Dihydroflavonol 4-Reductase (DFR) Gene in Ribes nigrum L. Enhances Cold and Drought Resistance by Promoting Flavonoid Synthesis

The ripe fruits of blackcurrant (Ribes nigrum L.) are rich in vitamin C, anthocyanins, and flavonoids. Besides being consumed fresh, the fruits can be processed into fruit juices, jams, wines, and other products, exhibiting considerable economic and nutritional value. Flavonoids are a class of important plant secondary metabolites with antioxidant, anti-inflammatory, and anti-cancer properties. Although previous studies have confirmed the involvement of multiple structural genes and transcription factors in flavonoid biosynthesis, the specific role of the dihydroflavonol 4-reductase (DFR) gene in regulating flavonoid accumulation during fruit development of blackcurrant remains to be clearly elucidated. In this study, we identified an RnDFR gene located in the nucleus and cytoplasm, which has the same expression trend as flavonoid content in fruit development stages. Overexpression of RnDFR improved the flavonoid accumulation and upregulated the expression levels of related structural genes (4CL, CHS, LDOX, ANR, and UFGT) in tomato. Transiently overexpressing RnDFR in blackcurrant fruit also increased the content of flavonoids and DFR enzyme activity, whereas silencing RnDFR resulted in the opposite effect. In addition, overexpression of RnDFR in tomato seedlings improved cold and drought tolerance by increasing flavonoid accumulation, reducing membrane lipid peroxidation damage and enhancing the activities of antioxidant enzymes. This study systematically reveals the key role of RnDFR in flavonoid biosynthesis and the enhancement of cold and drought tolerance, and offers an important theoretical basis for future efforts to optimize flavonoid content in blackcurrant and improve fruit nutritional quality.