An atlas of conserved transcription factor binding sites reveals the cell type-resolved gene regulatory landscape of flowering plants

Transcription factors (TFs) play a central role in regulating gene expression, a process fundamental to cellular function. Characterizing transcription factor binding sites (TFBSs) is essential for understanding TF functions and identifying TF target genes, but many predicted TFBSs lack clear biological significance. We generated an atlas of TFBSs across ten flowering plants and demonstrated that conserved TFBSs are strongly enriched for genomic and epigenomic signatures of functional regulatory elements. We uncovered conserved properties of TFBSs, including TF family-specific clustering of binding sites in distinct promoter and gene body locations. By integrating this atlas with single nuclei RNA and chromatin profiles, we elucidated conserved TF activity underlying development and stress responses and annotated key cell type-specific regulatory pathways. Finally, we found that TF ortholog binding specificity is preserved over long evolutionary times, suggesting that divergence of regulatory sequences, rather than TFs, is a primary driver of regulatory evolution.