Drought stress severely affects the growth and yield of alfalfa, and deciphering its drought-resistant molecular mechanisms is crucial for breeding drought-tolerant varieties. This study focused on the MfERF053 gene from Medicago falcata. By constructing overexpression (OE) and RNA interference (RNAi) lines, combined with physiological phenotype analysis, antioxidant enzyme activity determination, photosynthetic parameter detection, and transcriptome sequencing, the function and regulatory network of MfERF053 in drought response were revealed. The results showed that MfERF053 enhanced drought resistance of Medicago sativa through multiple mechanisms: In terms of stomatal regulation, it reduced leaf water loss rate by promoting stomatal closure to maintain water balance; in the antioxidant defense process, it increased the activities of catalase (CAT) and peroxidase (POD), thereby reducing the accumulation of reactive oxygen species (ROS) and alleviating membrane lipid peroxidation damage; in photosynthesis protection, it slowed down chlorophyll degradation, maintained the efficiency of photosystem II, and improved water use efficiency. Transcriptome analysis results indicated that MfERF053 was significantly enriched in the pathways of ”plant hormone signal transduction”, ”oxidoreductase activity”, and ”photosynthetic process”. It synergistically enhanced drought resistance by regulating the expression of genes involved in the ABA signaling pathway (such as the PYR/ PYL- SnRK2 cascade), antioxidant genes (such as CAT1 and APX7), and photosynthesis-related genes (such as FTSH6 and PPH/ PAO). This study confirmed that MfERF053 is a positive regulator of alfalfa’s drought response, providing key genetic resources and theoretical basis for molecular breeding of drought-resistant alfalfa.

Abscisic acid (ABA) is a vital stress resistant hormone of plant in coping with adverse environmental conditions, such as drought stress. Sensitivity of seed germination to exogenous ABA treatment could link to different drought tolerance ability of different plant species. Here, we selected alfalfa seedlings (S0-50) from seeds germinated under 50 μM ABA treatment. The S0-50 plant showed more sensitivity in stomatal closure to exogenous ABA and PEG treatments, and also stronger drought tolerance than the plant of ABA-sensitive seed during germination (S0-0). Testing of ABA content in leaf indicated that the S0-50 had a higher ABA content in normal and under drought stress growth conditions than that of the S0-0 plants. The seed of S0-50 next generation (S1-50) showed significantly higher germination ratio under 50 μM ABA treatment, and also had longer root after 15% PEG6000 treatment than the segregated ABA-sensitive seed (S1-0). We found a cytosol-nucleus dual-localized PPR protein gene MsSOAR1 was significantly highly expressed in S0-50 than in S0-0 plant. Overexpression of AtSOAR1, a negative regulator in ABA-mediated Arabidopsis seed germination inhibition, and also a homologous gene of MsSOAR1, significantly improved alfalfa drought tolerance, branch number and plant height, and reduced the expression level of ABA receptors MsPYL5 and MsPYL6. The results suggest that ABA-insensitive during seed germination could associate with repression of ABA signaling transduction. Selection of alfalfa seedling during seed germination with exogenous ABA could be a way to obtain drought tolerance germplasm, at least in ‘Zhongmu No.1’ alfalfa cultivar the plant material we used in the experiment.