Winter rapeseed (Brassica rapa) has good chilling and freezing tolerance. INDUCER OF CBF EXPRESSION1 (ICE) plays a crucial role in cold signaling in plant; however, its role in B. rapa remains unclear. In this study, we identified 41 ICE1 homologous genes from 6 widely cultivated Brassica species. These genes exhibited high conservation, with evolutionary complexity between diploid and allotetraploid species. Cold stress induced ICE1 homolog expression, with differences between strong and weak cold-tolerant varieties. Two novel ICE1 paralogs, BrICE1 and BrICE2 were cloned from B. rapa Longyou 6. Subcellular localization assays showed that they localized to the nucleus, and low temperature did not affect their nuclear localization. Overexpression of BrICE1 and BrICE2 increased cold tolerance in transgenic Arabidopsis, and enhanced reactive oxygen species (ROS) scavenging ability. Furthermore, our data demonstrate that overexpression of BrICE1 and BrICE2 inhibited root growth in Arabidopsis, and low temperatures could induce the degradation of BrICE1 and BrICE2 via the 26S-proteasome pathway. In summary, ICE1 ho-mologous genes exhibit complex evolutionary relationships in Brassica species, and are involved in the CBF pathway, and ROS scavenging mechanism in response to cold stress; these regulating mechanisms might be also responsible for balancing the development and cold defense of B. rapa.

Inducer of C-Repeat Binding Factor (CBF) expression (ICE), a well-known MYC-type bHLH transcription factor, plays a crucial role in the ICE-CBF-cold regulated (COR) cold signaling pathway. Brassica rapa (B. rapa), an important oilseed and vegetable crop, exhibits strong chilling and freezing tolerance. However, the exact molecular mechanisms by which ICE1 functions in B. rapa remain unclear. Here, 41 ICE1 homologous genes were identified from six widely cultivated Brassica species using a bioinformatics approach. These genes exhibit high conservation but reveal evolutionary complexity between diploid and allotetraploid species. Low-temperature stress induced ICE1 homolog expression, with patterns differing between strong and weak cold-tolerant varieties. Two novel ICE1 paralogs, BrICE1 and BrICE2, were cloned from Longyou 6. They positively regulate cold tolerance in B. rapa via a CBF-dependent pathway, contributing to reactive oxygen species (ROS) scavenfging and osmotic adjustment. Immunoblot analysis revealed that low temperatures induce BrICE1 and BrICE2 degradation via the 26S-proteasome pathway. In summary, ICE1 exhibits complex evolutionary relationships in Brassica species. BrICE1 and BrICE2 positively regulate cold tolerance via the CBF-dependent pathway and ROS scavenging mechanism and are also responsible for balancing the development and cold defense of B. rapa.