Molecular mechanisms of catechins regulation by EfMYB5b in Euryale ferox

Flavonoids are important secondary metabolites, which exist widely and play different roles in plants. Many kinds of flavonoid compounds have been identified in E. ferox seed kernels, but the main flavonoid components and biosynthesis mechanism of E. ferox are still unclear. In this study, UPLC-MS/MS was used to identify catechin as the main flavonoid compound in seed kernel of E. ferox . The 32 structural genes (2 EfDFRs and 30 EfANRs ) related to catechin synthesis were identified. Among them, key genes EfDFR-1 and EfANR-11 were screened by transcriptome, real-time fluorescence quantification and enzyme activity analysis. Further, in vitro enzyme activity assay demonstrated that EfANR-11 and EfDFR-1 could catalyze the formation of gallocatechin (GC) and epigallocatechin (EGC) from the substrate, respectively. Then, subcellular localization showed that EfDFR-1 and EfANR-11 were located in the Golgi apparatus. Transient overexpression of EfDFR-1 and EfANR-11 significantly increased catechin content in seed kernels of E. ferox . Subsequently, the EfMYB5b directly bind to TAACCA and ACCTAC in the EfDFR-1 and EfANR-11 promoter and promote their expression. Meanwhile, transientoverexpression of EfMYB5b showed that the expression of EfDFR-1 and EfANR-11 were significantly enhanced, and the content of catechin was increased in seed kernel E. ferox . Our findings clarified the molecular mechanism of transcription factor EfMYB5b regulating key genes EfDFR-1 and EfANR-11 in catechin synthesis pathway. It provides theoretical basis for improving the quality of E. ferox and breeding new varieties.