Periodic Reporting for period 3 - PCOMOD (Targeting the Plant Cysteine Oxidases to Regulate Plant Stress Tolerance)
Période du rapport: 2023-04-01 au 2024-09-30
Population growth and climate change mean that food security is an emerging global challenge. Crop loss due to flood, drought and other weather extremes is something that disproportionately affects the world's poor, but also has widespread international impact. There is an immediate and urgent need to develop tools and strategies to improve crop tolerance to such abiotic stress.
One effective mechanism towards this goal is molecular engineering of crops to withstand prolonged abiotic stress. Group VII Ethylene Response transcription Factors (ERF-VIIs) have a key role in plant stress tolerance, in particular flooding but also salinity, high temperature, drought and oxidative stress. ERF-VIIs are readily degraded, but their stabilisation has led to improved flood tolerance in model plants and crops. Consequently, ERF-VIIs are focal points for engineering abiotic stress resistance in crops.
ERF-VII degradation is triggered by the action of Plant Cysteine Oxidase enzymes (PCOs). These enzymes are oxygen-dependent; this means that ERF-VIIs are degraded in the presence of oxygen, but stabilised upon reduced oxygen availability, as is the case when plants become flooded. Manipulation of PCO activity is therefore a feasible mechanism to increase ERF-VII stability. However, complete and permanent inhibition of PCO function is detrimental to plant health, likely because of a role for these enzymes in other biological pathways. My team is therefore seeking to target PCO activity in a very specific manner. We are pursuing both chemical inhibition of PCO function which can be applied in a temporary manner, and targeted engineering of PCO function to change its oxygen sensitivity and/or substrate selectivity. This requires a detailed knowledge of the structure and function of these enzymes. Furthermore we are trying to understand confounding factors which can influence PCO-mediated ERF-VII degradation, for example the influence of reactive oxygen and reactive nitrogen species. With this knowledge in hand we can generate PCO variants with altered properties which result in improved flood tolerance. We will validate these biochemically before introducing them into model plants and crops.
One effective mechanism towards this goal is molecular engineering of crops to withstand prolonged abiotic stress. Group VII Ethylene Response transcription Factors (ERF-VIIs) have a key role in plant stress tolerance, in particular flooding but also salinity, high temperature, drought and oxidative stress. ERF-VIIs are readily degraded, but their stabilisation has led to improved flood tolerance in model plants and crops. Consequently, ERF-VIIs are focal points for engineering abiotic stress resistance in crops.
ERF-VII degradation is triggered by the action of Plant Cysteine Oxidase enzymes (PCOs). These enzymes are oxygen-dependent; this means that ERF-VIIs are degraded in the presence of oxygen, but stabilised upon reduced oxygen availability, as is the case when plants become flooded. Manipulation of PCO activity is therefore a feasible mechanism to increase ERF-VII stability. However, complete and permanent inhibition of PCO function is detrimental to plant health, likely because of a role for these enzymes in other biological pathways. My team is therefore seeking to target PCO activity in a very specific manner. We are pursuing both chemical inhibition of PCO function which can be applied in a temporary manner, and targeted engineering of PCO function to change its oxygen sensitivity and/or substrate selectivity. This requires a detailed knowledge of the structure and function of these enzymes. Furthermore we are trying to understand confounding factors which can influence PCO-mediated ERF-VII degradation, for example the influence of reactive oxygen and reactive nitrogen species. With this knowledge in hand we can generate PCO variants with altered properties which result in improved flood tolerance. We will validate these biochemically before introducing them into model plants and crops.
1. Temporary inhibition of PCO activity: We are screening a range of potential small molecule and peptidomimetic inhibitors of PCO activity, both in silico, in vitro and in vivo; some of these are revealing interesting features of the PCO catalytic mechanism.
