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

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 . 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 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 , three , 13 s, 18 , five , 14 , 12 , nine , and three s, and found that either tandem duplication or proximal duplication may have contributed to the expansion of key genes such as , , and . 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, < 0.05), while two other modules were associated with sieboldin accumulation (r = 0.59-0.74, < 0.05). We also identified , -like, , and 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 and , 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.