Fluorescent chemical tools to unravel the mechanisms underpinning vacuolar sequestration in plants

Supervisors: Marc Vendrell, Karl Oparka

Project description:

BACKGROUND: Every year, thousands of agrochemicals are produced with potential herbicidal, insecticidal and fungicidal properties. Many of these compounds fail because of our lack of knowledge on how agrochemicals transport and accumulate in plants. In particular, our group is interested in studying the chemical mechanisms by which agrochemicals can be sequestered into vacuoles, limiting their action. The mechanisms by which some of the most successful commercial herbicides (e.g. glyphosate and paraquat) are accumulated into the vacuoles are not fully understood. For example, glyphosate localizes partially in the vacuoles, with a major mechanism of resistance to glyphosate in weeds being increased vacuolar localization with a concurrent decrease in phloem mobility.

Our team has extensive expertise in the chemical synthesis and biological characterization of small fluorophores. These fluorophores include heterocyclic cores (e.g. coumarins), which share structural similarities with several agrochemicals. Notably, their inherent fluorescence properties allow us to monitor their localization directly in live cells or plants by simple fluorescence microscopy, opening a window of opportunities to investigate the mode of action (MoA) of agrochemical-like structures and the rules of engagement underpinning vacuolar sequestration. Using combinatorial chemistry methods, we will modify small fluorophores (initially coumarins and nitrobenzodiazoles but extendable to other cores) to accommodate wide structural diversity to cover broad physicochemical properties (i.e. clogP and pKa) and variations on chemical groups that we have recently identified as critical for vacuolar sequestration (e.g. free and substituted alcohols and phenols). These structures will also include weak carboxylic acids for enhanced diffusion through plants. The team also has extensive experience in microscopy-based assays using cell lines and models developed in the Oparka lab that will help to identify structures with and without resistance to vacuolar sequestration. Both predicted and experimental physicochemical properties (pKa, log P) for the chemical structures will be determined and we will generate physicochemical heat maps where groups or combinations of groups with resistance to vacuolar sequestration will be identified.

AIMS, METHODOLOGY AND IMPACT: The aim of this project is to identify key molecular features that determine the MoA and internalisation of agrochemicals into vacuoles. Collections of fluorophores resembling common cores found in some agrochemicals will be synthesized. Fluorescence measurements will enable systematic exploration of the structure-activity relationships (SAR) to compounds with resistance to vacuolar sequestration. Notably, the high spatial resolution achieved with fluorescence confocal microscopy will overcome some of the limitations of autoradiography or chromatography-based analysis, in which it is difficult to determine the localisation of chemicals with subcellular resolution. The identification of molecules that are not promptly sequestered into the vacuoles will have strong impact on the design of efficient agrochemicals with improved distribution profiles, accelerating the commercial development pipeline and helping to re-purpose valuable chemicals.