Abstract
BACKGROUND: Cytokinin oxidases/dehydrogenases (CKXs) are key enzymes regulating cytokinin homeostasis and plant development, yet their transcriptional regulation remains poorly characterized, especially in legumes. RESULTS: Here, we combined promoter truncation, transgenic analysis, and DNA-protein interaction assays to dissect the regulatory mechanism of MtCKX1 in Medicago truncatula. A 1789 bp promoter region was identified, harboring multiple cis-elements responsive to hormones (CK, IAA, ABA, JA, GA) and stresses. Truncation analysis and GUS assay revealed a core cytokinin-responsive region (− 1789 to − 1125 bp), with one construct (CKXp2) functioning as a strong, specific cytokinin-inducible promoter throughout plant development. Using DNA pull-down mass spectrometry, yeast one-hybrid assays, and dual-luciferase reporter, we further demonstrated that C2H2 and GRF transcription factors directly bind to the MtCKX1 promoter. CONCLUSIONS: These findings provide novel insights into the transcriptional regulation of CKX genes and highlight the potential of engineered promoters for precise spatiotemporal control of gene expression in biotechnology applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08265-8.