DUNNE, JON-PAUL,ANTHONY (2022) Beyond the Shikimate pathway: The novel role of photosynthetic electron transport in mediating hormesis and lethal responses to glyphosate. Masters thesis, Durham University.
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Author-imposed embargo until 22 September 2024.
Glyphosate is the most widely used herbicide in agriculture, with a truly global market dominance. The existence of a range of glyphosate-resistant crops has led to a surge in glyphosate usage in countries where genetically modified crops are deregulated. This makes glyphosate a key environmental chemical pollutant sourced from agriculture and understanding its impact on non-target organisms is paramount. With this high level of usage, an inevitably increasing number of glyphosate-resistant weeds emerged over the past few decades. Therefore, there is pressing need to design appropriate countermeasures for controlling such weeds to protect food production systems. This requires a clear understanding of how glyphosate kills cells. Glyphosate inhibits the shikimate pathway, resulting in aromatic amino acid depletion and subsequent protein starvation. However, recent studies identified a clear link between glyphosate toxicity and exposure to high levels of light, suggesting that glyphosate-induced cell death involves mechanisms far more complex than imagined previously. This presents a key research question, what is the mechanism underpinning glyphosate toxicity? Remarkably, shikimate pathway-possessing organisms bereft of chloroplasts have very high glyphosate lethal dose concentration, in comparison to equivalent organisms with chloroplasts. Therefore, our group proposed the hypothesis that glyphosate inhibition of the shikimate pathway activates cell death through generation of damaging reactive oxygen species (ROS) triggered by constricting photosynthetic electron transport (PET) in the presence of light. To test this hypothesis, we used two model organisms (Synechocystis sp. PCC 6803 and Chlamydomonas reinhardtii CCAP 11/32B) to evaluate the impact of glyphosate on cell growth. Growth assays revealed two distinct responses to glyphosate viz. (i) hormesis, characterised by activation of growth at sublethal glyphosate concentrations and (ii) growth arrest and cell death at lethal doses. Crucially, whether cells responded by hormesis or cell death was controlled by the presence of as yet unidentified extracellular signals secreted by cells into the growth medium. To unravel the molecular basis for the hormetic and lethal responses, a global proteomic analysis of glyphosate-treated Synechocystis cells was conducted using isobaric tags for relative and absolute quantitation (iTRAQ) technology. This highlighted the key proteins differentially-expressed in the two responses. Gene ontology analysis revealed that a majority of the identified proteins are photosynthetic proteins located in thylakoids. The overall pattern emerging from the protein data is that the lethal response was associated with a collapse in the abundance of key components of the electron transport chain, while in hormesis the same components remained unaffected or were increased. Furthermore, the protein profile in the hormetic response was consistent with redirecting fixed carbon away from the inhibited shikimate pathway to re-establish photosynthetic electron transport flow to prevent reduction of O2 and ROS generation. This study has revealed the light driven mechanism of glyphosate toxicity. This entails excessive ROS generation by the photosystems regulated by complex metabolic feedback signalling centred around reconfiguration of metabolite distribution from the Calvin-Benson cycle. The implications of this research are 3-fold. First, redirecting Calvin-Benson cycle metabolites to alternative pathways constitutes a viable escape from toxicity and may be a potential mechanism of glyphosate resistance in agriculture. Secondly, hormesis driven by secreted extracellular signals could be the basis for pollution-induced algal blooms, which have far-reaching environmental consequences. Finally, these results could be exploited in industrial algal cultivation to generate biomass for commercial applications.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||26 Sep 2022 12:39|