Positional grammar of transcription factor binding partitions developmental and stress-response regulation in plants

Understanding how transcription factor binding site (TFBS) location influences gene regulation remains a fundamental challenge in plants. Here we integrate conserved TFBS with single-nucleus chromatin accessibility and expression datasets to resolve how TFBS location relates to regulatory function. Although TF binding patterns are conserved and frequently enriched near the TSS, positional enrichment of TFBSs poorly predicts cell type-specific gene expression. Instead, cell type-specific gene expression aligns with conserved TFBSs that are embedded in cell type-restricted chromatin, which show TF family-specific distributions across distal promoter and genic regions. In contrast, TSS-proximal TFBSs are implicated in rapid, tissue-wide transcriptional stress responses, as supported by hormone-induced gene expression analysis. Finally, distal upstream regions contain conserved TF clusters that overlap rare cell type-specific accessible chromatin and are highly enriched for genes controlling embryonic and meristem patterning, including auxin and other hormone pathway components. This positional partitioning of regulatory function indicates that TFBS location encodes distinct regulatory programs for TFs.