Genome-wide identification of the m6A gene family and analysis of m6A 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. N⁶-methyladenosine (m⁶A) is the most prevalent and reversible internal modification found in eukaryotic mRNA, and is installed, removed, and recognized by methyltransferases (writers), demethylases (erasers), and m⁶A-binding proteins (readers), playing crucial roles in regulating plant growth, development, and environmental stress response. However, the role of m⁶A in alfalfa remains largely unexplored. In this work, we conducted comprehensive whole genome identification and expression analysis of the m⁶A 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 m⁶A 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 m⁶A methylome under drought stress. We identified 10,855 hyper-methylated peaks in shoots and 3,262 in roots, along with a global increase in m⁶A levels during drought stress, individually highlighting the significant role of m⁶A modification in drought stress responses. Notably, we found that ABA signaling-related genes, such as ABA2, SnRK2.1, SnRK2.3, and SnRK2.4, exhibited m⁶A hypermethylation in shoots after drought stress treatment. Furthermore, we demonstrated that m⁶A modifications of ABA2, SnRK2.1, SnRK2.3, and SnRK2.4 enhance their mRNA stability, suggesting that m⁶A 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 m⁶A modifications as a novel approach for alfalfa stress resistance breeding strategies.