Abstract

Cymbidium ensifolium exhibits highly specialized floral structures with considerable morphological diversity, including multi-tepal varieties that are distinguished by unique floral patterning and are of high ornamental value, making them a key breeding target. However, the molecular mechanisms underlying multi-tepal formation in this species remain poorly characterized. To address this, we employed an integrated approach, combining phenotypic observation, cytological examination, transcriptome sequencing, gene expression profiling, functional validation, yeast one-hybrid (Y1H), and dual-luciferase reporter (DLR) assays. The multi-tepal phenotype was characterized by increased numbers of sepals and petals and the absence of a gynostemium. Transcriptome analysis revealed several key regulatory genes, including CeLFY , CeAP1-1 , CeAP1-2 , CeAG-1 , CeAG-2 , and CeAG-3 . Expression analysis revealed a marked upregulation of CeLFY and CeAP1 s during early bud development in the multi-tepal variety, correlating with enhanced perianth organ formation. In contrast, persistent downregulation of CeAG s across floral stages was associated with gynostemium abortion. Transgenic assays confirmed that perturbation of these genes resulted in aberrant floral organ development, and subcellular localization supported the nuclear presence of their protein products. Y1H and DLR experiments indicated that CeLFY activates the promoters of CeAP1 s, promoting sepal and petal proliferation. Furthermore, promoter analysis identified structural variations in the CeAG s promoters that reduced CeLFY binding affinity, thereby repressing CeAG s expression and impairing gynostemium development. These findings provide mechanistic insights into the multi-tepal flower formation in C. ensifolium and contribute to the molecular basis for ornamental trait improvement in orchids.