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

Lead Research Organisation: University of Oxford
Department Name: Plant Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The project focuses on the role of the chloroplast outer envelope membrane protein SP2, which we have shown to function in the regulated degradation of other chloroplast envelope proteins, most notably the chloroplast protein import machinery. The proteolytic pathway in which SP2 acts we have called CHLORAD, for chloroplast-associated protein degradation.
Exploitation Route My manipulating SP2 and CHLORAD, we believe it will be possible to modify diverse aspects of chloroplast function, enabling novel crop improvement strategies; for example, improving the tolerance of crop plants to abiotic stress.
Sectors Agriculture, Food and Drink,Environment

URL http://www.ox.ac.uk/news/2019-02-26-discovery-new-pathway-may-help-develop-more-resilient-crop-varieties
 
Description The findings formed the basis of a patent application covering the use of SP2 (CHLORAD) to modify chloroplast functions in crop plants.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Societal

 
Description BBSRC Follow-On Funding Pathfinder: "Manipulation of the chloroplast-associated protein degradation pathway (CHLORAD) - applications in plant breeding and biotechnology" (Jan - Jul 2019)
Amount £10,897 (GBP)
Funding ID BB/S013873/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2019 
End 07/2019
 
Description Dr Enrique Lopez-Juez, Royal Holloway, University of London 
Organisation Royal Holloway, University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration on the import characteristics of newly identified mutant affecting the TIC machinery of chloroplasts
Collaborator Contribution Identification of the gene affected by the mutation
Impact Identification and characterization of a significant new mutant affecting the TIC machinery of chloroplasts; manuscript in preparation
Start Year 2012
 
Description Dr Gail Preston, Department of Plant Sciences, University of Oxford 
Organisation University of Oxford
Department Department of Experimental Psychology
Country United Kingdom 
Sector Academic/University 
PI Contribution We are collaborating on the role of SP1 in biotic stress responses in Arabidopsis and brassica, via a PhD studentship
Collaborator Contribution Expertise in plant pathology
Impact The project is on-going.
Start Year 2015
 
Description Dr Masanori Izumi 
Organisation Tohoku University
Department Graduate School of Life Sciences
Country Japan 
Sector Academic/University 
PI Contribution We are collaborating on the role of ubiquitination in chloroplast degradation by autophagy. Dr Izumi visited the group in Oxford for four months in 2015 on a Japanese government fellowship.
Collaborator Contribution Expertise in autophagy.
Impact The work is on-going.
Start Year 2015
 
Description Dr Mats Töpel, Department of Marine Sciences, University of Gothenburg, Sweden 
Organisation University of Gothenburg
Department Department of Marine Sciences
Country Sweden 
Sector Academic/University 
PI Contribution We have collaborated on the phylogenetic analysis of STIC2-related proteins, and other chloroplast proteins, as well as on the analysis of whole genome sequence data.
Collaborator Contribution Advanced expertise in bioinformatics and phylogenetic analysis.
Impact Ling, Q., Broad, W., Trösch, R., Töpel, M., Demiral Sert, T., Lymperopoulos, P., Baldwin, A. and Jarvis, R.P. (2019) Ubiquitin-dependent chloroplast-associated protein degradation in plants. Science 363: eaav4467. Bédard, J., Trösch, R., Wu, F., Ling, Q., Flores-Pérez, Ú., Töpel, M., Nawaz, F. and Jarvis P. (2017) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell 29: 1726-1747. Trösch, R., Töpel, M., Flores-Pérez, Ú. and Jarvis, P. (2015) Genetic and physical interaction studies reveal functional similarities between ALBINO3 and ALBINO4 in Arabidopsis. Plant Physiol. 169: 1292-1306.
Start Year 2015
 
Description Prof Simon Newstead, Dept. of Biochemistry, University of Oxford 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We are collaborating on the in vitro reconstitution of the CHLORAD system of chloroplast protein degradation.
Collaborator Contribution Provision of expertise in in vitro reconstitution analysis.
Impact The work is on-going.
Start Year 2018
 
Description Prof. Matthew Terry, University of Southampton 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing expertise in the area of chloroplast protein import, as well as seeds of relevant mutant genotypes. We will be conducting analyses of the levels of components of the protein import machinery in a new mutant identified by our collaborators that displays defective plastid signalling.
Collaborator Contribution Our collaborators are supplying the new plastid signalling mutant to us, and are conducting detailed analyses on the mutant seed lines that we are providing.
Impact This collaboration is on-going and so has not yet generated any outputs.
Start Year 2016
 
