A novel CsbZIP26–CsSEP4–CsSPL18 regulatory module governs gynostemium morphology and floral architecture in <i>Cymbidium sinense</i>

Floral organ formation plays an essential role in <i>Cymbidium sinense</i> reproductive development and serves as a key determinant of their ornamental traits. During the domestication and natural evolution of <i>C. sinense</i>, 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 <i>CsSEP4</i> closely linked to GV through genome-wide association studies. Functional analyses of <i>CsSEP4</i> 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 <i>CsSEP4</i> promoter and activates its expression in normal flowers, whereas the SNP mutations from ACGTG to ATGTG or ACGTA of the <i>CsSEP4</i> promoter were detected in GV lines, which resulted in the inability of CsbZIP26 to bind and regulate the expression of <i>CsSEP4</i>. Furthermore, DNA affinity purification sequencing (DAP-seq) and Y1H experiments identified <i>CsSPL18</i> as a direct downstream target of CsSEP4. Genetic evidence also demonstrated that CsSEP4 orchestrates gynostemium development by positively activating <i>CsSPL18</i> expression. Collectively, our results revealed that the <i>CsbZIP26-CsSEP4-CsSPL18</i> regulatory module governs the development of stamen gynostemium to regulate flower morphology in <i>C. sinense</i>. 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.