Heterologous expression of MfERF053 enhances alfalfa drought resistance by regulating ABA signaling, antioxidant defense, and photosynthetic protection

Medicago sativa (alfalfa), a vital perennial leguminous forage with economic and nutritional significance, is severely limited by drought stress. AP2/ERF transcription factors act as core modulators of plant responses to abiotic stresses. To improve alfalfa drought resistance, the MfERF053 gene cloned from Medicago falcata was introduced into the alfalfa genome. Its function and regulatory mechanism in alfalfa drought adaptation were investigated. We hypothesized that MfERF053 plays a pivotal role in drought resistance. Transgenic alfalfa lines overexpressing MfERF053 (OE) and ERF053 RNA interference (RNAi)-mediated alfalfa lines were developed. Drought resistance of OE, RNAi, and wild-type (WT) plants was assessed, alongside physiological phenotyping and RNA-seq profiling. The findings demonstrated that MfERF053 boosted alfalfa drought resistance. Specifically, OE lines exhibited a higher survival rate (68.05% vs. 12.96% in RNAi lines) and stronger water retention (29.45% leaf relative water content vs. 7.87% in RNAi lines). Their catalase and ascorbate peroxidase activities were also elevated, reactive oxygen species (ROS) accumulation was reduced, and photosynthetic function was stabilized (mitigated chlorophyll degradation and maintained PSII efficiency). RNA-seq analysis indicated that differentially expressed genes (DEGs) in OE plants were concentrated in three key pathways: abscisic acid (ABA) signaling, antioxidant defense, and photosynthetic pathways. Additionally, these DEGs synergistically regulate key genes within these pathways. This study verified the function of MfERF053 in drought resistance through multiple regulatory pathways. Furthermore, it provides novel insights into ERF-mediated drought resistance in alfalfa and offers a valuable molecular candidate for breeding drought-tolerant alfalfa varieties.