Description Prof. Ralf Bock, Max-Planck Institute of Molecular Plant Physiology, Potsdam, Germany 
Organisation Max Planck Society
Department Max Planck Institute of Molecular Plant Physiology
Country Germany 
Sector Charity/Non Profit 
PI Contribution We are providing expertise in the area of chloroplast protein import in order to understand the role of a plastid signalling mutant. We hosted a visiting postdoctoral researcher from Germany in order that skills in this area may be transferred to our collaborators.
Collaborator Contribution Our collaborators provided the initial observations and the mutant genotypes of interest, and are completing the analyses in readiness for publication.
Impact The collaboration is on-going and so has not yet generated any outputs.
Start Year 2015
 
Description Professor Felix Kessler, University of Neuchâtel, Switzerland 
Organisation University of Neuchatel
Country Switzerland 
Sector Academic/University 
PI Contribution A collaboration was established to share knowledge and optimize methods for the native purification of chloroplast translocon complexes using the tandem affinity purification (TAP) technique. As part of this collaboration, a researcher visited the Kessler laboratory for 10 days in 2010 (18th-29th January, 2010). Since then, the collaboration has continued to evolve and take new directions, for example related to the involvement of post-translation modification in the regulation of chloroplast protein import. As a result of the collaboration, Professor was invited visit our department in Oxford on 22 November 2018, and to give a seminar here.
Collaborator Contribution A collaboration was established to share knowledge and optimize methods for the native purification of chloroplast translocon complexes using the tandem affinity purification (TAP) technique. As part of this collaboration, a researcher visited the Kessler laboratory for 10 days in 2010 (18th-29th January, 2010). Since then, the collaboration has continued to evolve and take new directions, for example related to the involvement of post-translation modification in the regulation of chloroplast protein import. As a result of the collaboration, Professor was invited visit our department in Oxford on 22 November 2018, and to give a seminar here.
Impact Jarvis, P. and Kessler, F. (2014) Mechanisms of chloroplast protein import in plants. In: Advances in Plant Biology: Plastid Biology (S.M. Theg and F.-A. Wollman, eds.) Springer, New York, pp. 241-270. Aronsson, H., Combe, J., Patel, R., Agne, B., Martin, M., Kessler, F. and Jarvis, P. (2010) Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency. Plant J. 63: 297-311.
Start Year 2010
 
Title CONTROL OF PLASTID ASSOCIATED PROTEIN DEGRADATION 
Description The patent application covers the possibility of manipulating CHLORAD to modify diverse aspects of chloroplast function, enabling novel crop improvement strategies; for example, improving the tolerance of crop plants to abiotic stress. 
IP Reference GB1815206.6 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact Too early for impact to be assessed.
 
Title CONTROL OF PLASTID ASSOCIATED PROTEIN DEGRADATION I 
Description The patent application covers the possibility of manipulating SP2 and CHLORAD to modify diverse aspects of chloroplast function, enabling novel crop improvement strategies; for example, improving the tolerance of crop plants to abiotic stress. 
IP Reference GB1803833.1 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact Too early for impact to be assessed.
 
Title Deconvolution software for interpretation of gene sequence data from new CRISPR/Cas9 mutants 
Description We required the deconvolution of Sanger sequencing data to determine if the CRISPR-Cas9 induced mutations of genes of interest were true knockouts (as opposed to non-frameshifting point mutations). To achieve this we wrote a deconvolution program in Python programming language. This software enabled characterization of the heteroallelic CRISPR mutations without the necessity of cloning PCR products of the gene into bacteria (the time-consuming conventional method). It is in the process of being made freely available as an open source platform for other groups working on CRISPR-Cas9 to use. 
Type Of Technology Software 
Year Produced 2019 
Impact Too early to assess. 
 
