Probing the redox regulation of plant growth in response to environmental cues

Achieving global food security for a growing population under a changing climate and depleting resource base is amongst the greatest challenges of our time. Unlike animals, plants grow continuously, new cells providing the building blocks for growth. When plants perceive stress, specific signals block cell division and proliferation. Under extreme conditions this is beneficial, but accumulating evidence suggests that plants shut down cell division early as a precaution in response to stress. Therefore plants tend to stop growing, even though weather conditions are not really bad enough to require this drastic step. Sustaining the plant cell cycle and hence growth and crop yields under stress provides a significant opportunity to address this challenge and meet future food demand. While proliferative oxidative signals flow through intracellular signalling pathways to activate the cell-cycle, stress-related oxidative signals from within or outside the cell oppose proliferation. The aim of this project is to investigate how the mitotic cell cycle is regulated by environmental cues using state of the art molecular physiology and proteomic tools. Reduction-oxidation (redox) regulation is a crucial posttranslational mechanism (PTM) that controls protein functions. This project will use cutting‐edge reduction-oxidation (redox) proteomics technologies to characterise how environmental signals alter the redox state of dividing plant cells and identify the redox-sensitive proteins that are responsible for switching on the cell cycle in response to oxidative activation and off in response to over-oxidation. The specific objectives of this proposal are:
1. To characterize the site-specific and quantitative cysteine oxidation of the proteome in the G1 and G2 phases of the cell cycle in Arabidopsis thaliana cell cultures in the absence of oxidative stress. A sub-set of the redox modified proteins will be identified that have a profound and measurable influence on cell cycle regulation.
2. To determine the protein interaction networks modified by redox PTMs that respond to oxidative stress (exerted either by the addition of oxidants or in mutants with low antioxidant capacity) on the redox proteome in G1 and G2.
3. To characterise structurally and functionally conserved redox sensors associated with cell cycle regulation in crop plants.
The redox-sensitive proteins that are responsible for switching on the cell cycle in response to oxidative activation and off in response to over-oxidation will be identified in Arabidopsis cell suspension cultures, which provide a homogenous cell source, where the mitotic cell cycle can be readily synchronized by the addition of drugs blocking specific cell cycle steps, such as aphidicolin or hydroxurea. The G1 and G2 phases of the cell cycle will be determined by flow cytometry and the redox proteome will be characterized using four different approaches that will allow a systematic and comprehensive identification of redox modulated cell cycle regulators. These are: 1) OxICAT/biotin-switch: global redox modifications, 2) thioredoxome, 3) dimedone chemical probes: S-sulfenylation, and 4) YAP protein probe: S-sulfenylation. Structure/function analyses will be performed on selected proteins identified in the redox proteomics screens using a range of approaches including general protein chemistry methods e.g. recombinant protein expression in E.coli, protein purification, protein folding, and all standard redox biochemistry techniques such as redox potential determination, reductase/oxidase assays and protein stability measurements. Structures of selected newly identified proteins will be solved using X-ray crystallography. Taken together, the outcomes of this project will have profound implications for both fundamental and applied biology. The discovery and characterisation of redox PTMs that control plant cell division will advance scientific knowledge and provide new molecular markers for the selection of improved crop varieties that are better adapted to grow well when exposed to environmental stress.