Understanding specificity in auxin perception

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.

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