| Summary: | <i>Artemisiae argyi</i> Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in <i>A.</i><i>argyi</i> leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for <i>A. argyi</i> harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC–MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in <i>A. argyi</i> leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within <i>A. argyi</i> leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of <i>A. argyi</i> leaves, aiding in the selection of optimal timing for harvests of <i>A. argyi</i>.
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