Award details

Selective chemical regulation of plant metabolism with herbicide safeners

Reference	BB/D005620/1
Principal Investigator / Supervisor	Professor Robert Edwards
Co-Investigators / Co-Supervisors	Professor David Hodgson, Professor Patrick Steel
Institution	Durham University
Department	Chemistry
Funding type	Research
Value (£)	382,065
Status	Completed
Type	Research Grant
Start date	01/04/2006
End date	31/03/2009
Duration	36 months

Abstract

In an interdisciplinary 3 year programme to be carried out by chemists and biologists it is proposed to identify the mechanisms by which herbicide safeners initiate the selective reprogramming of plant primary and secondary metabolism. This intervention is chemical- and species-specific and involves the transcriptional activation of key proteins involved in glutathione biosynthesis and herbicide metabolism in cereal crops. We have determined that this safening activity extends to the model plant Arabidopsis thaliana and encompasses changes in endogenous phenolic metabolism as well as xenobiotic biotransforming enzymes. Based on their chemistries, we can group major classes of cereal-specific safeners into 3 chemical reactivity groups (chemotypes), all of which are all capable of covalently modifying proteins. Uniquely, all 3 chemotypes are differentially active in Arabidopsis in inducing herbicde detoxifying enzymes, making this an ideal species for identifying initial events in safening. In our paradigm of safener action we propose that these compounds covalently modify reactive residues on regulatory proteins. These selective chemical modifications then initiate a series of signalling events which lead to the transcrptional activation of an important sub-set of the plant antioxidant response which can confer tolerance to toxic chemicals such as herbicides. To test this paradigm we will first identify a full set of biomarkers of safening in Arabidopsis based on directed and global proteomics screens as well as metabolic profiling of antioxidant metabolites to fully define the associated biochemical phenotype. In parallel, we will prepare safeners which have been tagged with biotin/ fluorophores to identify target proteins in Arabidopsis using a combination of in vitro and in vivo labelling, the latter using click-based chemistries. The tagged proteins will then be identified by MS-based proteomics and the importance of the proteins in safener-recognition tested bygenerating the respective knock-out mutants using either available T-DNA collections or through the use of inducible RNAi technology. To confirm the importance of the respective proteins in agronomically important plants, the safener-probes developed in Arabidopsis will be used in tagging experiments in cereals. Finally we will explore the selectivity in chemical intervention of safeners in Arabidopsis and cereals by generating a library of related chemistries and testing each compound for its ability to differentially induce the safening biomarkers. Specific programme objectives are 1. Definition of a full range of biomarkers for safening in Arabidopsis based on changes in xenome, proteome and metabolite profiles (0- 6 months). 2. Synthesis of first generation safener in vitro labelling probes (0- 12 months). 3. In vitro screening of safener probes in Arabidopsis and identification, cloning and characterisation of labelled proteins using proteomics (6- 15 months). 4. Generation and evaluation of click-labelling probes for in vivo labelling (12- 24 months) 5. Isolation/ generation of Arabidopsis safener-binding protein knock outs (12 / 24 months). 6. Generation and biological evaluation of safener analogue series using high-throughput screens in Arabidopsis (wild-type and knock outs) and cereals (24- 36 months) 7. Proteomic studies with in vitro and in vivo labelling probes in cereals (18 / 30 months)

Summary

Safeners are used in conjunction with herbicides in major cereal crops such as wheat, maize and rice to enhance their selective weed control. This is achieved by the safeners acting to increase the expression of herbicide detoxifying proteins by up-regulating the expression of the respective genes in the crop. Recently, we have determined that safeners work in a species specific manner, with each crop responding to a specific class of safener chemistry. We have also determined that the effects of safeners are not restricted to herbicide detoxifying proteins, but extend to a subtle modification of endogenous plant metabolism, notably through altering the production of phenolics and thiol antioxidants. Safeners are therefore bioactive synthetic compounds which are able to selectively reprogramme elements of plant metabolism which are important in counteracting stress caused by chemicals and/or oxidative damage. While the mechanism of safener action is partly understood at the level of the metabolic responses, nothing is known as to how these synthetic chemicals are recognised by plants to initiate specific cellular signalling events. Based on chemical principles, we propose that modern safeners can be grouped into three major classes of reactivity, all of which will lead to the selective covalent modification of reactive residues on the surface of proteins. We propose that such modification of regulatory proteins causes changes in their activity or interactions which initiate signalling pathways leading to the selective activation of genes encoding antioxidant proteins. The objectives of this programme are to use a combination of synthetic chemistry and biochemistry to identify which signalling-proteins become selectively labelled with safeners . This will entail synthesising safeners labelled with recognition tags and then using these 'chemical hooks' to identify the proteins targeted, thereby identifying how these chemical selectively intervene in plant biochemistry. We will concentrate our efforts on identifying these initial binding events in the model plant species Arabidopsis thaliana, which uniquely among the plants we have studied is selectively responsive to all three classes of safener chemistry. Using different synthetic strategies, the chemical probes will be designed to work in protein extracts and in whole plants, with the tagged proteins then identified from their sequence using methods based on mass spectrometry. As part of the programme we will confirm the roles of these proteins in safener-recognition in Arabidopsis by determining the effect of knocking-out their expression on their responses to the three chemistries under test. We will also determine whether or not similar proteins become labelled in cereals to confirm our results in agriculturally important species and develop chemical variants of existing safeners to determine how this affects their selective enhancement of antioxidant responses in Arabidopsis and the crop species. At the conclusion of our interdisciplinary programme we will have determined for the first time how an important group of agrochemicals exert their selective action in plants. Defining their site of action has important implications in identifying new generations of safeners with improved activities to ensure the future efficient and sustainable use of herbicides in crop protection. In addition, understanding how safeners work will allow us to extend their use in promoting resistance to chemical and oxidative stress in other plants which has potential for applications in bioremediating contaminated land.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Crop Science, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	Selective Chemical Intervention In Biological Systems (SCIBS) [2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search