Genome‐wide identification of <i>MsAlkB</i> family genes and functional analysis of <i>MsALKBH2</i> as an RNA m ⁶ A eraser in alfalfa ( <i>Medicago sativa</i> L.) subject to drought stress

Abstract Background N 6 ‐methyladenosine (m 6 A), the most prevalent mRNA modification in plants, plays a pivotal role in developmental processes and stress responses. Proteins from the alpha‐ketoglutarate‐dependent dioxygenase (AlkB) family are implicated in the demethylation of m 6 A, and yet, the AlkB gene family remains poorly characterized in alfalfa. Methods Bioinformatic analysis was systematically performed to identify and characterize the MsAlkB gene family at the whole‐genome level in alfalfa. Functional validation of MsALKBH2 was subsequently conducted using Agrobacterium rhizogenes ‐mediated hairy root transient transformation assays. Results Twenty MsALKBH candidate genes were identified from the genome of autotetraploid alfalfa cultivar Xinjiang Daye. Phylogenetic analysis classified these genes into four distinct groups, and promoter region analysis revealed numerous cis ‐regulatory elements associated with hormonal signaling and abiotic stress responses. With the exception of MsALKBH20 , the remaining 19 MsALKBH genes were distributed across eight chromosomes. Gene duplication analyses indicated that segmental and tandem duplications were the primary drivers of the expansion of this gene family during evolution. Expression profiling of the 20 MsALKBH genes across various tissues and under salt, mannitol, and ABA treatments revealed differential expression patterns and stress‐induced regulation. Real‐time quantitative polymerase chain reaction analysis demonstrated that 12 MsALKBH genes were responsive to osmotic stress, with MsALKBH2 expression being notably suppressed under mannitol, salt, and ABA treatments. Overexpression of MsALKBH2 significantly reduced global m 6 A levels in alfalfa, mitigating oxidative damage and enhancing drought tolerance. These findings establish MsALKBH2 as an m 6 A demethylase that regulates drought tolerance in alfalfa. Conclusions This study provides the first comprehensive analysis of the MsAlkB gene family and functional characterization of MsALKBH2 , offering critical insights into the potential application of this gene family for crop improvement, particularly in legume species.