<i>Saussurea involucrata CML6</i> Enhances Freezing Tolerance by Activating Antioxidant Defense and the CBF-COR Pathway in Plants

Low-temperature stress severely limits plant growth and reduces agricultural productivity. Calmodulin-like (CML) proteins are crucial calcium sensors in plant cold responses. Transcriptome analysis of cold-stressed <i>Saussurea involucrata</i> identified seven differentially expressed <i>CML</i> genes. qRT-PCR confirmed <i>that SiCML6</i> was strongly induced at 4 °C and −2 °C. Bioinformatics analysis showed that <i>SiCML6</i> encodes a transmembrane protein containing an EF-hand domain. This protein carries a signal peptide and shows the closest phylogenetic relationship to <i>Helianthus annuus</i> CML3. Its promoter contains ABA, methyl jasmonate (MeJA), and cold-response elements. <i>Arabidopsis</i> plants overexpressing <i>SiCML6</i> showed significantly higher survival rates at −2 °C than wild-type plants. Under freezing stress, <i>SiCML6</i>-overexpressing lines exhibited reduced malondialdehyde content, relative electrolyte leakage, and ROS accumulation (H₂O₂ and O₂⁻), along with increased proline, soluble sugars, soluble proteins, and total antioxidant capacity (T-AOC). <i>SiCML6</i> elevated the expression of cold-responsive genes <i>CBF3</i> and <i>COR15a</i> under normal conditions and further upregulated <i>CBF1/2/3</i> and <i>COR15a</i> at 4 °C. Thus, low temperatures induced <i>SiCML6</i> expression, which was potentially regulated by ABA/MeJA. <i>SiCML6</i> enhances freezing tolerance by mitigating oxidative damage through boosted T-AOC and osmoprotectant accumulation while activating the CBF-COR signaling pathway. This gene is a novel target for improving crop cold resistance.