Floral organ formation plays an essential role in Cymbidium sinense reproductive development and serves as a key determinant of their ornamental traits. During the domestication and natural evolution of C. sinense, numerous floral organ variant cultivars have emerged, among which many floral morphological variations arise from abnormal development of the gynostemium, a reproductive organ. These gynostemium variant (GV) cultivars not only exhibit enhanced commercial appeal but also provide a unique model for investigating floral morphogenesis and evolutionary diversification. In this study, we identified single nucleotide polymorphisms (SNPs) in the promoter region of CsSEP4 closely linked to GV through genome-wide association studies. Functional analyses of CsSEP4 revealed that it played a crucial role in the development of gynostemium. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays indicated that the CsbZIP26 transcription factor binds to the CsSEP4 promoter and activates its expression in normal flowers, whereas the SNP mutations from ACGTG to ATGTG or ACGTA of the CsSEP4 promoter were detected in GV lines, which resulted in the inability of CsbZIP26 to bind and regulate the expression of CsSEP4. Furthermore, DNA affinity purification sequencing (DAP-seq) and Y1H experiments identified CsSPL18 as a direct downstream target of CsSEP4. Genetic evidence also demonstrated that CsSEP4 orchestrates gynostemium development by positively activating CsSPL18 expression. Collectively, our results revealed that the CsbZIP26–CsSEP4–CsSPL18 regulatory module governs the development of stamen gynostemium to regulate flower morphology in C. sinense. These findings provide insight into the molecular mechanisms underlying gynostemium development in orchids and establish a molecular framework for further elucidating orchid diversity and evolution.

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