Promoter Motif Profiling and Binding Site Distribution Analysis of Transcription Factors Predict Auto- and Cross-Regulatory Mechanisms in Arabidopsis Flowering Genes

The transition to flowering in Arabidopsis thaliana is governed by complex transcriptional regulatory networks, in which promoter-associated cis-regulatory elements integrate both developmental and environmental cues. To investigate these regulatory interactions, we analyzed promoter motifs of 18 flowering-related genes using curated motif resources, including the Eukaryotic Promoter Database (EPD) and JASPAR, applying stringent statistical thresholds. Transcription factors (TFs), which were predicted to bind across all examined promoters, were designated as putative master regulators, resulting in the identification of 36 candidates, predominantly belonging to the MADS-box, DOF, and IDD families. Positional analyses revealed both proximal and distal binding sites, including a notable motif at −1024 in PISTILLATA and at +466 in SEPALLATA3, potentially indicative of autoregulatory control. Comparative analysis further identified 96 gene-specific associations, reflecting a balance between shared and specialized regulatory mechanisms. Treatment with β-aminobutyric acid (BABA), which has a flowering delaying effect, repressed SQUAMOSA and increased DOF-type TFs, indicating a chromatin-associated reprogramming process, which may coordinate the transcriptional suppression of flowering activators. These findings refine current models of floral regulatory networks and provide testable hypotheses regarding autoregulatory and cross-regulatory circuits in the control of flower development.