Grafting-associated cold tolerance in pitaya involves ROS metabolism and the ICE1-CBF pathway

Low winter temperatures in subtropical regions adversely affect the growth and development of pitaya plants, making it essential to mitigate low temperature (LT) stress to ensure sustainable development of the pitaya industry. Although grafting has been shown to enhance stress resistance in various crops, the physiological and molecular basis for its association with improved cold tolerance in pitaya remains poorly understood. In this study, we used grafting combinations between ‘Taiwan No. 6’ (R, a cold-sensitive red-fleshed cultivar) and ‘Baishuijing’ (W, a cold-tolerant white-fleshed cultivar) to investigate the physiological responses and gene expression of different graft combinations under LT stress. The results showed that LT stress for 72 h significantly reduced root viability and chlorophyll content, while increasing malondialdehyde (MDA) content and relative electrical conductivity (REC), leading to a marked rise in the chilling injury index in all plants. Between the two cultivars, W exhibited significantly less damage than R. Notably, self-grafted R/R did not improve cold tolerance, whereas R/W grafting (R scion on W rootstock) significantly alleviated stress damage. In the R/W combination, the expression of superoxide dismutase (SOD) and peroxidase (POD) genes (at 6 h and 24 h) and their enzymatic activities (at 72 h) were significantly elevated, along with enhanced activities and upregulated expression of ascorbate–glutathione (AsA–GSH) cycle-related enzymes, including ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). These changes helped maintain higher ascorbate/dehydroascorbate (AsA/DHA) and reduced glutathione/oxidized glutathione (GSH/GSSG) ratios (at 72 h) and strengthened reactive oxygen species (ROS) scavenging capacity. Furthermore, the R/W combination induced stronger upregulation of ICE1 (inducer of CBF expression 1) and CBF (C-repeat binding factor) gene expression after 6 h and 24 h of LT stress compared with R/R. Collectively, these findings are consistent with a model in which grafting onto the cold-tolerant rootstock ‘Baishuijing’ is associated with activation of the ICE-CBF signaling pathway in the ‘Taiwan No. 6’ scion, coordinated upregulation of antioxidant enzyme genes and AsA–GSH cycle components, and enhanced ROS scavenging capacity. Additional functional genetic validation is necessary to elucidate the causal relationships between these pathways. Clinical trial number Not applicable.