Characterization of McCIPK6, a mitochondrial protein, positively regulating cold stress tolerance in bitter gourd (Momordica charantia L.).

As an economically important vegetable species, bitter gourd (Momordica charantia L.) is widely cultivated around the globe. However, its production is frequently threatened by late spring coldness, which severely impairs seedling establishment, delays flowering and fruit set, and reduces both yield and quality. Unraveling mechanisms of cold resistance in bitter gourd is therefore critical for breeding resilient cultivars and expanding its planting range. Here, we found a cold-inducible CIPK family gene, McCIPK6, from bitter gourd via transcriptome screening, and systematically characterized its function in cold stress responses using phenotypic, physiological, and molecular approaches. Expression analysis revealed that McCIPK6 was rapidly and strongly induced in the cold-tolerant genotype '0208' (R) under low temperature, whereas the cold-sensitive line '2206' (S) showed a delayed and dysregulated expression pattern. Mitochondrial localization analysis revealed that McCIPK6 is a mitochondria-targeted CIPK kinase. Heterologous overexpression of McCIPK6 in Arabidopsis significantly enhanced cold tolerance by simultaneously promoting mitochondrial ROS scavenging and activating the CBF-dependent cold signaling pathway. These effects were reflected by improved survival rates, reduced MDA accumulation, lower ROS levels, higher antioxidant enzyme activities (SOD, POD, CAT), and upregulated expression of CBF regulation genes as well as antioxidant genes. Further verification via yeast two-hybrid and luciferase complementation experiments revealed that McCIPK6 physically interacts with McCBL1, a core calcium sensor in bitter gourd. Collectively, our data establish that McCIPK6 functions as a positive regulator of cold tolerance by regulating ROS scavenging and the CBF-dependent cold signaling cascade, likely through forming a functional complex with McCBL1. This study reveals a novel regulatory module underlying cold adaptation in bitter gourd and offers a valuable candidate gene for the genetic enhancement of cold resistance in cucurbit crops.