Evolution of the Auxin Response Factors from charophyte ancestors.

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.

Capturing Auxin Response Factors Syntax Using DNA Binding Models.

Auxin is a key hormone performing a wealth of functions throughout the life cycle of plants. It acts largely by regulating genes at the transcriptional level through a family of transcription factors called auxin response factors (ARFs). Even though all ARF monomers analyzed so far bind a similar DNA sequence, there is evidence that ARFs differ in their target genomic regions and regulated genes. Here, we report the use of position weight matrices (PWMs) to model ARF DNA binding specificity based on published DNA affinity purification sequencing (DAP-seq) data. We found that the genome binding of two ARFs (ARF2 and ARF5/Monopteros [MP]) differ largely because these two factors have different preferred ARF binding site (ARFbs) arrangements (orientation and spacing). We illustrated why PWMs are more versatile to reliably identify ARFbs than the widely used consensus sequences and demonstrated their power with biochemical experiments in the identification of the regulatory regions of IAA19, an well-characterized auxin-responsive gene. Finally, we combined gene regulation by auxin with ARF-bound regions and identified specific ARFbs configurations that are over-represented in auxin-upregulated genes, thus deciphering the ARFbs syntax functional for regulation. Our study provides a general method to exploit the potential of genome-wide DNA binding assays and to decode gene regulation.

Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression.

Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.

Structural basis for oligomerization of auxin transcriptional regulators.

The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.