Biochemical synthesis of uniformly ¹³C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins <i>in planta</i>

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown <i>in planta</i>. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly ¹³C-labeled <i>ent</i>-cassadiene and <i>syn</i>-pimaradiene were enzymatically synthesized with structural verification by GC–MS and ¹³C-NMR. Application of the ¹³C-labeled substrates on rice leaves led to the detection of ¹³C-labeled metabolites using LC-MS/MS. Further application of this method in the moss <i>Hypnum plumaeforme</i> and the nearest out-group of <i>Oryza</i> species <i>Leersia perrieri</i>, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-¹³C₂₀] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds. Uniformly labeled ¹³C-diterpene: A powerful tool for tracing endogenous metabolites without dilution with naturally abundant unlabeled compounds