Periodic Reporting for period 1 - PCOMOD (Targeting the Plant Cysteine Oxidases to Regulate Plant Stress Tolerance)
Reporting period: 2020-04-01 to 2021-09-30
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.
2. Understanding PCO interaction with known and novel substrates: We are underway with a kinetic investigation of all PCO s from Arabidopsis towards all known substrates; this will help identify the most biologically relevant PCO-catalysed reactions. 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; again, 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; these are ongoing.
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 are in the middle of producing a further suite of variant enzymes, based on comparison with the active sites of other thiol dioxygenases, in order to screen for differences in oxygen sensitivity and substrate selectivity; we will generate additional variants as structural and functional information is generated from other parts of the project. We have also started introducing an additional variant into a plant model to test the effects of this variant on plant phenotype and stress tolerance.