DcmiR172c-DcNAC41-DcHSFA2d/DcHSP70 module collaborates with DcGOX1 to modulate thermotolerance in carnation.

Heat stress (HS) driven by global warming poses a challenge to the productivity and aesthetic value of carnation. To explore the molecular basis of thermotolerance, this study conducted integrated multi-omics analyses under HS, identifying a heat-responsive microRNA, DcmiR172c, whose expression exhibited an inverse relationship with the NAC transcription factor gene DcNAC41. Functional assays suggested that elevated DcmiR172c levels were associated with increased heat-induced membrane peroxidation and photosynthetic damage. Bioinformatic prediction, qRT-PCR analysis, dual-luciferase reporter assays, and protein-level experiments indicated that DcmiR172c may post-transcriptionally repress DcNAC41. In contrast, DcNAC41 acts as a transcriptional activator potentially involved in heat adaptation. It bound to and activated the promoters of the heat shock transcription factor DcHSFA2d and the heat shock protein gene DcHSP70-5, suggesting its involvement in the canonical HSF-HSP defense pathway. Notably, DcHSFA2d further amplified this response by activating DcHSP70-5, implying a possible feed-forward regulatory circuit. Moreover, protein interaction screening identified the glycolate oxidase DcGOX1 as a novel binding partner of DcNAC41. This interaction, supported by multiple in vivo and in vitro assays, appeared to enhance the transcriptional activity of DcNAC41 toward its targets, and contribute to heat tolerance. Collectively, this research outlines a multi-tiered regulatory framework in which a miRNA-guided NAC module coordinates with the HSF-HSP network and a metabolic enzyme to modulate heat acclimation. These findings highlight the potential importance of the DcmiR172c-DcNAC41-DcHSFA2d/DcHSP70-5 axis and offer valuable genetic targets for engineering climate-resilient carnation varieties.