Genome-wide identification of the mA gene family and analysis of mA methylome in alfalfa under drought stress.

Crops frequently endure diverse environmental stresses that negatively impact their growth and yield. Understanding the mechanisms underlying stress responses is crucial for developing stress-tolerant crop varieties, thereby improving their adaptability and productivity. N-methyladenosine (mA) is the most prevalent and reversible internal modification found in eukaryotic mRNA, and is installed, removed, and recognized by methyltransferases (writers), demethylases (erasers), and mA-binding proteins (readers), playing crucial roles in regulating plant growth, development, and environmental stress response. However, the role of mA in alfalfa remains largely unexplored. In this work, we conducted comprehensive whole genome identification and expression analysis of the mA gene family in alfalfa under abiotic stress conditions. There are 11 writers, 6 erasers, and 9 readers were identified in the alfalfa genome. Through comparative genomic analysis, we uncovered the evolutionary relationships of the mA gene family across different species. Additionally, we characterized the gene structures, conserved motifs, cis-regulatory elements, chromosomal localization, and expression patterns specific to tissues and under various abiotic stress treatments. Furthermore, we conducted a transcriptome-wide analysis of mA methylome under drought stress. We identified 10,855 hyper-methylated peaks in shoots and 3,262 in roots, along with a global increase in mA levels during drought stress, individually highlighting the significant role of mA modification in drought stress responses. Notably, we found that ABA signaling-related genes, such as ABA2, SnRK2.1, SnRK2.3, and SnRK2.4, exhibited mA hypermethylation in shoots after drought stress treatment. Furthermore, we demonstrated that mA modifications of ABA2, SnRK2.1, SnRK2.3, and SnRK2.4 enhance their mRNA stability, suggesting that mA modification may regulate drought stress responses through the ABA signaling pathway. Above all, these findings provide new insights into the adaptive mechanism of alfalfa under drought stress and highlight the potential of regulating mA modifications as a novel approach for alfalfa stress resistance breeding strategies.