A conserved ARF-DNA interface underlies auxin-triggered transcriptional response

Auxin Response Factor (ARF) plant transcription factors are the key effectors in auxin signaling. Their DNA-Binding Domain (DBD) contains a B3 domain that allows base-specific interactions with Auxin Response Elements (AuxREs) in DNA target sites. Land plants encode three phylogenetically distinct ARF classes: the closely related A- and B-classes have overlapping DNA binding properties, contrasting with the different DNA-binding properties of the divergent C-class ARFs. ARF DNA-binding divergence likely occurred early in the evolution of the gene family, but the molecular determinants underlying it remain unclear. Here, we show that the B3 DNA-binding residues are deeply conserved in ARFs, and variability within these is only present in tracheophytes, correlating with greatly expanded ARF families. Using the liverwort Marchantia polymorpha, we confirm the essential role of conserved DNA-contacting residues for ARF function. We further show that ARF B3-AuxRE interfaces are not mutation-tolerant, suggesting low evolvability that has led to the conservation of the B3-DNA interface between ARF classes. Our data support the almost complete interchangeability between A/B-class ARF B3 by performing interspecies domain swaps in M. polymorpha, even between ARF lineages that diverged over half a billion years ago. Our analysis further suggests that C-class ARF DNA-binding specificity diverged early during ARF evolution in a common streptophyte ancestor, followed by strong selection in A and B-class ARFs as part of a competition-based auxin response system.