Functional divergence of the duplicated BrrCBL2.1 and BrrCBL2.2 genes in turnip (Brassica rapa var. rapa)

Gene duplication is a core driver of plant genome diversification and adaptive evolution. Calcineurin B-like protein 2 (CBL2), a plant-specific calcium sensor, forms signaling complexes with CBL-interacting protein kinases (CIPKs) to regulate ion homeostasis and stress responses. Turnip ( Brassica rapa var. rapa ) is a traditional crop cultivated on the Qinghai-Tibet Plateau, yet the functional divergence and adaptive mechanisms of its CBL2 genes remain unclear. Using the turnip genome, we identified two segmentally duplicated CBL2 copies, designated BrrCBL2.1 and BrrCBL2.2 . Phylogenetic analyses placed the two copies in distinct clades and revealed sequence differences in the C-terminal PFPF motif, critical for CIPK interaction, suggesting functional divergence. Quantitative real-time PCR showed that BrrCBL2.1 is strongly up-regulated under ion stresses such as low potassium and high magnesium, whereas BrrCBL2.2 responds specifically to 4 °C cold stress. In vivo luciferase complementation (LUC) assays in tobacco demonstrated pronounced specificity in their interaction patterns with members of the BrrCIPK family, with BrrCBL2.2 forming complexes with a broader set of CIPKs. Transgenic validation in Arabidopsis thaliana further showed that BrrCBL2.1 effectively rescues the growth phenotype of the cbl2 mutant under low-potassium stress, retaining the ancestral CBL2 function in ion-homeostasis control; by contrast, BrrCBL2.2 markedly enhances cold tolerance in transgenic plants. These findings reveal that duplicated BrrCBL2 genes achieved functional division through divergence in the PFPF motif, specialized expression, and expanded interaction networks. This study offers insights into plant adaptation to high-altitude environments and provides genetic resources for stress-resilience breeding in Brassica crops.