Understanding specificity in auxin perception

Lead Research Organisation: University of Leeds
Department Name: Inst of Integrative & Comparative Biolog


Context: Plant growth and development are controlled in a defined, but dynamic way through the activity of hormones. Hormones are mobile molecules that carry signals to coordinate growth and initiate responses to environmental cues. Auxin is a very important hormone in plants because it is involved in mediating almost every aspect of a plant's life, from embryogenesis to responses to gravity. In order for auxin to trigger all these responses it needs a receptor, a protein which binds the auxin and in doing so acts as a molecular switch, initiating a chain of events that ultimately leads to an appropriate change in the plant's development. We have recently identified a protein called TIR1 as an auxin receptor and this proposal describes a set of experiments that allows us to link whole plant responses to auxin back to the molecular events of auxin perception by TIR1. Aims and objectives: As noted above, auxin is involved in many very different developmental responses. Therefore an important question in plant biology is; how can this one molecular signal give rise to so many outcomes correctly? TIR1 turns out to be a member of a small family of related auxin receptors in the model plant Arabidopsis and it is apparent that there are differences in the receptor activity of these family members. To begin to understand these differences, once we have described auxin binding to TIR1, we will compare the rates and affinities of binding to each family member to identify the molecular basis of these differences. Once auxin is bound to TIR1 and its family members they act by binding another family of proteins, caled Aux/IAAs, which are then labelled for degradation. This degradation allows auxin-regulated genes to be switched on and a cascade of secondary reactions then proceeds. To determine whether or not each receptor has a preference for these Aux/IAA targets we will measure the reaction rates for association and dissociation with a representative sets of Aux/IAAs in both the presence and absence of auxin. These data will give us a matrix of interaction preferences and indicate a molecular basis for specificity, with high affinities in the presence of auxin likely to maximise the chances that this target will be degraded. To be sure that these measurements are relevant in intact, living plants the lifetimes of these target proteins will be measured. We will also generate mutant versions of Arabidopsis which lack particular receptor proteins, or present altered versions of receptors, and relate the auxin responsiveness of these plants to their genetic makeup and our biochemical data. These complementary datasets will add robustness and allow us to quantify the contribution played by selectivity at the receptor complex in specifying particular auxin responses. Potential applications and benefits: The data collected above all relate to the action of the natural plant auxin IAA. However, there are many synthetic auxins used in research and in agriculture and they each have slightly different activities. Examples of agricultural use include treatments to flowers, fruits and nuts and also as selective weedkillers. Auxinic weedkillers kill broadleaved plants, not cereals and grasses and are of great agricultural value. In order to address how these auxins can be selective we will perform experiments on cereal versions of TIR1 and compare both genetic and biochemical similarities and differences. Further, using both types of receptor we will establish a test system to help design and screen new compounds with auxin-like or antagonistic behaviour with the aim of creating a new generation of safe, selective and low dosage agricultural tools.

Technical Summary

We address how specificity is determined in the initial events of auxin perception and how this variation relates to the ability of auxin to control such remarkably diverse developmental processes. Objectives are: 1. To establish a reference set of kinetic and thermodynamic rules for the binding of auxin and Aux/IAAs to the TIR1 receptor: Wheatgerm in vitro transcription translation and baculovirus driven expression will be used to produce purified epitope-tagged TIR1. Interaction dynamics between TIR1, auxin and the TIR1 substrates for ubiquitination (Aux/IAA proteins) will be quantified using Biacore analyses. Binding interactions will be evaluated in BIAevaluate to derive kinetic constants. Thermodynamic experiments will test the 'molecular glue' model of receptor binding 2. To determine the extent and basis of specificity among TIR1/AFB receptors: The binding affinities of the 6 TIR1 family members for 4 representative Aux/IAAs will be quantified by Biacore, +/- auxin 3. To understand the basis of variation in Aux/IAA stability: The kinetic data of 2 will be extended to include mutant versions of Aux/IAAs using just TIR1 and AFB5 (most divergent). Kinetic data will be related to the protein structure. Our hypothesis is that affinity relates to Aux/IAA ubiquitination and reduced lifetime. We will measure Aux/IAA lifetimes using pull-downs and IAA:LUC fusions, relating lifetimes to affinities. Receptor mutants will be used to test preferences for Aux/IAAs 4. To understand the basis of auxinic herbicide selectivity across cereal/broadleaf species: Five classes of auxin give overlapping but distinct responses. AFB ligand specificity will be quantified by Biacore. Auxins are broadleaf herbicides so rice TIR1 orthologues will be expressed, modelled and BIAevaluated for comparison. To test predictions, a herbicide-resistant At mutant will be rescued by receptor engineering. The Biacore will be used to devise a screen for novel auxinic herbicides


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Description Key findings:

- A quantitive understanding of the binding preferences and affinities between TIR1/AFB-auxin-Aux/IAA auxin receptor complexes.

- Data underpinning the development of novel anti-auxin molecules.

Detailed summary:
The natural plant hormone known as auxin (also as indole-3-acetic acid or IAA) is a small mobile molecule that regulates many growth and developmental processes. The list includes root hair formation formation, the extent of lateral branching in the both the root and the shoot, and the angle at which plant organs grow with respect to gravity. These are all traits which are becoming increasingly important targets in crop improvement programs. Further understanding of how auxin signals are recognized and translated into this array of responses will therefore help in the battle for future food security. This project has helped us understand how one such a tiny chemical signal can mediate in such a huge variety of responses.

