Integrated metabolomic and transcriptomic analysis reveals the biosynthesis mechanism of dihydrochalcones in sweet tea (Lithocarpus litseifolius)

The demand for plant-based, low-calorie natural sweeteners is increasing. Four dihydrochalcones (DHCs), namely phloretin, phlorizin, trilobatin, and sieboldin, have been identified in the leaves of Lithocarpus litseifolius . These compounds serve as natural flavor sweeteners with potential health-promoting effects. However, the biosynthetic pathways of these DHCs are not yet fully understood. In this study, the content of four DHCs was quantified using LC-MS/MS across five developmental stages (S1–S5) of L. litseifolius leaves. Our results revealed an accumulation pattern where DHC levels peaked at stage S3, followed by a sharp decrease at stages S4 and S5, with the exception of sieboldin, which maintained high levels. We elucidated the complete biosynthetic pathway of DHCs, involving 82 candidate enzyme-encoding genes, including five PALs , three C4Hs , 13 4CL s, 18 CDBRs , five CHSs , 14 P2’GTs , 12 P4’GTs , nine F3’Hs , and three CH3H s, and found that either tandem duplication or proximal duplication may have contributed to the expansion of key genes such as CDBR , P2’GT , and P4’GT . Furthermore, we reconstructed 11 regulatory networks of DHCs, two modules were positively related to the contents of phloretin, phlorizin, and trilobatin (r = 0.54–0.69, P < 0.05), while two other modules were associated with sieboldin accumulation (r = 0.59–0.74, P < 0.05). We also identified MYB , WD40 -like, WRKY , and bHLH transcription factors as potential regulators in the biosynthesis of four DHCs. We found two biosynthetic gene clusters of DHCs, including nine and four genes encoding CDBR and P2’GT , respectively. Syntenic and phylogenetic analyses revealed that these two BGCs may have experienced independent evolutionary processes within the Fagaceae family. Our study provides a theoretical foundation for the resource development and utilization of sweet tea. It also paves the way for the development of high-quality natural sweeteners.