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The development of tools and resources for the identification of inhibitors of pigment biosynthetic pathways in the chloroplast
Closing date
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Supervisors
Professor Paul Fraser, p.fraser@rhul.ac.uk, Professor of Biochemistry, Royal Holloway, University of London
Project Details

Background. The global population is projected to increase by 30% to 9.2 billion in 2050. In order to feed this population a 70% increase in food production is required [1]. This increased production must occur with less land availability, less water, less energy input, fertilizers and chemicals. Although environmental concerns surround the use of herbicides and there are moves towards the use of biologicals [2], herbicides will remain an essential component of integrated pesticide management practices; if food production is to meet demand. No new herbicides have been commercialised with a novel mode of action for over 30 years now [3], hence the increased threat from resistance, and the need to be able to focus efforts on Modes of action (MOA) at an earlier stage in herbicide discovery. Bleaching herbicides are designated as chemicals that cause white bleaching in leaf material. Their effectiveness resides in their ability to cause light-dependant generation of singlet oxygen which damages lipids and proteins leading to plant death [4]. Bleaching herbicides are chemically diverse, which reflects the different chloroplastic biosynthetic pathways and the individual components within these pathways they effect. The common phenotypic traits between bleaching herbicides often causes confusion in assigning MOAs to specific herbicidal inhibitors. For example, Norflurazon, is an inhibitor of the carotenoid biosynthesis, specifically the enzyme phytoene desaturase (PDS), [5]. The carotenogenic enzyme phytoene desaturase is responsible for the desaturation of 15-cis phytoene to cis -z-carotene via phytofluene. Inhibition of PDS results in the precursor phytoene accumulating. However, phytoene can also accumulate in vegetative tissues from the inhibition of plastoquinone/tocopherol biosynthesis. This is because plastoquinone acts as a cofactor involved in phytoene desaturation. . Therefore it is important to have robust procedures in place to differentiate the mode of action used by the different existing and potentially new inhibitors. This includes confirmatory enzyme assay procedures.

 

Proposed programme of work. The proposed studentship programme will develop enabling technologies/procedures to ensure the identification of carotenoid biosynthesis inhibitors, while confirming and characterising inhibition at the enzyme level. This academic/Syngenta alliance will establish a pipeline of procedures for the unambiguous identification of inhibitors affecting the carotenoid biosynthetic pathway components. To achieve this goal: (i) Robust HPLC/UPLC methodologies enabling; (i) the differentiation of inhibitors affecting carotenoid formation from other pathways leading to bleaching and (ii) detailed separations of phytoene desaturase, precursors and products, including geometric isomers; (ii) procedures for the preparation of authentic carotenes/carotenoids to be used as analytical standards and enzyme assay precursors; (iii) In vitro phytoene desaturase assays for different weed species. The system will involve crude E. coli preparations and the purified enzyme, utilising direct precursor application assays, to enable a higher-throughput biochemical assay and (iv) validation of phytoene desaturase activity assays through; (i) the determination of kinetic properties and mode of inhibition for different inhibitor chemical classes.

 

The host laboratory. Prof Fraser’s group of 20 researchers is a vibrant, well equipped and funded environment. The group has dedicated analytical apparatus; GC-MS (x3), GC-FID, HPLC-PDA (x2), HPLC-PDA-radiodetector, UPLC-PDA and real-time PCR machines. Plant growth facilities include glasshouses, controlled tissue culture room, chambers and polytunnels. A state of the art analytical suite has been established with complementary MS platforms; 2x LC-accurate mass Q-TOF-MS/MS (Agilent IM-6560, Agilent iFunnel 6550), serving both metabolomic and proteomic applications and LC-QQQ -MS (Agilent 6470) has been established.

Industrial partner. Syngenta is one of the world’s leading Agri-businesses with a history of commercialising new crop protection and seed products. Innovative research is the cornstone to Syngenta’s strategy. In 2019 Syngenta’s annual sales in crop protection were $10.6 bn, with Syngenta re-investing up to 10% of its annual turnover in R & D.

The proposed studentship is directly aligned with Syngenta’s interest in crop protection, especially herbicides. Syngenta sells over 100 different crop protection brands to farmers worldwide (www.syngenta.com/what-we-do/crops-and-products/brands). No herbicide with a new MOA has been commericalised for thirty years, hence the threat of resistance and need to identify novel MOAs at an early stage in herbicide discovery.

References

1]. FAO (2009) Feeding the world in 2050. World agricultural summit on food security 16–18 November 2009. Food and Agriculture Organization of the United Nations, Rome,

[2]. Duke et al (2019) Pest Manag. Sci. 75, 314,

[3]. Duke, (2012) Pest Manag. Sci. 68, 505.

[4]. Ahrens, Herbicide Handbook 7th Ed., (1994) Weed Science Society of America, Champaign, IL,

[5]. Brausemann et al (2017) Structure 25, 1222.

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