One of the key players in the perception machinery is the small family of auxin receptor proteins known as the TIR1/AFBs. We have isolated representatives of this family and used biophysical and biochemical measurements to watch how fast they bind auxin and how fast they separate again. In fact, we found that a third party, a substrate protein, was also required to make up a co-receptor complex. These co-receptors are called Aux/IAA repressor proteins, transcription factors that act to repress the expression of auxin-regulated genes. After binding in the auxin co-receptor complex the Aux/IAAs are modified to carry a signal that marks them for rapid breakdown. In this way auxins regulate the expression of many hundreds of genes. Understanding the molecular detail of this three-way association of hormone (auxin) and two, distinct co-receptor partners that each contribute to the active site for the hormone is novel. It may also help us understand how other plant signals work.

A number of studies have shown that individual TIR1/AFB proteins are specialized with respect to their biochemical properties and biological function. We have tested a range of different Aux/IAAs against different TIR/AFBs to work out the relative preferences of complex association. Some bind weakly, others strongly and we have been able to rank a matrix of interactors. By also comparing whole Aux/IAA proteins with selected sections of the same proteins we have been able to specify parts of the structure that are important for co-receptor binding. Understanding which complexes may form in vivo helps explain how auxin may contribute to such a wide range of developmental responses and why different responses arise from different doses of hormone.

A number of synthetic auxins are used commercially, especially as selective herbicides. We have used a diversity of these and other, related compounds to establish that there is binding site selectivity conferred by the TIR/AFB co-receptor and, to a lesser extent by some Aux/IAAs. We have also developed a set of novel small molecules that can regulate the formation of the TIR1/AFB-Aux/IAA receptor complex and this work has revealed new possibilities for the development of novel, more specific agrochemicals that can act as herbicides or even as beneficial plant growth enhancers. Further, we have adapted our biophysical techniques to devise a novel, fast laboratory screen to help discover new auxin-like and anti-auxin molecules. The screen has been validated using a small library of compounds from our commercial project partner. The need for new, effective, selective herbicides remains an important goal if we are to maximise food production from existing agricultural land areas.

We are left in no doubt over the complexity and diversity of the auxin perception mechanism, and many questions remain, but by being able to assign parameters to some of the interactions at the centre of auxin binding we have greatly improved our understanding of auxin perception and our ability to translate decades of research on auxin into meaningful opportunities for improving crop production and so food security.
Exploitation Route This work has laid the foundation for the development of novel agrochemicals for both crop protection (i.e. herbicides) and crop yield improvement
Sectors Agriculture, Food and Drink,Chemicals,Energy

Description To date impacts have been principally academic although, given the commercial value of auxinic herbicides, it is likely that our findings are in use by Ag-Biotech companies in the development of new herbicides chemistries.
First Year Of Impact 2011
Sector Agriculture, Food and Drink,Chemicals,Environment
Impact Types Societal,Economic

Description Member of the Advisory Committee of the UK Plant Science Federation
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Description Beeckman lab collaboration 
Organisation Flemish Institute for Biotechnology
Country Belgium 
Sector Academic/University 
PI Contribution We have provided research materials and shared data ahead of publication. We performed biochemical experiments for the papers listed below.
Collaborator Contribution They have provided research materials and shared data ahead of publication.
Impact De Rybel, B., Audenaert, D., Xuan, W., Overvoorde, P., Strader, L.C., Kepinski, S., Hoye, R., Brisbois, R., Parizot, B., Vanneste, S., Liu, X., Gilday, A., Graham, I.A., Nguyen, L., Jansen, L., Njo, M.F., Inzé, D., Bartel, B., Beeckman, T. (2012) A role for the root cap in root branching revealed by the non-auxin probe naxillin. Nature Chemical Biology 8: 798-805 De Rybel, B., Audenaert, D., Beeckmann, T., Kepinski, S. (2009) The past, present and future of chemical biology in auxin research. ACS Chemical Biology 4(12), 987-998
Start Year 2009
Description Estelle lab collaboration 
Organisation University of California, San Diego (UCSD)
Country United States 
Sector Academic/University 
PI Contribution We have provided research materials and shared data ahead of publication. We performed biochemical/biophysical experiments for the papers listed below.
Collaborator Contribution They have provided research materials and shared data ahead of publication.
Impact alderon Villalobos, L-I., Lee, S., Armitage, L., Parry, G., Mao, H., De Oliveira, C., Ivetac, A., Brandt, W., McCammonn, A., Zheng, N., Napier, R., Kepinski, S., Estelle, M. (2012) TIR1/AFBs and Aux/IAAs constitute a combinatorial co-receptor system to perceive auxin with differential sensitivities. Nature Chemical Biology 8: 477-485
Start Year 2007
Description Hayashi lab collaboration 
Organisation Okayama University of Science
Country Japan 
Sector Academic/University 
PI Contribution We have provided research materials and shared data ahead of publication. We performed biochemical experiments for the papers listed below.
Collaborator Contribution They have provided research materials and shared data ahead of publication.
Impact Hayashi, K., Neve, J., M., Hirose, M., Kuboki, A., Shimada, Y., Kepinski, S., Nozaki, H. (2012) Rational design of an auxin antagonist of the SCFTIR1 auxin receptor complex. ACS Chemical Biology 7: 590-98 Hayashi, K., Tan, X., Zheng, N,. Hatate, T., Kimura,Y., Kepinski, S., Nozaki. H. (2008) Small- molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling. Proc. Natl. Acad. Sci. U S A 105, 5632-5637
Start Year 2007
Description Discovery Zone 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact The feedback from schools is always positive.

Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015