CmNAC29 Positively Regulates Chilling Tolerance in Melon Seedlings by Activating Antioxidant Defense, Proline Biosynthesis, and the ICE-CBF-COR Pathway

Low-temperature stress severely limits early-spring melon (Cucumis melo L.) production. However, the regulatory roles of NAC transcription factors in melon responses to abiotic stress remain insufficiently understood. In this study, the melon cultivar ‘Xizhoumi No. 17’ was used as the experimental material, and an Agrobacterium-mediated root transformation system was employed to generate CmNAC29-overexpressing and empty-vector control plants. Phenotypic analysis, physiological measurements, transcriptome sequencing, and molecular interaction assays were performed to systematically investigate the regulatory mechanism by which CmNAC29 mediates chilling tolerance in melon roots. The results showed that CmNAC29 overexpression significantly alleviated cold stress-induced growth inhibition in melon seedlings, reduced membrane lipid peroxidation, and enhanced antioxidant enzyme activities and proline accumulation. Transcriptome analysis revealed that differentially expressed genes associated with CmNAC29 overexpression were significantly enriched in functional categories related to oxidoreductase activity. Further validation showed that CmNAC29 upregulated the expression of antioxidant enzyme genes, the key proline biosynthesis gene CmP5CS1, and core components of the Inducer of CBF Expression–C-repeat Binding Factor–Cold-Regulated pathway. Molecular assays confirmed that CmNAC29 possesses transcriptional activation activity and directly binds to the promoters of CmP5CS1-1 and CmCOR413 by recognizing NAC-binding sites, thereby activating their transcription. Taken together, these findings demonstrate that CmNAC29 positively regulates chilling tolerance in melon seedlings by coordinately enhancing antioxidant defense, osmotic adjustment, and cold signal transduction. This study provides an important genetic resource and theoretical basis for the molecular breeding of cold-tolerant melon cultivars.