Understanding specificity in auxin perception

背景：植物的生长和发育是通过激素的活动以一种确定的、动态的方式控制的。荷尔蒙是一种可移动的分子，它携带信号来协调生长并启动对环境提示的反应。生长素是植物中一种非常重要的激素，因为它几乎参与了植物生活的方方面面，从胚胎发育到对重力的反应。为了让生长素触发所有这些反应，它需要一个受体，一种与生长素结合的蛋白质，在这样做的过程中扮演着分子开关的角色，启动一系列事件，最终导致植物发育的适当变化。我们最近发现了一种名为TIR1的蛋白质是生长素受体，这个提议描述了一系列实验，允许我们将整个植物对生长素的反应与TIR1感知生长素的分子事件联系起来。目的和目的：如上所述，生长素参与许多非常不同的发育反应。因此，植物生物学中的一个重要问题是：这一个分子信号如何正确地产生如此多的结果？在模式植物拟南芥中，TIR1是相关生长素受体小家族的成员之一，很明显，这些家族成员的受体活性存在差异。为了开始了解这些差异，一旦我们描述了生长素与TIR1的结合，我们将比较与每个家族成员的结合速度和亲和力，以确定这些差异的分子基础。一旦生长素与TIR1及其家族成员结合，它们就会结合另一个蛋白质家族，称为AUX/IAA，然后标记这些蛋白质进行降解。这种降解允许生长素调节的基因被激活，然后进行一连串的二级反应。为了确定每个受体是否对这些AUX/IAA靶标有偏好，我们将测量在生长素存在和不存在的情况下与一组具有代表性的AUX/IAA结合和解离的反应速率。这些数据将给我们一个相互作用偏好的矩阵，并表明特异性的分子基础，在生长素存在的情况下具有高亲和力可能会最大化这个目标被降解的机会。为了确保这些测量在完整的活植物中是相关的，这些目标蛋白质的寿命将被测量。我们还将产生缺乏特定受体蛋白的拟南芥的突变版本，或呈现受体的改变版本，并将这些植物的生长素响应与它们的基因组成和我们的生化数据联系起来。这些互补的数据集将增加稳健性，并允许我们量化受体复合体的选择性在指定特定生长素反应中所起的作用。潜在的应用和好处：以上收集的数据都与天然植物生长素IAA的作用有关。然而，有许多合成生长素用于研究和农业，它们各自的活性略有不同。农业用途的例子包括处理花卉、水果和坚果，也用作选择性除草剂。奥辛克除草剂杀死的是阔叶植物，而不是谷类和草，具有很大的农业价值。为了解决这些生长素是如何选择性的，我们将在谷物版本的TIR1上进行实验，并比较遗传和生化上的相似和差异。此外，使用这两种类型的受体，我们将建立一个测试系统，以帮助设计和筛选具有生长素样行为或拮抗行为的新化合物，目的是创造新一代安全、选择性和低剂量的农具。

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.