Award details

Chloroplast-Associated Degradation (CHLORAD): Molecular definition of a ubiquitin-dependent system for plastid protein removal in plants

Reference	BB/R009333/1
Principal Investigator / Supervisor	Professor Paul Jarvis
Co-Investigators / Co-Supervisors	Professor Shabaz Mohammed
Institution	University of Oxford
Department	Biology
Funding type	Research
Value (£)	537,125
Status	Completed
Type	Research Grant
Start date	03/04/2018
End date	02/04/2022
Duration	48 months

Abstract

We previously identified the chloroplast-localized E3 ligase SP1 as a regulator of the chloroplast protein import ('TOC') machinery that is vital during plant development (Science, 2012) and abiotic stress adaptation (Curr. Biol., 2015). Now, we have identified two new components that work together with SP1 in TOC protein degradation. We believe that SP1 and these new factors form the core of a novel ubiquitin-proteasome system pathway, termed Chloroplast-Associated Protein Degradation (CHLORAD). We propose that the new components mediate substrate protein 'retrotranslocation' to the cytosol, enabling delivery to the proteasome. We will study this system in detail in Arabidopsis, and explore possibilities for its application in crops. Specifically, we will: 1. Define the functions of the two new CHLORAD components. Different experiments will be applied in each case, depending on the proteins' predicted functions. These will include assessments of: functional relationships with SP1 and the proteasome; involvement in the retrotranslocation step; localization and topology; involvement in factor-recruitment to the chloroplast envelope; physical interactions with each other, SP1 and substrate proteins; and, physiological significance (e.g., in abiotic stress tolerance). 2. Identify targets of the CHLORAD system, by using quantitative proteomics to analyse chloroplasts isolated from CHLORAD-defective mutants. Selected putative substrates identified in this way will be verified in immunoblot and turnover assays. This will yield a comprehensive picture of CHLORAD's role in chloroplast regulation. 3. Identify new components of the CHLORAD system. Two complementary approaches will be used: (i) targeted analysis of candidates identified bioinformatically; (ii) co-immunoprecipitation with the known CHLORAD components followed by proteomics. Selected new factors will be studied in respect of localization, interactions with known components, and biochemical function.

Summary

The human population is growing rapidly and set to reach 9bn by 2050, and there is ever increasing pressure on natural resources. Thus, the drivers for increased crop yields and resilience to climate change and sub-optimal growing conditions are stronger than ever. To meet these demands it will be essential to develop improved crop varieties. Through research on the model plant thale cress, we recently made a significant breakthrough: We discovered a gene called SP1 that controls important aspects of plant growth, including plant responses to adverse environmental conditions such as water stress and high salinity (collectively, abiotic stresses). Thale cress plants can be made more tolerant of such stresses by modifying SP1 expression. Recently, we identified two new genes that function in the same regulatory pathway as SP1 - a pathway which we now term CHLORAD (for "Chloroplast-Associated Degradation"). In this project, we will study these new genes in detail, to elucidate their functions and understand how they work together with SP1, and in doing so we hope to identify new strategies for crop improvement. Like SP1, the two new CHLORAD genes regulate the development of structures inside plant cells called chloroplasts. Chloroplasts are normal cellular constituents (i.e., they are organelles), and in many ways they define plants. They contain the green pigment chlorophyll and are responsible for photosynthesis, capturing sunlight energy and using it to power the activities of the cell. As photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of huge importance, not just to plants but to all life on Earth. Chloroplasts also have critical roles in plant responses to abiotic stress, and so are ideal targets for engineering stress tolerance in crops. Chloroplasts are composed of thousands of different proteins, and most of these are encoded by genes in the cell nucleus and so are synthesized outside of the organellein the cellular matrix known as the cytosol. As chloroplasts are each surrounded by a double-membrane envelope, sophisticated machinery is needed to bring about the import of these proteins into the organelle. This comprises two molecular machines, one in each membrane, called TOC (for "Translocon at the Outer membrane of Chloroplasts") and TIC. Each machine is composed of several different proteins that work cooperatively. The SP1 gene encodes a regulatory factor called a "ubiquitin E3 ligase". Such regulators work by labelling-up unwanted proteins to target them for removal. The SP1 E3 ligase mediates the removal of TOC components, and thereby controls TOC functions so that only the desired proteins are imported by chloroplasts. Such control enables major functional changes of chloroplasts during development and in adaptation to stress. But TOC proteins are deeply embedded in the chloroplast outer membrane, presenting a physical obstacle to their removal following labelling by SP1. Our discovery of the new CHLORAD genes provides a clue as to how this obstacle is overcome: our unpublished data strongly suggest these genes encode key components of a molecular motor that drives the extraction of unwanted TOC proteins. We will study this CHLORAD machinery to understand more clearly how unwanted chloroplast proteins are removed. Moreover, the role of CHLORAD in environmental stress tolerance will be studied. We will explore how manipulating the activity of the pathway may be used to improve stress tolerance in plants. The pathway appears to operate in many different plant species, including major crops, and so our results have the potential to see broad application. Drought and salinity are among the most significant factors affecting crop yields, with annual global losses due to drought alone estimated at $10bn. We believe that our work on CHLORAD may help to alleviate such losses.

