role of diterpenoid formation in belowground tissue. Together, these findings expand the known chemical and functional space of diterpenoid metabolism in monocot crops.