High-throughput sequencing and analysis of target genes uncover microRNA-mediated responses to low-temperature stress in Medicago falcata

Low temperature severely restricts plant growth and development. Medicago falcata is an important germplasm resource for alfalfa breeding because of its strong adaptability and tolerance to abiotic stresses, particularly low-temperature stress. MicroRNAs (miRNAs) are key regulators of stress-responsive genes and modulate plant adaptation to stress by directing transcript cleavage or translational repression. However, the roles of miRNAs in low-temperature response mechanisms in M. falcata remain largely unclear.

Here, we constructed miRNA libraries from M. falcata roots grown under normal or low-temperature conditions. High-throughput sequencing identified 442 miRNAs, including 245 known and 197 novel miRNAs. Among these, 74 were putatively upregulated and 37 were putatively downregulated under chilling and freezing treatments. Based on degradome sequencing, 193 target genes involved in diverse biological processes were predicted. The M. falcata MfbZIP transcription factor family, consisting of 41 members across six subfamilies, responded to the cold treatments. Of these, MfbZIP10, a nuclear-localized protein homologous to a bZIP transcription factor in M. truncatula, showed the most significant response to cold stress in stems and roots. miR408-5p was responsive to chilling and freezing, and could cleave MfbZIP10 transcripts in a transient assay, as determined by RLM-RACE analysis. Physiological analyses and gene expression profiling of transgenic lines further demonstrated that the miR408-5p-MfbZIP10 module plays a vital role in low-temperature stress responses, with MfbZIP10 functioning as a key regulator by modulating physiological traits and the expression of genes in multiple signaling pathways.

Our results identified candidate cold-responsive miRNAs in M. falcata and predicted their target genes involved in diverse biological processes. miR408-5p was found to target MfbZIP transcription factors in response to cold stress. Physiological characterization and quantitative expression analyses of MfbZIP10 transgenic lines showed that the miR408-5p-MfbZIP10 module plays a crucial regulatory role in low-temperature adaptation. miRNA sequencing and target gene identification provide new insights into miRNA-mediated regulatory mechanisms in M. falcata. These findings may facilitate the development of cold-tolerant forage legume crops through genetic improvement.