Impact Summary

Beneficiaries will include: 1) the academic community and research staff employed by the project; 2) commercial stakeholders in agriculture; and 3) the wider public and government. How these groups will engage with and benefit from the research is summarized below. 1. Academic community and research staff. Academic impact will be large due to the work's interdisciplinarity and fundamental significance, as detailed under Academic Beneficiaries. This will manifest itself in several ways: a) The work will provide new knowledge with relevance in numerous fields, inspiring new lines of investigation. b) The project will contribute to the health of UK plant science by generating publicity, fostering interactions, and enabling engagement activities designed to stimulate enthusiasm for plant biology among school students and teachers. c) The research staff will receive advanced training in bioscience research, further contributing to the health of UK plant science, reinforcing the UK's position as a leading country for academic research, and aiding transition to a Knowledge Based Bio-Economy. Training will also result from our supervision of (under)graduate students with related projects, who will have daily interaction with the PI and research staff. 2. Commercial stakeholders in agriculture. Manipulating SP1 expression improves abiotic stress tolerance, and has the potential to do so without compromising growth under normal conditions. Abiotic stresses have major adverse effects on crop yields: annual global crop losses due to drought alone are estimated at US$10bn. Owing to human population growth and increasing pressure on natural resources, the drivers for increased crop yields and resilience to climate change and sub-optimal growing conditions are stronger than ever. The CHLORAD system has considerable potential as a technology for the mitigation of stress-related crop losses. As well as potentially offering more efficient food production in the UK and other developed economies, translation of our work into crops may bring public good benefits to food production in developing countries by enhancing subsistence agriculture. Current IP associated with SP1 is protected by a patent application and licensed to Plant Bioscience Ltd. (PBL) who are promoting the technology globally. We expect new IP pertaining to the broader CHLORAD system to be generated here, and we will work with PBL and Oxford University Innovation (the University's technology transfer company) to ensure that this is similarly protected, and to promote uptake by the agbiotech industry. At an appropriate time, we may seek BBSRC Follow-on Funding to facilitate development and commercialization of CHLORAD as a technology. 3. Wider public and government. Scientific information has enriching and educational quality of life benefits for society. Thus, we will work in partnership with the Oxford Botanic Garden and Harcourt Arboretum, Oxford Natural History Museum, and the Oxford Sparks online resource, which are all excellent avenues for science-related outreach, to develop and deliver a range of innovative, high-quality engagement activities and educational resources centred on the themes of the project. These activities will not only inform and educate the public, but will also benefit the aforementioned partner organizations by promoting their bilateral engagement with the academic community and public. Through publications and associated press releases and media coverage, and via our presence at the STEM for Britain event attended by Members of both Houses of Parliament at Westminster, we will engage government. Opportunities for political dialogue that arise through the Oxford Martin Programme on the Future of Food will also be exploited. Our aim will be to highlight the importance of scientific research and plant biotechnology in relation to major societal challenges such as food security and climate change, and to influence policy in a positive way.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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