A specific 296 bp insertion in the <i>DFR1</i> promoter activates ethylene-mediated anthocyanin biosynthesis during autumn leaf senescence in <i>Quercus aliena</i>

Red-leaf ornamental trees are commonly characterized by the enrichment of anthocyanins. Quercus aliena, a dominant native oak species in China, displays striking autumn foliage with yellow to red colors. However, the molecular regulation of anthocyanin biosynthesis during leaf senescence remains largely unknown. In this study, we identified a 296 bp insertion in the promoter of Dihydroflavonol 4-Reductase (DFR1) from the red-leaf Q. aliena cultivar 'Qiuyun', which was significantly associated with high anthocyanin accumulation. We established a highly efficient method for genetic transformation in Q. aliena calli and verified the function of DFR1. Yeast one-hybrid library screening identified ETHYLENE INSENSITIVE 3 (EIN3) that can directly bind to the EIN3-binding site in the 296 bp insertion and activate DFR1 expression. Together with a dual-luciferase assay and EMSA, we reported for the first time that EIN3 could directly activate the structural gene DFR1 during anthocyanin biosynthesis. DFR1 expression is markedly induced in EIN3-overexpressing oak calli compared with the wild type. In addition, exogenous ethephon induced anthocyanin accumulation in autumn leaves in the red cultivar, rather than in the yellow cultivar without the insertion. In summary, our findings demonstrate a practical and effective approach for validating gene function in vivo, and provide new insight into the regulatory mechanisms involved in anthocyanin biosynthesis during leaf senescence.