Integrative physiology, transcriptomic, and metabolomic analysis reveals the response mechanism of melon seedlings to cold stress

Early-spring cultivation enables melons to be marketed ahead of the regular season, yet cold stress during this critical growth stage severely suppresses seedling growth and development, ultimately causing substantial yield losses. To date, the physiological shifts, molecular regulatory cascades and metabolic reprogramming triggered by cold stress in melon seedlings have not been fully characterized. In this work, we combined physiological assays, transcriptome, and metabolome profiling to dissect cold-responsive regulatory networks at the seedling stage using two melon inbred lines with divergent cold tolerance: NM3 (cold-tolerant, Netted muskmelon) and M40-1 (cold-sensitive, Hami melon). Our phenotypic and physiological data revealed that cold treatment drastically retarded seedling growth and triggered leaf wilting, alongside elevated reactive oxygen species (ROS) and malondialdehyde (MDA) concentrations in both inbred lines. Relative to cold-sensitive M40-1, NM3 maintained superior growth performance, accompanied by greater accumulation of sensitive and stronger antioxidant enzymatic activities. Transcriptome comparison uncovered sets of differentially expressed genes (DEGs) enriched in phenylalanine metabolism, flavone/flavonol/isoflavonoid biosynthesis, glutathione (GSH) metabolism and fatty acid metabolic pathways. Metabolite quantification further indicated that flavonoids, soluble sugars, lipids and terpenoids accumulated to markedly higher abundances in NM3 after 6 h of cold treatment. Weighted gene co-expression network analysis (WGCNA) integrating transcriptomic and metabolomic datasets pinpointed hub genes and signature metabolites tightly linked to cold tolerance, such as MELO3C017481 (encoding xyloglucan endotransglucosylase-hydrolase 23, XTH23) and MELO3C021100 (encoding heat shock protein 70, HSP70), as well as numerous transcription factors (TFs) and functional genes participating in sugar and flavonoid metabolism. Integrated transcriptomic and metabolomic profiling further verified that genes and metabolites governing flavonoid synthesis and GSH metabolism serve as central modulators of melon cold tolerance. We also found that cold stress robustly activated GSH-related gene expression, and exogenous GSH supplementation effectively relieved cold-induced seedlings injury. Collectively, this study deepens our mechanistic understanding of cold tolerance in melon seedlings, confirms that flavonoids and GSH metabolites act as core components facilitating plant stress adaptation, and supplies valuable genetic and metabolic resources to accelerate the breeding of cold-tolerant melon varieties.