Volatilomic and transcriptomic analyses uncover the mechanisms underlying flavor-related VOC changes during the harvest period of Citrus reticulata ‘Chachi’

Harvest timing is a critical determinant of the quality of Citrus reticulata ‘Chachi’ (CRC), the sole raw material for Guang Chenpi. However, the molecular mechanisms underlying the dynamics of flavor-related volatile organic compounds (VOCs) across the commercial harvest period remain largely uncharacterized. Here, by integrating volatilomic and transcriptomic analyses, we systematically profiled dynamic changes in VOCs and dissected their potential transcriptional regulation in CRC flavedo across five consecutive harvest stages. A total of 334 VOCs were annotated, among which 161 were identified as odor-active VOCs, and the transition from 100 to 150 days after flowering (S1 to S2) was identified as the critical window for CRC aroma formation, during which 72.67% of the odor-active VOCs exhibited differential accumulation patterns. Through gene-metabolite co-expression analysis, we identified four core structural genes ( CrLOX2/4/7 , CrADH4 ) and 35 transcription factors (predominantly from the NAC, MYB, and AP2/ERF families) associated with fatty acid-derived VOC biosynthesis, as well as 10 candidate terpene synthase ( TPS ) genes linked to terpenoid accumulation. Most importantly, in vivo functional validation confirmed that CrTPS19 functions as a key enzyme mediating the biosynthesis of d -limonene, the signature aroma compound of CRC. Integration of cis -acting element prediction and co-expression network analysis narrowed down a set of candidate upstream transcription factors potentially regulating CrTPS19 expression. Collectively, this study provides a comprehensive dynamic atlas of flavor-related VOCs across the CRC commercial harvest cycle, identifies key candidate genes underlying aroma biosynthesis, and offers both a chemical basis for precise harvest timing optimization and candidate targets for future flavor quality improvement in CRC.