Description Animation illustrating the problems posed by abiotic stress to crops and food security, and the way our research may help (2019) 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact In conjunction with Oxford Sparks (https://www.oxfordsparks.ox.ac.uk/), we are preparing an animation illustrating the problems posed by abiotic stress to crop yields and food security, and explaining in simple terms the way in which our research on chloroplasts may help to address these issues.
Year(s) Of Engagement Activity 2019
URL https://www.oxfordsparks.ox.ac.uk/
 
Description Article in the BBSRC Business Magazine, Winter 2018 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This article highlighted our latest results on the regulated proteolysis of chloroplast proteins in plants, via a new pathway which we have termed CHLORAD, for chloroplast-associated protein degradation. Manipulating the CHLORAD pathway can alter diverse aspects of plant performance, including abiotic stress tolerance.
Year(s) Of Engagement Activity 2018
URL https://bbsrc.ukri.org/documents/bbsrc-business-winter-2018-pdf/
 
Description Attendance of research associate at Public Engagement Masterclass, Oxford Botanic Garden (2018) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact This was an educational session designed to develop public engagement and outreach skills.
Year(s) Of Engagement Activity 2018
 
Description Established a group Twitter account 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A Twitter account (Jarvis Lab, @PaulJarvisLab) for reporting the activities of the group was established in January 2019. We use this to disseminate our research to the general public and a range of different audiences, and to connect with other groups with similar interests in plant biology. During the first two months we had 38 tweets and accumulated 184 followers.
Year(s) Of Engagement Activity 2019
URL https://twitter.com/PaulJarvisLab
 
Description Invited speaker at EMBO Workshop on "Current advances in protein translocation across membranes" (Spain, 2019) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an invited speaker at this prestigious international meeting, which took place during 23-27 March 2019, at Sant Feliu de Guixols, Spain.
Year(s) Of Engagement Activity 2019
URL http://meetings.embo.org/event/19-protein-translocation
 
Description Invited speaker at GRC Chloroplast Biotechnology Meeting entitled "Redesigning Plastids for Novel Functions" (California, USA, 2019) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an invited speaker at this prestigious international meeting, which took place during 6-11 January 2019, at Ventura Beach Marriott, Ventura, CA, USA.
Year(s) Of Engagement Activity 2019
URL https://www.grc.org/chloroplast-biotechnology-conference/2019/
 
Description Invited speaker at GRC Protein Transport Across Cell Membranes Meeting entitled "Nanoscale Imaging and Molecular Mechanisms of Protein Transport Systems" (Texas, USA, 2018) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an invited speaker at this prestigious international meeting, which took place during March 11 - 16, 2018, at Hotel Galvez, Galveston, TX, USA.
Year(s) Of Engagement Activity 2018
URL https://www.grc.org/protein-transport-across-cell-membranes-conference/2018/
 
Description Invited speaker at International Symposium on Photosynthesis & Chloroplast Biogenesis (Kurashiki, Japan, 2018) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was an invited speaker at this prestigious international meeting, which took place during 7-10 November 2018, at Kurashiki, Japan.
Year(s) Of Engagement Activity 2018
URL http://www.rib.okayama-u.ac.jp/ISPCB/
 
Description Participation in Fascination of Plant Day (May 2019) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Our group will participate in the Fascination of Plants Day on 11 May 2019. In particular, together with the University of Oxford Botanic Garden, we are preparing an outreach activity explaining the problem of salinity and land degradation. We will have a stand at the "Westgate Oxford" shopping centre on the day.
Year(s) Of Engagement Activity 2019
URL https://epsoweb.org/all-events/fascination-of-plants-day-2019/
 
Description Press release associated with Science paper on the discovery of the CHLORAD pathway 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Press release associated with Science paper on the discovery of the CHLORAD pathway. The press release was coordinated by the University of Oxford, and was additionally disseminated by BBSRC and AAAS.
Year(s) Of Engagement Activity 2019
URL http://www.ox.ac.uk/news/2019-02-26-discovery-new-pathway-may-help-develop-more-resilient-crop-varie...
 
Description Talk by research associate (Dr Pablo Pulido) to A-level students as part of Study Day at Oxford's Museum of Natural History (Dec 2018) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This talk (on 4 December 2018) was part of an engagement event (a Study Day) organized by Oxford's Museum of Natural History and Botanic Garden. It was a special day for A-level biologists and it focused on cells; our contribution was a presentation by research associate Dr Pablo Pulido entitled "The future of food", as part of the program of "The Cellular World". Approximately 300 students attended from a mixture of local schools. All students were between 16 and 18 years old and had chosen to study biology.
Year(s) Of Engagement Activity 2018
URL https://www.oumnh.ox.ac.uk/learn