Recruitment, rewiring and deep conservation in flowering plant gene regulation.

Transcription factors (TFs) are proteins that bind DNA to control where and when genes are expressed. In plants, dozens of TF families interact with distinct sets of binding sites (TFBSs) that reflect each TF's role in organismal function and species-specific adaptations. However, defining these roles and understanding broader patterns of regulatory evolution remain challenging, as predicted TFBSs may lack a clear impact on transcription, and experimentally derived TF binding maps to date are modest in scale or restricted to model organisms. Here we present a scalable TFBS assay that we leveraged to create an atlas of nearly 3,000 genome-wide binding site maps for 360 TFs in ten species spanning 150 million years of flowering plant evolution. We found that TF orthologues from distant species retain nearly identical binding preferences, while on the same timescales the gain and loss of TFBSs are widespread. Within lineages, however, conserved TFBSs are over-represented and found in regions harbouring signatures of functional regulatory elements. Moreover, genes with conserved TFBSs showed striking enrichment for cell-type-specific expression in 14 single-nucleus RNA atlases, providing a robust marker of each TF's activity and developmental role. Finally, we compare distant lineages, illustrating how ancient regulatory modules were recruited and rewired to enable adaptations underlying the evolutionary success of grasses.