Understanding specificity in auxin perception

Lead Research Organisation: University of Warwick
Department Name: Warwick HRI

Abstract

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

Publications

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Calderón Villalobos LI (2012) A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. in Nature chemical biology

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Kowalska M (2011) Prussian Blue acts as a mediator in a reagentless cytokinin biosensor in Analytica Chimica Acta

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Sadanandom A (2010) Biosensors in plants. in Current opinion in plant biology

 
Description The most significant achievements



1. We have recorded in real-time the kinetics of forming the three-partner auxin receptor complex using SPR technology. We have examined the molecular glue hypothesis and looked at how specificity is determined.

2. We can explain some of the differential activity of auxins and auxinic herbicides by the association and (particularly) the dissociation rates of the receptor complex. Different members of the receptor family and different substrate Aux/IAA proteins add to the multiplicity of outcomes.

3. We have established SPR as a rapid, in vitro screen for (novel) auxins and tested a blind compound (sub)library from partner Syngenta.



We have delivered on all of the original project objectives.



1. To establish a reference set of kinetic rules for the binding of auxin and Aux/IAAs to the TIR1 receptor: Done. Using an SPR assay based on the immobilization of IAA degron peptides and auxin-dependent binding of TIR1 or AFB5 we have established rate constants and KDs for the associations. Different auxin affinities are found to be governed primarily by the dissociation rate of each complex. As long as auxin remains in the wash solutions, the interaction of the complex is stable and long-lived.



2. To determine the extent and basis of specificity among TIR1/AFB receptors: Done. AFB5 shows considerably higher off-rates than TIR1 for all compounds tested. The binding characteristics for a series of Aux/IAA degron peptides were collected with a series of auxins for TIR1 and AFB5.



3. To understand the basis of variation in Aux/IAA stability: Done. Binding of IAA proteins to TIR1 and AFB5 is dependent on the degron sequence with data agreeing for both SPR and pull-down methods. AXR3/IAA17 mutant phenotypes may be understood in terms of receptor complex binding and dissociation kinetics.



4. To understand the basis of auxinic herbicide selectivity. Developing a proof of concept screen for novel compounds with auxinic activity with IPA partners Syngenta. Done: Picloram's affinity relative to that of eg IAA increases for AFB5 and is comparatively poor for TIR1. A novel SPR-based, ast throughput, in vitro screen has been established, tested and validated with data and many compounds exchanged with Syngenta.
Exploitation Route The information gained is pertinent to the discovery of novel agrochemicals and ways to manage arable cropping systems to improve food security. The results are being published in peer-reviewed scientific journals.

As an industrial partner, Syngenta are a conduit for industrial communication of the new understanding. We have a contract with Syngenta covering IP rights and exploitation.
Sectors Agriculture, Food and Drink,Chemicals,Environment

 
Description 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. Because of this pivotal role in plant development, synthetic auxins are used commercially in agriculture, mainly as herbicides, but also in horticultural applications. This project allowed us to define some of the basic rules of recognition of auxins and this information is helping to underpin new advances in understanding of how this hormone works and to inform molecule discovery programmes in agropharma businesses.
First Year Of Impact 2014
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Leeds 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution we have drafted grant proposals We have developed protein purification protocols We have purified target proteins and run activity assays We have drafted publications (2 published so far, more due)
Collaborator Contribution They have refined proposals They brought partner Syngenta to the table They created expression cassettes They input critical ideas
Impact Publications (2 so far), more pending Presentations to national and international audiences Initial IPA award New responsive mode award Industrial CASE studentship
Start Year 2006
 
Description Partnership with Syngenta 
Organisation Syngenta International AG
Department Syngenta Crop Protection
Country United Kingdom 
Sector Private 
PI Contribution Syngenta became a partner in the IPA grant proposal to BBSRC. The funded grant allowed the partnership to flourish. Contact has continued through a consultancy since the original project completion.
Collaborator Contribution Syngenta brought a series of specialists (chemists, molecular biologists, structural biologists etc) to review and planning meetings They supplied recombinant baculovirus lines, which included cloning and preparation of transfer vectors. They supplied fine chemicals
Impact Joint publication Multidisciplinary (chemistry, biology, protein chemistry, agronomy)
Start Year 2008
 
Description Biochem Soc 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact discussions led to improved efficiency

new network links formed
Year(s) Of Engagement Activity 2010
 
Description Euphorus 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact workshops for growers sparked discussions and sharing of information

press releases and reports
Year(s) Of Engagement Activity 2011,2012
 
Description Foresight Brussels 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact talk sparked questions and discussion

Briefing paper developed
Year(s) Of Engagement Activity 2010
 
Description Gatsby 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Talk and open discussion forum

many students become engaged with plant sciences
Year(s) Of Engagement Activity 2009
 
Description Ghent ISHS 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact talk followed by questions and discussions

led to application for joint EU funding and a research partnership
Year(s) Of Engagement Activity 2011
 
Description Host commonwealth fellow 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Training selected fellows in new technologies and providing skills and experience for subsequent teaching

Two Fellows returned to India to pursue teaching careers with improved understanding and experience
Year(s) Of Engagement Activity 2010,2011,2013,2014
 
Description OPen days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact engagement sparked applications for degrees in Biosciences

engagement left visitors enthused
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013
 
Description Rotary talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact talk followed by discussions

Public enthused and questioning.
Year(s) Of Engagement Activity 2010,2011,2013
 
Description SEB conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact talk and posters led to formative discussions, partnerships and new ideas

Higher number of requests for collaboration
Year(s) Of Engagement Activity 2010,2011,2012
 
Description School talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact talk sparked discussions and questions

invited back
Year(s) Of Engagement Activity 2010
 
Description work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact students made informed decisions about degree courses and career paths

the students involved chose science courses at university
Year(s) Of Engagement Activity 2009,2011