LAUSR

Alfalfa represents one of the most important legume forages, and it is also applied as an organic fertilizer to improve soil quality. However, this perennial plant is native to warmer temperate regions, and its valuable cold-acclimation-related regulatory mechanisms are still less known. In higher plants, the bHLH transcription factors play pleiotropic regulatory roles in response to abiotic stresses. The recently released whole genome sequencing data of alfalfa allowed us to identify 469 MsbHLHs by multi-step homolog search. Herein, we primarily identified 65 MsbHLH genes that significantly upregulated under cold stress, and such bHLHs were classified into six clades according to their expression patterns. Interestingly, the phylogenetic analysis and conserved motif screening of the cold-induced MsbHLHs showed that the expression pattern is relatively varied in each bHLH subfamily, this result indicating that the 65 MsbHLHs may be involved in a complex cold-responsive regulatory network. Hence, we analyzed the TFBSs at promoter regions that unraveled a relatively conserved TFBS distribution with genes exhibiting similar expression patterns. Eventually, to verify the core components involved in long-term cold acclimation, we examined transcriptome data from a freezing-tolerant species (cv. Zhaodong) in the field and compared the expression of cold-sensitive/tolerant subspecies of alfalfa, giving 11 bHLH as candidates, which could be important for further cold-tolerance enhancement and molecular breeding through genetic engineering in alfalfa.