Abstract:

Post-anthesis color change（PACC），a process in which Lotus filicaulis flowers transition from yellow to red，is an adaptive strategy to enhance pollination efficiency. While existing studies have primarily focused on anthocyanins，the regulatory role of flavonols and their synergistic relationship with anthocyanins remain unclear. In this study，flavonol-targeted metabolomics and transcriptomic analyses were integrated to identify flavonols and genes related to flavonol biosynthesis during PACC in L. filicaulis. The functions of flavonol synthase（FLS）genes were validated through prokaryotic enzyme activity assays and transgenic experiments in tobacco. Additionally，transient protoplast transfection assays were employed to investigate the regulatory mechanisms by which MYB transcription factors control FLS expression. The results revealed that flavonols and anthocyanins accumulate synchronously during PACC in L. filicaulis，with total flavonols significantly increased in the red flowers. Two FLS genes，LfFLS1 and LfFLS2，exhibited differential expression patterns：LfFLS1 was highly expressed in yellow flowers，whereas LfFLS2 was specifically upregulated in red flowers. Moreover，both LfFLS proteins displayed catalytic activity but showed distinct substrate preference：LfFLS1 exclusively utilized dihydrokaempferol（DHK）and dihydroquercetin（DHQ），while LfFLS2 efficiently catalyzed all three types of dihydroflavonols. Additionally，LfFLS1 was co-activated by subgroup 7 MYB transcription factor LfMYB12 and subgroup 19 MYB transcription factor LfMYB21，whereas LfFLS2 was exclusively regulated by LfMYB21. This differential expression pattern aligns with the metabolic demands of distinct PACC stages，highlighting a sophisticated regulatory network that modulates flavonol metabolism to support adaptive color transitions.

Key words: Lotus filicaulis, flower color, flavonol, transcription regulation