Characterisation of the unique lambda class of plant glutathione transferases

谷胱甘肽转移酶（GST）是一组适应性强的蛋白质，存在于所有需氧生物体中，在代谢和对抗氧化应激中具有多种作用。在人类中，它们的保护活性是公认的，GST在解毒摄入的天然和合成毒素方面很重要。有时，这种活性是有问题的，因为肿瘤中高水平的GST可以阻止化疗药物杀死癌细胞。因此，药物化学家已经开发了一系列选择性抑制剂，以抑制癌组织中存在的GST，而不是健康组织中存在的GST，从而克服耐药性。我们对GST的兴趣与这些蛋白在植物中的多种功能有关。在进化过程中，植物GST已被招募以在外源化合物解毒、氨基酸和抗氧化剂代谢以及细胞周围反应性天然产物的运输中发挥多种作用。然而，这些功能是知之甚少，和复杂的调节GST在植物发育和暴露于压力表明，这些酶的许多其他重要功能尚未确定。一个很好的例子是lambda类GST（= GSTL）。这些植物特异性蛋白质在小麦中被一组称为除草剂安全剂的农用化学品上调。然后，它们的表达增加与除草剂耐受性增强相关。更令人不安的是，当野草开始过度表达GSTL时，它们也会对除草剂产生抗性，这可能导致杂草由于失去选择性化学控制而与谷类作物竞争。因此，确定这些GSTL在谷物和杂草中的作用并利用这些信息制定新的作物保护策略将是非常有趣的。然而，小麦和禾本科杂草都含有编码GSTL的多个基因，建立它们的功能是有问题的，特别是在我们无法获得必要的遗传信息和工具来测试它们的活性的杂草中。相反，从药物化学的领先地位，我们建议测试GSTL功能使用化学探针，选择性地抑制这些酶，从而破坏它们的功能。通过测试针对不同GST库的一组化学物质，我们已经鉴定了一类选择性地使GSTL失活的抑制剂。以此为起点，我们建议合成一系列GSTL抑制剂，并通过在模式植物拟南芥中使用它们来测试它们为我们提供关于GSTL功能的准确信息，在拟南芥中，我们也有能力使用常规遗传方法破坏这些蛋白质的表达。在每种情况下，我们将用抑制剂处理植物，并寻找代谢物和蛋白质的变化。通过显示抑制剂与当相应的GSTL基因被“敲除”时所看到的那些产生相同的生化反应，我们有一种有力地验证其用途的方法。一旦确信它们的选择性，我们就可以在小麦和禾本科杂草中使用抑制剂，分别确定GSTL在除草剂安全性和抗性中的作用。因此，该项目代表了一个有用的例子，说明我们如何使用来自研究模式植物基因功能的信息和工具来研究作物和杂草的重要农艺性状。这项工作的长期目标是利用这些信息来抵消禾本科杂草的除草剂抗性，提高谷物的抗逆性和作物产量。

The glutathione transferases (GSTs) are an adaptable group of proteins found in all aerobic organisms with multiple roles in metabolism and counteracting oxidative stress. In man, their protective activity is well recognised, with GSTs being important in detoxifying ingested natural and synthetic toxins. On occasions, this activity is problematic, as high levels of GSTs in tumours can prevent chemotherapeutic agents from killing cancer cells. As such medicinal chemists have developed a range of selective inhibitors to inactivate GSTs present in carcinomas, but not in healthy tissue, thereby over-coming drug resistance. Our own interests in GSTs relate to the multiple functions of these proteins in plants. In the course of evolution, plant GSTs have been recruited to fulfil multiple roles in foreign compound detoxification, amino acid and antioxidant metabolism and the transport of reactive natural products around the cell. However, these functions are poorly understood, and the complex regulation of GSTs during plant development and exposure to stress suggests that many other important functions for these enzymes are yet to be determined. An excellent example is seen with the lambda class GSTs (=GSTLs). These plant specific proteins are up-regulated in wheat by a group of agrochemicals called herbicide safeners. Their increased expression is then associated with an enhanced tolerance of herbicides. More disturbingly, when wild grasses start to over-express GSTLs they also become resistant to herbicides and this can result in the weeds out-competing the cereal crops due to the loss of selective chemical control. It would therefore be very interesting to determine the role of these GSTLs in both cereals and weeds and use this information to develop new crop protection strategies. However, both wheat and grass weeds contain multiple genes encoding GSTLs and establishing their functions is problematic, especially in weeds where we do not have access to the necessary genetic information and tools to test their activities. Instead, taking a lead from medicinal chemistry, we propose to test for GSTL function using chemical probes which selectively inhibit these enzymes, thereby disrupting their function. By testing a panel of chemistries against a library of different GSTs, we have identified a class of inhibitor which selectively inactivates GSTLs. Using this as a starting point, we propose to synthesise a series of GSTL inhibitors and test that they give us accurate information about GSTL function by using them in the model plant Arabidopsis thaliana, where we also have the ability to disrupt the expression of these proteins using conventional genetic methods. In each case we will treat the plants with the inhibitors and look for changes in metabolites and proteins. By showing that the inhibitors give the same biochemical responses to those seen when the respective GSTL gene is 'knocked out' we have a means of robustly validating their use. Once confident of their selectivity, we can then use the inhibitors in wheat and grass weeds, determining the roles of GSTLs in herbicide safening and resistance respectively. This project therefore represents a useful example of how we can use information and tools derived from investigating the functions of genes in model plants to studying important agronomic traits in crops and weeds. The long-term objectives of this work are to use this information to counteract herbicide resistance in grass weeds and improve stress tolerance and crop yields in cereals.