Molecular identification of chalcone synthase-encoding genes EfCHSs and EfGATA8 as a negative regulator for flavonoid biosynthesis in Euryale ferox

Chalcone synthase is a crucial rate-limiting enzyme for flavonoid biosynthesis in plants. Despite the abundance of flavonoid resources in Euryale ferox, resolving their biosynthesis and regulatory pathways is remained scarce. Five putative CHS genes were obtained in E. ferox through genome-wide characterization. Phylogenetic, structural analysis and sequence alignment were performed to determine EfCHS1 and EfCHS2 as candidate genes for flavonoid biosynthesis. Enzyme activity assays and transient overexpression demonstrated that EfCHS1 and EfCHS2 exhibited potent activities. Interestingly, it was found that there was strong expression of EfCHS1 in the seed shells and EfCHS2 in the leaves. Moreover, the transcription factor EfGATA8 was screened in the E. ferox yeast library using a yeast one-hybrid system. Subcellular localization analysis displayed that EfGATA8 only existed fluorescent signals in the nucleus. It was revealed that EfGATA8 binds directly to the promoter of EfCHS2 and represses its expression according to further Y1H assay and luciferase (LUC) activity analysis. EfGATA8 transient transformation into tobacco leaves resulted in significantly reduced accumulation of flavonoids. Taken together, these findings clarify the position of EfCHS1 and EfCHS2 as key genes for flavonoid biosynthesis in E. ferox, while perfecting the regulatory network for flavonoid biosynthesis in plants.