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.

NAC family transcription factors (TFs) are important regulators in plant development and stress responses. However, the biological functions of NAC TFs in wheat are rarely studied. In this study, 43 putative drought-induced NAC genes were identified from de novo transcriptome sequencing data of wheat following drought treatment. Twelve wheat NACs along with ten known stress-related NACs from Arabidopsis and rice were clustered into Group II based on a phylogenetic analysis. TaNAC48, which showed a higher and constitutive expression level in Group II, was selected for further investigation. TaNAC48 transcript was up-regulated by drought, PEG, H₂O₂ and abscisic acid (ABA) treatment and encoded a nuclear localized protein. Overexpression of TaNAC48 significantly promoted drought tolerance with increased proline content, and decreased rates of water loss, malondialdehyde (MDA), H₂O₂ and O₂⁻ content. Root length and a stomatal aperture assay confirmed that TaNAC48-overexpression plants increased sensitivity to ABA. Electrophoretic mobility shift assay (EMSA) and luciferase reporter analysis indicated that TaAREB3 could bind to a cis-acting ABA-responsive element (ABRE) on TaNAC48 promoter and activate the expression of TaNAC48. These results suggest that TaNAC48 is essential in mediating crosstalk between the ABA signaling pathway and drought stress responses in wheat.