摘要The Lamiaceae family is renowned for its terpenoid-based medicinal components,but Leonurus,which has traditional medicinal uses,stands out for its alkaloid-rich composition.Leonurine,the principal active com-pound found in Leonurus,has demonstrated promising effects in reducing blood lipids and treating strokes.However,the biosynthetic pathway of leonurine remains largely unexplored.Here,we present the chromosome-level genome sequence assemblies of Leonurus japonicus,known for its high leonurine production,and Leonurus sibiricus,characterized by very limited leonurine production.By integrating ge-nomics,RNA sequencing,metabolomics,and enzyme activity assay data,we constructed the leonurine biosynthesis pathway and identified the arginine decarboxylase(ADC),uridine diphosphate glucosyltrans-ferase(UGT),and serine carboxypeptidase-like(SCPL)acyltransferase enzymes that catalyze key reac-tions in this pathway.Further analyses revealed that the UGT-SCPL gene cluster evolved by gene duplica-tion in the ancestor of Leonurus and neofunctionalization of SCPL in L.japonicus,which contributed to the accumulation of leonurine specifically in L.japonicus.Collectively,our comprehensive study illuminates leonurine biosynthesis and its evolution in Leonurus.