Stefan Kepinski

The Arabidopsis F-box protein TIR1 is an auxin receptor

Despite 100 years of evidence showing a pivotal role for indole-3-acetic acid (IAA or auxin) in plant development, the mechanism of auxin perception has remained elusive. Central to auxin response are changes in gene expression, brought about by auxin-induced interaction between the Aux/IAA transcriptional repressor proteins and the ubiquitin–ligase complex SCFTIR1, thus targeting for them proteolysis. Regulated SCF-mediated protein degradation is a widely occurring signal transduction mechanism. Target specificity is conferred by the F-box protein subunit of the SCF (TIR1 in the case of Aux/IAAs) and there are multiple F-box protein genes in all eukaryotic genomes examined so far. Although SCF–target interaction is usually regulated by signal-induced modification of the target, we have previously shown that auxin signalling involves the modification of SCFTIR1. Here we show that this modification involves the direct binding of auxin to TIR1 and thus that TIR1 is an auxin receptor mediating transcriptional responses to auxin.

Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins

The plant hormone auxin is central in many aspects of plant development. Previous studies have implicated the ubiquitin-ligase SCFTIR1 and the AUX/IAA proteins in auxin response. Dominant mutations in several AUX/IAA genes confer pleiotropic auxin-related phenotypes, whereas recessive mutations affecting the function of SCFTIR1 decrease auxin response. Here we show that SCFTIR1 is required for AUX/IAA degradation. We demonstrate that SCFTIR1 interacts with AXR2/IAA7 and AXR3/IAA17, and that domain II of these proteins is necessary and sufficient for this interaction. Further, auxin stimulates binding of SCFTIR1 to the AUX/IAA proteins, and their degradation. Because domain II is conserved in nearly all AUX/IAA proteins in Arabidopsis, we propose that auxin promotes the degradation of this large family of transcriptional regulators, leading to diverse downstream effects.

A novel sensor to map auxin response and distribution at high spatio-temporal resolution

Auxin is a key plant morphogenetic signal but tools to analyse dynamically its distribution and signalling during development are still limited. Auxin perception directly triggers the degradation of Aux/IAA repressor proteins. Here we describe a novel Aux/IAA-based auxin signalling sensor termed DII-VENUS that was engineered in the model plant Arabidopsis thaliana. The VENUS fast maturing form of yellow fluorescent protein was fused in-frame to the Aux/IAA auxin-interaction domain (termed domain II; DII) and expressed under a constitutive promoter. We initially show that DII-VENUS abundance is dependent on auxin, its TIR1/AFBs co-receptors and proteasome activities. Next, we demonstrate that DII-VENUS provides a map of relative auxin distribution at cellular resolution in different tissues. DII-VENUS is also rapidly degraded in response to auxin and we used it to visualize dynamic changes in cellular auxin distribution successfully during two developmental responses, the root gravitropic response and lateral organ production at the shoot apex. Our results illustrate the value of developing response input sensors such as DII-VENUS to provide high-resolution spatio-temporal information about hormone distribution and response during plant growth and development.

The auxin signalling network translates dynamic input into robust patterning at the shoot apex

The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large‐scale analysis of the Aux/IAA‐ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin‐based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA‐ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.

A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding to the F-box protein TIR1 and promotes the degradation of the Aux/IAA transcriptional repressors. Here, we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity appears to be largely determined by the Aux/IAA. As there are 6 TIR1/AFBs and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin sensing properties. We also demonstrate that the AFB5-Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.

Small-molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling.

The regulation of gene expression by the hormone auxin is a crucial mechanism in plant development. We have shown that the Arabidopsis F-box protein TIR1 is a receptor for auxin, and our recent structural work has revealed the molecular mechanism of auxin perception. TIR1 is the substrate receptor of the ubiquitin–ligase complex SCFTIR1. Auxin binding enhances the interaction between TIR1 and its substrates, the Aux/IAA repressors, thereby promoting the ubiquitination and degradation of Aux/IAAs, altering the expression of hundreds of genes. TIR1 is the prototype of a new class of hormone receptor and the first example of an SCF ubiquitin-ligase modulated by a small molecule. Here, we describe the design, synthesis, and characterization of a series of auxin agonists and antagonists. We show these molecules are specific to TIR1-mediated events in Arabidopsis, and their mode of action in binding to TIR1 is confirmed by x-ray crystallographic analysis. Further, we demonstrate the utility of these probes for the analysis of TIR1-mediated auxin signaling in the moss Physcomitrella patens. Our work not only provides a useful tool for plant chemical biology but also demonstrates an example of a specific small-molecule inhibitor of F-box protein–substrate recruitment. Substrate recognition and subsequent ubiquitination by SCF-type ubiquitin ligases are central to many cellular processes in eukaryotes, and ubiquitin-ligase function is affected in several human diseases. Our work supports the idea that it may be possible to design small-molecule agents to modulate ubiquitin-ligase function therapeutically.

Structural Basis for DNA Binding Specificity by the Auxin-Dependent ARF Transcription Factors

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.

Rational Design of an Auxin Antagonist of the SCFTIR1 Auxin Receptor Complex

The plant hormone auxin is a master regulator of plant growth and development. By regulating rates of cell division and elongation and triggering specific patterning events, indole 3-acetic acid (IAA) regulates almost every aspect of plant development. The perception of auxin involves the formation of a ternary complex consisting of an F-box protein of the TIR1/AFB family of auxin receptors, the auxin molecule, and a member the Aux/IAA family of co-repressor proteins. In this study, we identified a potent auxin antagonist, α-(phenylethyl-2-oxo)-IAA, as a lead compound for TIR1/AFB receptors by in silico virtual screening. This molecule was used as the basis for the development of a more potent TIR1 antagonist, auxinole (α-[2,4-dimethylphenylethyl-2-oxo]-IAA), using a structure-based drug design approach. Auxinole binds TIR1 to block the formation of the TIR1-IAA-Aux/IAA complex and so inhibits auxin-responsive gene expression. Molecular docking analysis indicates that the phenyl ring in auxinole would strongly interact with Phe82 of TIR1, a residue that is crucial for Aux/IAA recognition. Consistent with this predicted mode of action, auxinole competitively inhibits various auxin responses in planta. Additionally, auxinole blocks auxin responses of the moss Physcomitrella patens, suggesting activity over a broad range of species. Our works not only substantiates the utility of chemical tools for plant biology but also demonstrates a new class of small molecule inhibitor of protein-protein interactions common to mechanisms of perception of other plant hormones, such as jasmonate, gibberellin, and abscisic acid.

Defining auxin response contexts in plant development

The apparent domination of so much of plant development by the hormone auxin raises important questions about how this simple, generic signalling molecule can give rise to such an enormous range of very specific developmental outputs. What is becoming increasingly clear is that alongside the tight control of auxin distribution within the plant, the cellular and developmental context in which the auxin signal is received is of utmost importance. Recent work has highlighted that these distinct auxin response contexts are defined by complex and inter-dependent relationships between auxin metabolism, transport and response that can be modulated at many levels by inputs from both endogenous and environmental signals.

A role for the root cap in root branching revealed by the non-auxin probe naxillin

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.