Brassica rapa BrICE1 and BrICE2 Positively Regulate the Cold Tolerance via CBF and ROS Pathways, Balancing Growth and Defense in Transgenic Arabidopsis

Winter rapeseed (<i>Brassica rapa</i>) has a good chilling and freezing tolerance. inducer of CBF expression 1 (ICE1) plays a crucial role in cold signaling in plants; however, its role in <i>Brassica rapa</i> remains unclear. In this study, we identified 41 <i>ICE1</i> homologous genes from six widely cultivated Brassica species. These genes exhibited high conservation, with evolutionary complexity between diploid and allotetraploid species. Cold stress induced <i>ICE1</i> homolog expression, with differences between strongly and weakly cold-tolerant varieties. Two novel <i>ICE1</i> paralogs, <i>BrICE1</i> and <i>BrICE2</i>, were cloned from <i>Brassica rapa</i> Longyou 6. Subcellular localization assays showed that they localized to the nucleus, and low temperature did not affect their nuclear localization. The overexpression of <i>BrICE1</i> and <i>BrICE2</i> increased cold tolerance in transgenic Arabidopsis and enhanced reactive oxygen species' (ROS) scavenging ability. Furthermore, our data demonstrate that overexpression of <i>BrICE1</i> and <i>BrICE2</i> inhibited root growth in Arabidopsis, and low temperatures could induce the degradation of BrICE1 and BrICE2 via the 26S-proteasome pathway. In summary, <i>ICE1</i> homologous genes exhibit complex evolutionary relationships in Brassica species and are involved in the C-repeat/DREB binding factor (CBF) pathway and ROS scavenging mechanism in response to cold stress; these regulating mechanisms might also be responsible for balancing the development and cold defense of <i>Brassica rapa</i>.