Genetic suppressors of Arabidopsis chloroplast protein import mutations
Lead Research Organisation:
University of Leicester
Department Name: Biology
Abstract
Chloroplasts are normal components of plant cells (such sub-cellular components are called organelles) that in many ways resemble free-living bacteria. They contain the green pigment, chlorophyll, and are exclusively responsible for the reactions of photosynthesis (the process that enables the capture of sunlight energy and its use to make organic products). Since photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of inestimable importance, not just to plants but to all life on Earth. Chloroplasts are also important in many other ways, since they play important roles in the biosynthesis of lipids, amino acids and starch. Although chloroplasts do contain DNA (a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and are therefore able to encode some of their own proteins, >90% of the 3000 or so proteins required to build a fully functional chloroplast are encoded on DNA within the cell nucleus. The majority of chloroplast proteins are therefore synthesized outside of the chloroplast, in the cellular matrix known as the cytosol. Since chloroplasts are each surrounded by a double membrane, or envelope, that is impervious to the passive movement of proteins, this presents a significant problem. In order to overcome the problem, chloroplasts have evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol, across the envelope, and into the chloroplast interior. This protein import apparatus comprises two molecular machines: one in the outer envelope membrane called Toc (an abbreviation of 'Translocon at the outer envelope membrane of chloroplasts'), and one in the inner envelope membrane called Tic. Over the last decade, a great deal of progress has been made in our understanding of how this protein import apparatus works. In particular, it seems likely that most of the main components of the machinery have now been identified. Nevertheless, substantial gaps remain in our knowledge. For example, while it is known that the import process is regulated throughout plant development, very little is known about the mechanisms that underlie this regulation. To fill in these gaps in our knowledge, completely new experimental approaches will need to be employed. The experiments described in this proposal are an entirely new way of studying chloroplast protein import. In previous work, we identified plants carrying genetic mutations that affect the efficiency of protein import; what we propose to do here is to identify the genes affected by these mutations. Because chloroplasts carry out essential functions, and because protein import is essential for chloroplast development, it should come as no surprise to learn that plants with defective chloroplast protein import machinery are unable to survive beyond the embryo stage. Thus, chloroplast protein import is an essential process for plants. Similarly, since we are all ultimately dependent upon plant products for survival, it follows that chloroplast protein import is essential on a global scale. What is more, since chloroplasts play an instrumental role in the synthesis of many economically important products (such as lipids and starch), a more complete understanding of how these organelles develop may enable us to enhance the productivity of crop plants, or otherwise manipulate their products.
Technical Summary
To identify new loci involved in plastid protein import, or its regulation, we conducted screens for second-site suppressors of two fundamentally different protein import mutations, ppi1 and tic40. We identified five recessive suppressor of ppi1 (sp) mutations, at two loci, and ten suppressor of tic40 (stic) mutations (five recessive, five semi-dominant). These screens were done using previous BBSRC funding. We seek new support to take these projects forward: the proposed work is a logical continuation of the previous work. The sp1 locus maps to the bottom of chr 1. We will identify sp1 by map-based cloning, and then characterize the gene using different approaches; the approaches taken will depend on the nature of the gene. We will map the sp2 locus with a view to its cloning in a future project. Characterization of the sp mutants was started previously (project 91/P12928). We will complete a more detailed study of two mutants, sp1-3 and sp2. Specifically, we will: (i) compare expression levels of key translocon genes in sp1, sp2 and ppi1; (ii) measure protein import efficiency in sp2 (sp1 is already known to import more efficiently than ppi1); (iii) quantify chloroplast ultrastructural recovery in the suppressors; (iv) investigate the specificity of suppression mediated by sp1 and sp2, by crossing them to tic40. We will map the stic loci at low resolution, and test for allelism by crossing the mutants in all combinations. We will identify one stic locus by map-based cloning. Selection of the locus for cloning will be influenced by the mapping, allelism and characterization experiments. The cloned gene will be studied using a range of approaches, which will be determined by the nature of the gene. Characterization of the stic mutants was started previously (project 91/C18638). We will complete a more detailed study of two stic mutants (one recessive, one semi-dominant). The experiments will be very similar to those described for the sp mutants.
