Elucidating the role of SP2 and the SP1-SP2 machinery in chloroplast protein degradation

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 another gene called SP2 that functions in the same regulatory pathway as SP1. In this project, we will study the SP2 gene in detail, to elucidate its function, to understand how it works together with SP1, and to investigate its potential use for crop improvement by conducting studies in rice.

The SP1/2 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 organelle in 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 SP2 provides a clue as to how this obstacle is overcome. The SP2 gene encodes a channel across the chloroplast outer membrane, and our evidence suggests that it forms the exit gate for the removal of unwanted TOC proteins. In fact, we believe that the SP1 and SP2 factors are stably associated in a complex to enable coordinated protein labelling and removal. We will study this SP1-SP2 machinery to understand more clearly how unwanted chloroplast proteins are removed.

Moreover, the role of SP2 in environmental stress tolerance will be studied. In particular, we will manipulate activity of the SP1-SP2 pathway with the aim of improving stress tolerance in rice. The SP1-SP2 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 with SP1/2 may help to alleviate such losses.

We previously identified the chloroplast-localized E3 ligase SP1 as a regulator of the chloroplast protein import (TOC) machinery (Science, 2012) which is particularly important during abiotic stress (Curr. Biol., 2015). Now, we have identified a new component called SP2, and found that it works together with SP1 in the degradation of TOC proteins. We hypothesize that SP2 and SP1 exist at the core of a novel ubiquitin-proteasome system (UPS) pathway, termed Chloroplast-Associated Protein Degradation (CHLORAD), in which SP2 mediates substrate retrotranslocation. We will characterize this system in detail in the model plant Arabidopsis (Obj. 1-4), and explore potential applications in crops using rice as a model (Obj. 5). Specifically, we will:

1. Functionally define SP2 and its partnership with SP1. We will characterize the physical interactions between SP2 and SP1 and its substrates, and investigate SP2 (and SP1) involvement in the critical retrotranslocon function in vivo.

2. Characterize the SP1-SP2 core complex in relation to overall size, composition, and component stoichiometry, using sucrose gradient centrifugation and BN-PAGE. Novel components will be identified by mass spectrometry.

3. Identify SP2 interactors to define new components of the SP1-SP2 machinery. To do this, we will employ co-immunoprecipitation coupled with proteomic analysis. The most interesting new components identified will be characterized.

4. Reconstitute the SP1-SP2 system in vitro. We will recapitulate the CHLORAD reaction using purified proteins, chemically defined lipids, and cytosol extract to demonstrate that SP2 and SP1 are (amongst) the minimal set of membrane factors (i.e., core components) required for CHLORAD, and to elucidate molecular mechanisms of the system.

5. Investigate whether SP2 promotes abiotic stress tolerance, as the SP1 protein does, and explore possibilities for the application of SP2 in promoting stress tolerance in crops, using rice as a model.

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 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 SP1-SP2 system (CHLORAD) 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 agricultural 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 SP2 and the broader CHLORAD system to be generated, 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 Follow-on Funding to facilitate development and commercialization of SP2 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 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.