Exogenous Abscisic Acid Enhances Water Use Efficiency and Drought Tolerance in Alfalfa (Medicago sativa L.)

Alfalfa (<i>Medicago sativa</i> L.) is a key forage crop, but its production is severely limited by water scarcity. This study evaluated the effects of exogenous abscisic acid (ABA) root application on the drought tolerance of the alfalfa cultivar "Xinmu No. 4", focusing on water use efficiency (WUE), physiological traits, and transcriptome expression. The highest WUE was achieved at 25 μM ABA and 10% polyethylene glycol-6000 (PEG-6000). Physiological parameters, including proline (Pro), malondialdehyde (MDA), hydrogen peroxide (H₂O₂), peroxidase (POD), catalase (CAT), and methyl jasmonate (MeJA) in both leaves and roots, showed strong positive correlations with the WUE (<i>p</i> < 0.01), with leaf POD showing a significant correlation (<i>p</i> < 0.05). Regression analysis showed that 34.42 µM ABA under 10% PEG-6000 significantly optimized the WUE. Transcriptomic analysis revealed that ABA enhanced the WUE by enriching genes involved in root defense response and leaf oxidoreductase activity. These results demonstrate that root-applied ABA under drought stress significantly improves the WUE and drought tolerance in alfalfa, offering a promising strategy to enhance forage crop resilience to water scarcity.