The characterisation of bleaching inhibitors

In order to sustain a predicted global population of 9.0 billion by 2050, it is estimated that a 70% increase in food production is required. These increases must be achieved with less land, water, energy input, fertilisers and chemicals. Herbicides will remain part of the food security solution despite stricter regulations on the use of chemicals and more prevalent resistance or tolerance to herbicides. Herbicide discovery and determination of their mode of action (MOA) is often very expensive and labour-intensive, with phenotypes from whole organism screening not always revealing the target site. Very few new herbicides have been discovered over the last thirty years. Bleaching herbicides enable the light activation of reactive oxygen species in leaf material upon application. However, diverse chemistries can be responsible for this bleaching. This phenotypic similarity makes it difficult to determine the precise molecular target, as multiple herbicide classes can induce bleaching through distinct mechanisms. The present objectives for this project will be to (i) use metabolite profiling to differentiate between the chemically diverse classes of bleaching herbicides, (ii) identify potential secondary targets of inhibition from adaptive mechanisms and (iii) concurrently, to establish an in vitro enzyme assay for the desaturation of phytoene to validate phytoene desaturase (PDS) as the target site.

Several sources of PDSs were used, including plant-type enzymes from Synechococcus and resistant mutants. The results from investigations into PDS activity by in vivo assays generated an active recombinant plant-type PDS in Escherichia coli (E. coli). Following the in vivo activity for PDS, yielding expected desaturation products phytofluene and ζ-carotene when co-transformed with a phytoene producer, enabling the establishment of an in vitro enzyme assay, characterised in a cell-free system. The enzyme activity was characterised, and the following properties were identified. Notably, increasing the lipid concentration to 25 mg/ml increases product formation by 10- to 100-fold. Additionally, a designed mutant PDS exhibited increased desaturated product accumulation compared to the wild-type enzyme at 100 µM Norflurazon treatment, providing evidence of resistance.
To conclude, this work culminated in generating a robust in vitro enzyme assay for PDS. This assay enabled the accurate determination of inhibition by target molecules and validated the suspected MOA proposed by in vivo treated plant metabolic profiling data. Thus, it provides a selection of valuable tools for determining PDS inhibition and carotenoid biosynthesis.