Multi-omics provides insights into the genetic influence on characteristic aroma formation of Xinhui Chenpi

Abstract

Xinhui Chenpi, the dried mature fruit peel of Citrus reticulata ‘Chachi’ (Cha-Zhi-Gan, CZG), is a geo-authentic materia medica widely used in traditional Chinese medicine. Despite its distinctive aroma, the influence of genetic background on volatile metabolite accumulation has not been fully understood. In this study, 11 volatile metabolites, including methyl 2-(methylamino)benzoate and γ-terpinene, were found at significantly higher concentrations in Xinhui Chenpi than in other Chenpi resources. A complete telomere-to-telomere (T2T) genome assembly of CZG (326.6 Mb) was generated, and genome sequences of 33 mandarin accessions were compiled to elucidate the origin and domestication of CZG. Comparative metabolomic and transcriptomic analyses across five developmental stages of C. reticulata ‘Unshiu’ (Guo-Qing-Yi-Hao, GQYH) peels revealed significantly elevated levels of 20 volatile compounds and differential expression of 2525 genes in CZG peels. Notably, higher levels of methyl 2-(methylamino)benzoate were associated with the upregulation of CreBSMT1, CreCHD1 and CrePAL1, whereas increased γ-terpinene levels correlated with the upregulation of CreGPPS2, CreMTSL61, and CreMTSL3. Integration of metabolomic and transcriptomic data enabled the construction of a regulatory network for volatile metabolite biosynthesis in CZG. A NAC transcription factor, CreNAC8, was identified as a key regulator of both methyl 2-(methylamino)benzoate and γ-terpinene accumulation. Transient overexpression and virus-induced gene silencing (VIGS) assays confirmed that CreNAC8 regulated the accumulation of γ-terpinene and methyl 2-(methylamino)benzoate in CZG fruit peels through the regulation of CreMTSL61 and CreCHD1, respectively. These findings offer novel insights into the molecular mechanisms underlying the characteristic aroma formation of Xinhui Chenpi.