To bind or not to bind: how AUXIN RESPONSE FACTORs select their target genes

Abstract

Most plant growth and development processes are regulated in one way or another by auxin. The best-studied mechanism by which auxin exerts its regulatory effects is through the nuclear auxin pathway (NAP). In this pathway, Auxin Response Factors (ARFs) are the transcription factors that ultimately determine which genes become auxin regulated by binding to specific DNA sequences. ARFs have primarily been studied in Arabidopsis thaliana, but recent studies in other species have revealed family-wide DNA binding specificities for different ARFs and the minimal functional system of the NAP system, consisting of a duo of competing ARFs of the A and B classes. In this review, we provide an overview of key aspects of ARF DNA binding such as auxin response elements (TGTCNN) and tandem repeat motifs, and consider how structural biology and in vitro studies help us understand ARF DNA preferences. We also highlight some recent aspects related to the regulation of ARF levels inside a cell, which may alter the DNA binding profile of ARFs in different tissues. We finally emphasize the need to study minimal NAP systems to understand fundamental aspects of ARF function, the need to characterize algal ARFs to understand how ARFs evolved, how cutting-edge techniques can increase our understanding of ARFs, and which remaining questions can only be answered by structural biology.

Keywords: AuxRE; Auxin; Auxin Response Element; Auxin Response Factor; DNA binding; dimerization; gene expression; transcription; transcription factors.

Fig. 1.

Overview of the nuclear auxin pathway and the anatomy of an ARF. (A) The minimalistic nuclear auxin pathway. Under low auxin conditions (upper panel), both A-ARFs (green) and B-ARFs (red) act as repressors. A-ARFs bind with a repressive cofactor, Aux/IAA (bordeaux), that recruits TPL, while B-ARFs recruit TPL directly via their middle region. Under increasing auxin conditions [indole-3-acetic acid (IAA) gold], IAA acts as a molecular glue and allows TIR1/AFB to sequester Aux/IAA away from the A-ARFs, which then become transcriptional activators. B-ARFs act as repressors in either condition. (B) The anatomy of an ARF. Top panel shows the general genetic sequence of an ARF, the lower panel shows the atomic structure of the DNA-binding domain in complex with an IR7 motif (pdb: 6ycq). Domains indicated and discussed in this review are: α1 (pear), the α-helix tethering the B3 domain and acting as a molecular hinge; N-terminal dimerization domain (yellow); B3 (green), the domain interacting with the DNA; αD (purple), the α-helix and loop that facilitates dimerization; and the C-terminal dimerization domain and ancillary domain (AD, cyan). The middle region (MR, white) and Phox and Bem1 domain (PB1, blue) are omitted.

Fig. 2.

Atomic interface between AuxRE and ARF-B3. Residues are numbered according to AtARF1 and DNA bases are numbered according to the first nucleotide of TGTCTC. (A and C) Schematic representation of DNA contact sites of ARF-B3 for TGTCTC (A) and high affinity TGTCGG (C). Contacting residues are indicated with ovals, green for residues only contacting the phosphate backbone and blue for residues contacting specific DNA bases. The figures are composites of three structures (4ldx, 6ycq, and 6sdg; cut-off for contacts of 3.5 Å) and, as such, some contacts are only found in a single structure, or compensated for by other residues. For instance, either K126 makes contacts alone, or T129 and S140 contact together, but never all three at the same time. (B and D) Close up of AtARF1–H136 conformation change with AuxRE bases of either TGTCTC (B) or TGTCGG (D). Based on Freire-Rios et al. (2020).

Fig. 3.

Spacing motifs determine ARF specificity. (A) Definition of a direct repeat (DR), inverted repeat (IR), and everted repeat (ER), following the definition set by Freire-Rios et al. (2020). N denotes A, T, C, or G, while dots represent variable numbers of intermittent nucleotides between two TGTCNN elements. (B) Schematic figure representing single-molecule FRET analysis of DNA binding. Arrow thickness represents affinity. A- (green) and B-ARFs (red) are competing with each other for DNA binding. Both ARFs have an affinity for IR7 motifs that allows for in vivo competition on the same locus. For DR5, both ARFs have a weaker affinity, with B-ARF affinity being so weak that their in vivo binding is likely to be negligible (Freire-Rios et al., 2020; Kato et al., 2020). (C) The proposed caliper model (Boer et al., 2014). The A-ARF AtARF5 (green) was found to be able to bind IR5–IR9 motifs, whereas the B-ARF AtARF1 (red) was limited to IR7/8.