Genome-Wide Identification of the AP2/ERF Family in Alfalfa and Its Association with Antioxidant Regulation Under Drought Stress

Abstract

In this study, Alfalfa (Medicago sativa L.) was used as the experimental material to systematically identify members of the AP2/ERF transcription factor family in its genome and to elucidate the response patterns and potential regulatory networks of this family under drought stress. A total of 756 AP2/ERF genes were identified and classified into four subfamilies: AP2, ERF, DREB, and RAV, among which the DREB and ERF subfamilies exhibited significant expansion. Evolutionary analysis indicated that whole-genome duplication (WGD)/segmental duplication was the primary driving force underlying the expansion of this family. Under graded drought treatments, multiple AP2/ERF genes were significantly upregulated, with MsERF210 showing continuously increasing expression as drought intensity escalated. WGCNA revealed that MsERF210 was highly positively correlated with antioxidant-related modules, including “Glutathione metabolism,” “Peroxisome,” and “Ascorbate and aldarate metabolism.” Further analysis showed that several antioxidant genes, such as SOD, GST, ALDH, and IDH, were significantly upregulated under drought stress, and their promoter regions were predicted to be bound by MsERF210. Physiological indicator measurements demonstrated that drought stress led to increased accumulation of H2O2 and MDA, along with elevated contents of AsA, DHA, GSH, and GSSG, while SOD activity initially increased and then decreased. qRT-PCR validated the expression trends of key genes, confirming the reliability of the transcriptome data. In summary, this study reveals the evolutionary characteristics of the AP2/ERF family in alfalfa and identifies MsERF210 as a key regulator that may enhance alfalfa’s adaptability to drought stress by modulating the antioxidant defense system and redox homeostasis. These findings provide important candidate gene resources and a theoretical basis for breeding new drought-resistant alfalfa varieties.