Flavonol synthase gene MsFLS13 regulates saline-alkali stress tolerance in alfalfa

Alfalfa (Medicago sativa L.) is one of the most extensively grown leguminous forage worldwide. Environmental saline-alkali stress significantly influences the growth, development, and yield of alfalfa, posing a threat to its agricultural production. However, little is known about the potential mechanisms by which alfalfa responds to saline-alkali stress. Here, we investigated these mechanisms by cloning a saline-alkali-induced flavonol synthase gene (MsFLS13) from alfalfa, which was previously reported to be significantly upregulated under saline-alkali stress, and examining its function in the saline-alkali response. Overexpression of MsFLS13 in alfalfa promoted plant tolerance to saline-alkali stress by enhancing flavonol accumulation, antioxidant capacity, osmotic balance, and photosynthetic efficiency. Conversely, MsFLS13 inhibition using RNA interference reduced flavonol synthase activity and inhibited hairy root growth under saline-alkali stress. Yeast one-hybrid and dual-luciferase reporter assays indicated that the R2R3-MYB MsMYB12 transcription factor activates MsFLS13 expression by binding to the MBS motif in the MsFLS13 promoter. Further analysis revealed that abscisic acid mediates the saline-alkali stress response partially by inducing MsMYB12 and MsFLS13 expression, which consequently increases flavonol levels and maintains antioxidant homeostasis in alfalfa. Collectively, our findings highlight the crucial role of MsFLS13 in alfalfa in response to saline-alkali stress and provide a novel genetic resource for creating saline-alkali-resistant alfalfa through genetic engineering.