The <scp>ATAF</scp> 1‐ <scp>AP</scp> 2/ <scp>ACE</scp> 1‐ <scp>MYB</scp> regulatory cascade negatively orchestrates flower color and scent formation in <i>Freesia hybrida</i>

Floral traits, including color, scent, morphology and longevity, are critical for pollinator attraction and aesthetic value. Notably, floral color and scent often exhibit synergistic variation during development, yet the molecular mechanisms coordinating these traits remain poorly understood. This study aimed to elucidate the regulatory network underlying their co-regulation in Freesia hybrida. The study employed integrated approaches including gene expression analysis, protein-protein interaction assays, promoter activity validation and transient overexpression experiments in Freesia petals and Arabidopsis protoplasts to characterize transcriptional regulation and functional interactions of regulators underlying anthocyanin and linalool biosynthesis. Two negative regulators, FhAP2 (AP2/ERF) and FhACE1 (bHLH), were identified to suppress anthocyanin and linalool biosynthesis by physically interacting with MYB activators FhPAP1 and FhMYB21L2, respectively. This interaction disrupts the binding of MYB regulators to their target gene promoters in a dose-dependent manner. In addition, FhAP2 and FhACE1 could be directly activated by an upstream NAC regulator (FhATAF1) via promoter binding. These interactions form a multi-layer mechanism driving trait co-regulation. A hierarchical regulatory cascade, ATAF1-AP2/ACE1-PAP1/MYB21L2, was uncovered to fine-tune floral color and scent dynamics during flower development. This mechanism provides insights into floral trait coordination and establishes a foundation for targeted breeding strategies to enhance ornamental qualities.