Ichang papeda (Citrus ichangensis), a wild primitive citrus species, exhibits remarkable cold tolerance, yet the transcriptional regulation of cold-responsive genes in C. ichangensis remains poorly explored. In this study, we identified the small heat shock protein (sHSP) CiHSP26.5 as a crucial member that was substantially induced under cold stress and played a positive role in cold tolerance. Furthermore, two APETALA2/Ethylene Responsive Factor (AP2/ERF) family transcription factors, CiERF023 and CiERF041, were found to directly bind to the dehydration-responsive element (DRE) motifs in the promoter of CiHSP26.5 and function as its negative and positive regulators, respectively. In addition, CiERF023 was proven to repress CiERF041 through interacting with the DRE element in the promoter. These two TFs were differentially induced during cold stress, with CiERF023 peaking at early stages and CiERF041 at later stages. Consistently, CiERF023 acts as a negative regulator, whereas CiERF041 serves as a positive regulator, of cold tolerance by regulating CiHSP26.5. Taken together, our findings reveal that CiERF023 and CiERF041 form a hierarchical transcriptional cascade that antagonistically regulates CiHSP26.5 expression under cold stress in C. ichangensis. This study provides fresh insights into the regulatory network governing HSP-mediated cold adaptation and identifies potential targets for enhancing cold tolerance in plants.

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