CmABF3-CmTPP2/5 modulates trehalose-mediated hydrogen peroxide signaling and CBF-dependent pathway to enhance cold tolerance in melon seedlings

Cold stress inhibits the growth and development of melon. Exogenous trehalose (Tre) has been reported to alleviate cold damage in melon seedlings; however, it remains unclear whether plants respond to cold stress by activating endogenous Tre synthesis and the associated upstream and downstream pathways. Here, the results show that cold stress markedly increases endogenous Tre levels in melon leaves. Transcriptome analysis identified two trehalose-6-phosphate phosphatase (key enzymes in Tre synthesis, TPP) genes, CmTPP2 and CmTPP5, which were strongly induced by cold stress. Silencing CmTPP2/5 significantly reduced Tre content, lowered the activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase, and exacerbated cold-induced lipid peroxidation damage; meanwhile, it inhibited apoplastic H₂O₂ accumulation, restricted H₂O₂ transport into the cytoplasm, and downregulated the expression of C-repeat binding factor 1/2 (CmCBF1/2) under cold stress. Protein-DNA interaction assays demonstrated that the transcription factor CmABF3 binds to the ABRE motif in the CmTPP2/5 promoter to activate their transcription. Silencing CmABF3 gene significantly reduced CmCBF1/2 and CmTPP2/5 expression and reduced Tre accumulation. Inhibition of trehalase activity by validamycin A elevated Tre content, enhanced antioxidant capacity under cold stress, promoted apoplastic H₂O₂ production and transport, and upregulated CmCBF1/2 expression. Silencing CmCBF1/2 also inhibited the production and transport of apoplastic H₂O₂ and reduced melon cold tolerance. These findings reveal that cold stress triggers CmABF3-mediated activation of CmTPP2/5, leading to Tre accumulation, which then enhanced antioxidant defenses via regulation of apoplastic H₂O₂ production and transport, and coordination with the CBF-dependent pathway to improve cold tolerance in melon seedlings.