Reactive oxygen species (ROS) and ROS processing enzymes and proteins have been co-opted during evolution into the regulation of plant growth, development and defence. ROS and oxidation/reduction signals arising from metabolism or phytohormone-mediated processes control almost every aspect of plant biology. This project is part of a larger interdisciplinary project that addresses the role of redox dependent post translational modifications on catalase. Specifically how these modifications affect interaction with different protein binding partners to control subcellular localisation of catalase, and how this in turn affects the balance between growth and programmed cell death. The specific objectives for PhD sub project are as follows:
1. Evaluate the suitability of protoplasts as an experimental system. (0-9 months)
Protoplasts are easy to obtain, uniform and easy to manipulate but represent a wounded system. Initial experiments will explore the extent to which the redox state of Arabidopsis cells is changed by the process of protoplast formation using Arabidopsis lines that express redox sensitive (RO-) green fluorescent protein (GFP). Working with the post doc the effect of protoplasting on the cysteine oxidation state of catalase compared to the seedlings from which the protoplast are derived will be established. This will determine whether protoplasts or seedlings are used in subsequent experiments
2. Genetic and chemical perturbation of redox state in chosen system. (9-18 months)
Using a range of mutants (e.g vtc2, cad2-1, ntra and ntrb) and chemical inhibitors (e.g paraquat, SHAM, Antimycin A) that are known to perturb redox state we will determine the associated changes in the abundance of key marker transcripts for defence and cell death pathways. This will guide the selection of treatments to be used in the redox proteomics experiments which will be performed by the post doc.
3. Determine the effect of redox perturbation on catalase localisation (18-30 months)
Using seedlings from transgenic plants that co express sfGFP1-10 and CATGFP11 (or protoplasts derived from these plants) the effect of the chemical treatments established in (2) on the location of catalase will be determined using live cell imaging confocal microscopy. Mutations of key residues identified by the proteomics and protein interaction experiments carried out by the post doc will be tested for effects on catalase location
4. Determine the effect of catalase relocation to the nucleus (18-40 months)
Transgenic plants expressing catalase with a nuclear localisation signal and a control catalytically inactive catalase targeted to the nucleus will be produced and characterised in terms of growth phenotype, including developmental and cell death phenotypes, redox perturbation and changes in expression of the same key marker transcripts of defence and cell death pathways used in (2).
5. Thesis dissertation (months 40-48)
These studies will establish 1) whether catalase isoforms relocate within plant cells upon perturbation of redox state, and 2) whether forcibly relocating catalase to the nucleus as occurs with some plant pathogens affects developmental and cell death decisions and 3) whether catalase enzymatic activity is required or whether this is a moonlighting function.