Award details

Critical Roles for Cytochromes P450 in Sustainable Wheat Production

Reference	BB/S005617/1
Principal Investigator / Supervisor	Professor Robert Edwards
Co-Investigators / Co-Supervisors	Dr Melissa Brazier-Hicks
Institution	Newcastle University
Department	Sch of Natural & Environmental Sciences
Funding type	Research
Value (£)	585,557
Status	Completed
Type	Research Grant
Start date	01/04/2019
End date	31/07/2022
Duration	40 months

Abstract

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for the primary detoxification of selective herbicides in wheat and competing weeds. In this project we will characterise the key CYPs active toward a range of herbicide chemistries in wheat and the problem wild grasses blackgrass, ryegrass, wild oat and sterile brome. Particular attention will be focussed on the CYPs that underpin the elevated tolerance to herbicides invoked by treatment with safeners in wheat (but not in weeds), and those enzymes associated with metabolism-based multiple herbicide resistance (MHR) in the wild grasses. CYP genes will be identified through next generation sequencing and enzymes of interest expressed and screened for their ability to metabolise herbicides using a yeast expression system, coupled to LC-MS detection; a platform that has been developed by the applicants for the routine screening of CYPs. Having linked expression to function, we will then assess the roles of CYPs in the responses of safeners and MHR using a combination of transcriptomics and proteomics, the latter being an important filter in identifying proteins rather than genes showing altered expression in planta. We will then examine the link between CYP expression profiles in MHR weed populations which are known to show variations in their sensitivity to different herbicides, linking this to the detoxification of the respective chemistries. The expression of key CYPs in wheat, blackgrass and ryegrass plants will then be studied by immunolocalisation and the functioning of these enzymes with their cognate glucosyltransferases and ATP-binding cassette transporters kinetically determined by transient co-expression in Nicotiana. Finally, we will screen for natural product substrates for CYPs linked to safening and MHR using LC-MS metabolomic screening linked to catalytic assays and metabolic profiling following of CYP knock-downs.

Summary

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.

Impact Summary

The impacts of this work are grounded in its foundational research that link to practical crop protection and as such, they focus on commercial outputs that are wealth creating, knowledge exchange with professionals working in the agricultural and chemical industries and engagement with members of the public interested in learning more about the pros and cons of pesticides. The commercial impact of the projects includes the national benefits of the University partner working with one of the world's largest companies in operating a major herbicide development research facility here in the UK. The outputs from the project directly feed into the value of Syngenta being based in the UK with it close proximity to specialist academic groups working on herbicide biochemistry. Furthermore the project and its outputs are particularly impactfull to the UK, as they direct the research focus of the industry partner onto a crop protection issue in wheat, the dominant national arable crop. In the wider commercial setting, the project will benefit from the close ties between the university research group and the DBeiS funded agritech Centre for Crop Health and Protection (CHAP), of which Newcastle is a founding member. CHAP offers the potential for rapid networking with other industry partners who might benefit from the technical approach adopted in the project. Through subsequent joint projects, these further collaborations will then build commercializable opportunities in smaller and more specialist crop protection companies, who by adopting similar approaches in understanding the roles of detoxification, will accelerate new lead development. In terms of knowledge exchange, advantage will be taken of the unique resources established at Newcastle in operating the CHAP mobile laboratories that bring the science from the laboratory to the farming industry. By demonstrating similar innovation at field trials and trade and agricultural shows, we have established an effective route to fast-track knowledge of new agritechnologies to the farming sector. As an added bonus and example of the value of two-way knowledge exchange these fora are instrumental in gaining valuable insights into new weed control issues emerging on-farm, that are suggestive of interesting science we can direct solutions to. Using approaches we have already tried and tested, a tiered approach is envisaged in which a set of interested stakeholders will be established through targeting the different farm industry practitioners attending the different forums. Finally we will engage with the public, targeting those most likely to have an interest in agriculture and food production by targeting county shows and using forums such as the regional Farmer Scientist Network, of which the Principal Investigator is the current chair, to host seminars and information events. This will culminate in a specific debate and associated workshop at the University research farm on the 'Future for Agrochemicals in the UK' , with is policy facing implications post-Brexit, attended by practitioners and environmentalists.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Industrial Partnership Award (IPA)

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