Physiological and biochemical basis of flower coloration in Aquilegia oxysepala with a functional study of AoDFR08

Aquilegia oxysepala, the most widespread columbine in northeast China, has considerable potential for future applications. It is imperative to elucidate the potential physiological and molecular mechanisms underlying flower coloration, which could facilitate the innovative breeding process of Aquilegia. A comprehensive analysis was conducted to elucidate the physio-biochemical basis of flower coloration in A. oxysepala and A. oxysepala f. pallidiflora. This analysis encompassed a range of parameters, including color parameters, vacuolar pH, total anthocyanin concentration, and metal ion content. In addition, the shape of the epidermal cells in the sepals and the accumulation of pigments in the epidermal cells were observed by light microscopy. Correlation analysis revealed a highly significant positive correlation between total anthocyanin, Al and Fe concentrations in sepals and the transition to red and blue. While K, Ca, Na, Mg, Mn, Cu and Zn exhibited a high correlation with the transition to green and yellow. The effect of vacuolar pH on flower color was found to be minimal. With the consideration of anthocyanin was the dominant substance that decided flower color, subsequent research was conducted into the dihydroflavonol 4-reductase (DFR) genes, as DFR is the key rate-limiting enzyme in the anthocyanin biosynthesis pathway. A total of 33 AoDFRs were identified in A. oxysepala. Based on a combined analysis of protein characteristics, expression patterns and phylogenetic relationships, AoDFR08 was selected as the key candidate gene. AoDFR08 contained a NADP-domain and was classified as an Asn-type DFR. Tobacco has been selected as a model plant due to the easily observable floral organs. The corollas of AoDFR08 overexpression tobacco were found to accumulate more anthocyanin, and the LBGs were found to be more highly expressed than in the wild type. Anthocyanin along with Al and Fe were observed to dominate in flower coloration. Moreover, AoDFR08 was found to play a crucial role in promoting anthocyanin biosynthesis. These findings provided a theoretical foundation for the breeding of A. oxysepala with innovative flower colors. Subsequent research will focus on the upstream regulatory mechanism of AoDFR08 and the potential involvement of methylation modification in this process.