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 <i>Lithocarpus litseifolius</i>. 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 <i>L. litseifolius</i> 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 <i>PALs</i>, three <i>C4Hs</i>, 13 <i>4CL</i>s, 18 <i>CDBRs</i>, five <i>CHSs</i>, 14 <i>P2'GTs</i>, 12 <i>P4'GTs</i>, nine <i>F3'Hs</i>, and three <i>CH3H</i>s, and found that either tandem duplication or proximal duplication may have contributed to the expansion of key genes such as <i>CDBR</i>, <i>P2'GT</i>, and <i>P4'GT</i>. 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, <i>P</i> < 0.05), while two other modules were associated with sieboldin accumulation (r = 0.59-0.74, <i>P</i> < 0.05). We also identified <i>MYB</i>, <i>WD40</i>-like, <i>WRKY</i>, and <i>bHLH</i> 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 <i>CDBR</i> and <i>P2'GT</i>, 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.