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

Juriaan Rienstra, Vanessa Polet Carrillo-Carrasco, Martijn de Roij, Jorge Hernandez-Garcia, and Dolf Weijers

PNAS; April 1, 2025; 122 (14) e2501915122; https://doi.org/10.1073/pnas.2501915122

Significance

Auxin response evolved nearly half a billion years ago in the earliest land plants. Auxin response is mediated by a family of DNA-binding ARF transcription factors. It has been unclear if and how the ARF family has evolved. In this paper, the authors show that the central protein–DNA interface that defines the genes that are under auxin control has remained essentially unchanged throughout auxin response evolution.

Abstract

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.

See https://www.pnas.org/doi/10.1073/pnas.2501915122

Figure 1: Extended ARF DNA-binding interface of AtARF1 and conservation of residues and structure. (A) Schematic overview of AtARF1 residues (ovals) and their interactions. Residues bind nucleobases in the major groove (blue), the phosphate backbone (green), or only make water-mediated contacts (purple). See SI Appendix, Fig. S1. (B) Conservation analysis of AtARF1 B3 domain residues using ConSurf (28). Individual residues are colored according to their conservation score ranging from variable to conserved (score 1 to 9). See SI Appendix, Fig. S2 for the full DBD. (C) Alignment of predicted protein structures of Marchantia polymorpha ARF1 (MpARF1, cyan, A-class), MpARF2 (green, B-class), and MpARF3 (magenta, C-class) using AlphaFold2 (29). The AtARF1 homologous DNA-binding residues are represented as sticks, with major groove-binding residues indicated for each ARF. (D) Alignment of the DNA-binding loops from M. polymorpha and Arabidopsis ARFs. Major groove binding residues are colored. (E) Table with all variation found in the land plant (embryophyte) A, B, and C-class ARFs from the phylogenetic tree of Mutte et al. (20).