2. Understanding PCO interaction with known and novel substrates: Kinetic investigation of all PCO s from Arabidopsis towards all known substrates is revealing which are the most kinetically competent targets; we are now seeking to understand how the molecular interactions between enzymes and substrates enable these kinetic preferences. Modelling work has suggested some key areas of interaction and mutagenesis of these parts of the enzymes is underway. We are also working to find ways to understand the molecular interactions involved when PCO binds its substrates, which will reveal where to concentrate our molecular engineering efforts; currently we are optimising conditions to enable crystallisation of a PCO:substrate complex. Finally, we are using molecular probes to help identify novel PCO substrates from plant tissue; this requires optimisation of experimental conditions to ensure PCO substrates can accumulate and be identified.
3. Understanding how reactive oxygen species (ROS) and reactive nitrogen species (RNS) impact on PCO function: We are conducting biochemical analysis to define and quantify the effects of ROS and RNS on both ERF-VII oxidation status and PCO function. We are currently working to correlate biochemical and biological observations.
4. Targeted engineering of PCO function to generate variants with altered properties: We have published the results of an initial screen of selected PCO variants, some of which showed dramatic differences in function compared to the wild type enzyme, including in a 4pco knockout model plant. We have produced a further suite of variant enzymes, based on comparison with the active sites of other thiol dioxygenases, and are screening these for differences in oxygen sensitivity and substrate selectivity. Recently reported data combined with molecular dynamic studies are suggesting novel areas of interest to examine in PCO4 regarding interactions with oxygen. We are therefore generating additional variants to test these novel hypotheses. We are concurrently working with in vivo models to introduce variants of interest into a biological context and examine the effects of this variant on plant phenotype and stress tolerance.
2. Understanding PCO interaction with known and novel substrates: Kinetic investigation of all PCO s from Arabidopsis towards all known substrates is revealing which are the most kinetically competent targets; we are now seeking to understand how the molecular interactions between enzymes and substrates enable these kinetic preferences. Modelling work has suggested some key areas of interaction and mutagenesis of these parts of the enzymes is underway. We are also working to find ways to understand the molecular interactions involved when PCO binds its substrates, which will reveal where to concentrate our molecular engineering efforts; currently we are optimising conditions to enable crystallisation of a PCO:substrate complex. Finally, we are using molecular probes to help identify novel PCO substrates from plant tissue; this requires optimisation of experimental conditions to ensure PCO substrates can accumulate and be identified.
3. Understanding how reactive oxygen species (ROS) and reactive nitrogen species (RNS) impact on PCO function: We are conducting biochemical analysis to define and quantify the effects of ROS and RNS on both ERF-VII oxidation status and PCO function. We are currently working to correlate biochemical and biological observations.
4. Targeted engineering of PCO function to generate variants with altered properties: We have published the results of an initial screen of selected PCO variants, some of which showed dramatic differences in function compared to the wild type enzyme, including in a 4pco knockout model plant. We have produced a further suite of variant enzymes, based on comparison with the active sites of other thiol dioxygenases, and are screening these for differences in oxygen sensitivity and substrate selectivity. Recently reported data combined with molecular dynamic studies are suggesting novel areas of interest to examine in PCO4 regarding interactions with oxygen. We are therefore generating additional variants to test these novel hypotheses. We are concurrently working with in vivo models to introduce variants of interest into a biological context and examine the effects of this variant on plant phenotype and stress tolerance.
We are developing a rigorous understanding of structure-function correlations for the PCO enzymes through our biochemical work as well as of ways in which PCO function can be inhibited. Our published work (White M et al (2020) Proc Natl Acad Sci 117: 23140) has demonstrated a correlation between the biochemical results we observe when we manipulate PCO structure and function and the biological outcome when the same changes are made to PCO in the context of a plant model. This provides a platform to study a range of PCO variants in planta with the confidence that the observed effects are likely directly due to the manipulation of the PCO enzymes. We are now in the process of implementing our biochemical findings into biological models; by the end of the study we should be able to correlate a range of impacts on PCO function with biological outcomes.