Critical Roles for Cytochromes P450 in Sustainable Wheat Production

小麦是英国的主要作物，也是全球重要的主食谷物，小麦的高效生产高度依赖于选择性除草剂的使用，这些除草剂控制竞争性杂草的生长，特别是麻烦的野草，如黑麦草、野燕麦、黑种草和雀麦。如果不能控制这些杂草，就会造成重大的作物损失，这可能会对已经面临困境的部门造成严重影响。被称为细胞色素P450（CYP）的酶超家族在控制除草剂选择性方面发挥着重要作用，因为它们可以通过催化氧化代谢来解毒各种化学物质，从而使化合物进一步修饰和安全储存。通常情况下，在小麦中，CYP快速解毒除草剂，而在竞争的禾本科植物中，这些反应发生得更慢，这是对杂草不同毒性的基础。这种选择性可以通过用安全剂处理小麦来进一步增强，安全剂是一种增强作物中除草剂表达并加速除草剂解毒的化合物。由于重复使用相对少量的化学物质，禾本科杂草已经开始进化出多重除草剂抗性（MHR），这是由于选择了具有提高的解毒酶表达的种群。在MHR的这些解毒过程中，CYP似乎起着主导作用。MHR现在在英国广泛存在，特别是在黑种草和黑麦草中，并造成主要作物损失，并威胁到冬小麦的持续生产。在这个项目中，我们将与农业化学公司先正达合作，在小麦和竞争性禾本科杂草中补充CYP，控制对除草剂的选择性和抗性。我们所采用的方法是基于申请人实验室的最新进展，其使得有可能常规地从植物克隆和表达CYP，并使用质谱的能力测定它们对除草剂的解毒活性以灵敏地检测代谢物。利用最新的RNA测序技术，我们将研究小麦和四种问题草中表达的MHR基因的范围，特别感兴趣的是与杂草中的MHR和小麦中的安全化相关的CYP。在酵母中表达编码序列后，我们将检测重组酶对除草剂的活性。有了这个特征CYP的清单，我们将研究它们在作物和杂草中的功能。优先考虑的问题将是哪些CYP与作物的安全化特别相关，杂草中的反应有什么差异，以及不同的CYP表达模式是否可以解释目前在野草中报道的MHR的不同亚型。然后，我们将研究这些酶在小麦和杂草中的表达，因为它与植物中除草剂解毒的发生有关，然后评估CYP如何与其他参与外源化合物代谢的酶和转运蛋白一起发挥作用。最后，我们将进行研究，寻找CYP的天然底物，前提是它们最初不是为了解毒除草剂而进化的。在该项目的结论，我们将在一个位置，第一次，在小麦和竞争杂草除草剂代谢选择性和抗性的CYP的作用进行概述。从这项研究中获得的信息将有助于开发新的选择性除草剂和抗性破坏增效剂，这是英国谷物行业现在迫切需要的，以抵消MHR草杂草的稳步上升，并提高英国小麦的生产力和盈利能力。

The efficient production of wheat, the major crop in the UK and a globally important staple cereal, is highly dependent on the use of selective herbicides that control the growth of competing weeds, particularly troublesome wild grasses such as ryegrass, wild oat, blackgrass and bromes. Failure to control these weeds causes major crop losses that can be crippling to an already hard-pressed sector. A superfamily of enzymes called cytochromes P450 (CYPs) play essential roles in controlling herbicide selectivity, as they can detoxify a wide range of chemistries through catalysing their oxidative metabolism, thereby allowing the compounds to be further modified and safely stored. Normally, in wheat, CYPs rapidly detoxify herbicides, whereas in the competing grasses these reactions occur more slowly and this is the basis of differential toxicity to the weeds. This selectivity can be further enhanced by treating the wheat with safeners, compounds that enhance CYP expression in crops and accelerate herbicide detoxification. Due to the repeated use of a relatively small number of chemistries, grass weeds have started to evolve multiple herbicide resistance (MHR), due to the selection of populations that have elevated expression of detoxifying enzymes. Among these detoxification processes in MHR, CYPs appear to play a dominant role. MHR is now widespread in the UK, particularly in blackgrass and ryegrass and is causing major crop losses and threatens the continued production of winter wheat. In this project, working with the agrochemical company Syngenta we will characterise the complement of CYPs in wheat and competing grass weeds that control selectivity and resistance to herbicides. The approach we have adopted is based on recent advance in the applicant's laboratory that have made it possible to routinely clone and express CYPs from plants and assay them for detoxifying activities toward herbicides using the power of mass spectrometry to sensitively detect metabolites. Using the latest RNA sequencing technology we will characterise the range of CYP genes expressed in wheat and the four problem grasses, with those CYPs associated with MHR in weeds and safening in wheat being of particular interest. After expressing the coding sequences in yeast, we will assay the recombinant enzymes for their activities toward herbicides. Armed with this inventory of characterised CYPs we will then investigate their functions in crops and weeds. Prioritised questions will be which CYPs are particularly associated with safening in crops and what differences there are with the responses seen in weeds and can different patterns of CYP expression account for the distinct sub-types of MHR now being reported in wild grasses. We will then investigate where these enzymes are expressed in wheat and weeds as it relates to where herbicide detoxification occurs in plants and then assess how CYPs function with other enzymes and transporters involved in foreign compound metabolism. Finally we will carry out studies to look for the natural substrates of CYPs, on the premise that they did not originally evolve to detoxify herbicides. At the conclusion of the project we will be in a position, for the first time, to overview the roles of CYPs in herbicide metabolism selectivity and resistance in wheat and competing weeds. The information derived from the study will help in the development of new selective herbicides and resistance busting synergists that are now desperately needed by the UK cereal industry to counteract the steady rise of MHR grass weeds and improve wheat productivity and profitability in the UK.