Evolutionary Dynamics of CBL Genes in Six Magnoliaceae Species and Conserved Function of CBL4 in Species-Specific Cold Inducibility.

Calcineurin B-like proteins (CBLs) are calcium sensors, and clarifying their evolutionary patterns and stress responses is critical for understanding adaptation in Magnoliaceae. Here, we characterize the CBL family in Magnoliaceae and test whether CBL4 shows conserved function in species-specific cold responses. We identified 38 CBL genes from six Magnoliaceae species. Evolutionary analysis revealed that CBLs likely originated in green algae and mainly expanded in eudicots, while copy number in Magnoliaceae remained stable (6-7). Phylogenetic and structural analyses classified Magnoliaceae CBLs into four clades and revealed conserved EF-hand domains and FPSF motifs. Analyses revealed purifying selection with frequent duplications, whereas positively selected lineages had fewer, suggesting functional divergence. GO/KEGG annotations linked Magnoliaceae CBLs to Ca2+-mediated signaling, stress responses, and metabolism. In three Magnoliaceae species with contrasting cold tolerance (Magnolia biondii, Liriodendron chinense, and Magnolia sinica), qRT-PCR showed an inverse cold-induction gradient of CBL4: cold-resistant M. biondii had the weakest upregulation, L. chinense was in the middle, and cold-sensitive M. sinica had the strongest upregulation. Other CBL members exhibited species-specific patterns. After equal heterologous expression of the CBL4 homologs from the three species in Arabidopsis, all lines exhibited similar enhancement of cold resistance. The results showed reduced ROS accumulation and electrolyte leakage, increased antioxidant enzyme activities, and improved photosynthetic performance under cold stress. CBL4 overexpression also enhanced the cold-induced expression of ICE1-CBF-COR pathway genes. Together, our study refines the evolutionary framework of Magnoliaceae CBLs and highlights CBL4 expression-level tuning as a practical route to enhance cold tolerance in woody plants.