Integrated metabolome and transcriptome profiling unveil the mechanisms of tissue-specific polyphenol synthesis in Chinese chestnut (Castanea mollissima BL.)

• Generated transcriptome and metabolome datasets for chestnut tissues. • Flavonoids are predominantly enriched in flowers, whereas proanthocyanidins specifically accumulate in chestnut shells. • Expansion of the UDP-glycosyltransferase gene family drives the diversification of Fagaceae-specific metabolites. • CmFLS, which is localized in the cytosol, redirects metabolic flux toward flavonol synthesis. Chinese chestnut ( Castanea mollissima BL.), an important woody nut tree species of the Fagaceae family, contains abundant polyphenols in its tissues.To date, the pathways and key genes involved in polyphenol biosynthesis in Chinese chestnut remain poorly characterized. In our study, polyphenol content ranked as follows: bur > peel > shell > leaf > flower > root > stem > embryo, and polyphenol content was positively correlated with antioxidant capacity. We integrated metabolomic and transcriptomic approaches to identify metabolites and genes associated with polyphenol biosynthesis. Metabolomic profiling identified 2,016 metabolites, including 344 flavonoids and 76 tannins, which exhibited distinct tissue-specific accumulation patterns. Transcriptomic data revealed that phenylpropanoid pathway genes ( PAL, C4H, 4CL, CHS ) were highly expressed in flower and bur, while proanthocyanidin biosynthesis genes ( DFR, LDOX, ANR ) were upregulated in shell, aligning with metabolite profiles. Tissue-specific transcription factors (e.g., MYB5, TT8, TT16 ) further regulated polyphenol biosynthesis. We propose a stepwise biosynthetic model of castanoside B from kaempferol, involving sequential glycosylation and dual acylation reactions. The UGT gene family in Chinese chestnut exhibited significant expansion (258 genes), which may drive Fagaceae-specific polyphenol diversification. Functional characterization of CmFLS (flavonol synthase) revealed it localizes to the cytosol. Overexpressing CmFLS in tobacco increased flavonol content, decreased anthocyanins, and lightened corolla color, confirming its role in diverting metabolic flux to flavonol biosynthesis. These findings provide a foundation for targeted metabolic engineering to enhance polyphenol production and optimize the utilization of chestnut tissues. This could significantly improve the economic and ecological value of chestnut and related Fagaceae species through applications in food, pharmaceutical, and industrial sectors.