The CBF-Like Gene Family in Alfalfa: Expression Analyses and Identification of Potential Functional Homologs of Arabidopsis CBF3

xWe recently identified the alfalfa (Medicago sativa L.) germplasm River Side (RS) and Foster Ranch (FR), both naturally adapted to the Grand River National Grassland environment in South Dakota and showing superior freezing tolerance. To understand the molecular basis of freezing tolerance in RS and FR, we examined expression of the C-repeat binding factor-like (CBFl) genes in alfalfa. Eighteen CBFl genes were identified after examining the genome of Medicago truncatula Gaertn., a close relative of alfalfa. Phylogenetic analysis clustered Medicago CBFs into four subgroups. Expression profiling of these genes in alfalfa seedlings revealed diverse cold-induction patterns. Four of the genes that showed an early induction as CBF3 in Arabidopsis under cold stress were selected for detailed expression analyses. These genes varied in expression patterns, in different tissues and at different developmental stages, and exhibited different diurnal regulation without cold treatment. Two of the genes, MsCBFl-17 and MsCBFl-18, showed an early and high induction under cold stress in RS and Apica, a cold-tolerant cultivar, when compared with a nonfreezing tolerant germplasm, suggesting that these two genes are potentially the functional homologs of CBF3. On the other hand, MsCBFl-11 was the only gene that was induced in all three cold-tolerant germplasm, including FR, but the induction was relatively late compared with MsCBFl-17 and MsCBFl-18. Together, these findings suggest that the CBFs may play an important role in the regulation of freezing tolerance in alfalfa, and additional mechanisms exist to explain the superior freezing tolerance in RS and FR.