Identification of Brassica rapa BrEBF1 homologs and their characterization in cold signaling

EIN3-binding F-box 1 (EBF1) is involved in cold tolerance in Arabidopsis; however, its exact roles in cold signaling in Brassica rapa remain uncertain. Herein, we demonstrated that EBF1 homologs are highly conserved in Brassica species, but their copy numbers are diverse, with some motifs being species specific. Cold treatment activated the expression of EBF1 homologs BrEBF1 and BrEBF2 in B. rapa; however, their expression schemas were diverse in different cold-resistant varieties of the plant. Subcellular localization analysis revealed that BrEBF1 is a nuclear-localized F-box protein, and cold treatment did not alter its localization but induced its degradation. BrEBF1 overexpression enhanced cold tolerance, reduced cold-induced ROS accumulation, and enhanced MPK3 and MPK6 kinase activity in Arabidopsis. Our study revealed that BrEBF1 positively regulates cold tolerance in B. rapa and that BrEBF1-regulated cold tolerance is associated with ROS scavenging and MPK3 and MPK6 kinase activity through the C-repeat binding factor pathway.