People |
ORCID iD |
Paul Jarvis (Principal Investigator) |
Publications
Aronsson H
(2010)
Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency.
in The Plant journal : for cell and molecular biology
Huang W
(2013)
The ubiquitin-proteasome system regulates chloroplast biogenesis.
in Communicative & integrative biology
Huang W
(2011)
In vivo analyses of the roles of essential Omp85-related proteins in the chloroplast outer envelope membrane.
in Plant physiology
Jarvis P
(2013)
Biogenesis and homeostasis of chloroplasts and other plastids.
in Nature reviews. Molecular cell biology
Kasmati AR
(2011)
Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts.
in The Plant journal : for cell and molecular biology
Kasmati AR
(2013)
Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts.
in PloS one
Ling Q
(2013)
Dynamic regulation of endosymbiotic organelles by ubiquitination.
in Trends in cell biology
Ling Q
(2015)
Regulation of Chloroplast Protein Import by the Ubiquitin E3 Ligase SP1 Is Important for Stress Tolerance in Plants.
in Current biology : CB
Ling Q
(2015)
Functions of plastid protein import and the ubiquitin-proteasome system in plastid development.
in Biochimica et biophysica acta
Ling Q
(2011)
Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana.
in Photosynthesis research
Description | The most significant result of this project was the molecular identification of the SP1 gene. This project revealed that SP1 encodes a type of regulatory protein called a ubiquitin E3 ligase. Such regulators act by bringing about the targeted removal and breakdown of proteins that are no longer required. What made this discovery so novel and impactful was the fact that SP1 is a chloroplast protein (it resides in the outer envelope membrane of chloroplasts where it regulates the protein import machinery), as it was not previously known that regulation by ubiquitin can act directly on chloroplasts. |
Exploitation Route | Others, like us, may wish to further explore the role of SP1 and ubiquitin in the regulation of plastid functions in plants, which may ultimately find commercial applications in crops. |
Sectors | Agriculture, Food and Drink,Energy |
Description | This work led to the realization that multiple, client-specific protein import pathways operate in plastids, and that these pathways are enabled by the existence of a diversity of client-specific protein import pathways, and by the operation of a regulator of the import machinery called SP1. We believe that manipulation of these import pathways will find beneficial applications in crops, by enabling manipulation of plastid functions and plastid development in a variety of different ways. This technology is covered by patent applications and is being promoted commercially. |
First Year Of Impact | 2012 |
Sector | Agriculture, Food and Drink,Energy |
Impact Types | Societal |
Description | Application of the plastidic E3 ligase SP1 in crop improvement, using tomato and rice as models |
Amount | £152,584 (GBP) |
Funding ID | BB/R005591/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2018 |
End | 03/2019 |
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 | BBSRC Responsive Mode |
Amount | £378,569 (GBP) |
Funding ID | BB/H008039/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2010 |
End | 12/2012 |
Description | BBSRC/BBR-Funded BRACT Crop Transformation Facility: CRISPR/Cas9 targeted knockouts of SP1 and SP2 in wheat (2018) |
Amount | £1 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 02/2018 |
End | 12/2019 |
Description | BBSRC/BBR-Funded BRACT Crop Transformation Facility: CRISPR/Cas9 targeted knockouts of SP1 in Brassica oleracea (2016-2018) |
Amount | £1 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 12/2016 |
End | 05/2018 |
Description | Chloroplast-Associated Degradation (CHLORAD): Molecular definition of a ubiquitin-dependent system for plastid protein removal in plants |
Amount | £537,125 (GBP) |
Funding ID | BB/R009333/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2018 |
End | 04/2022 |
Description | Elucidating the role of SP2 and the SP1-SP2 machinery in chloroplast protein degradation |
Amount | £498,394 (GBP) |
Funding ID | BB/R016984/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2018 |
End | 09/2022 |
Title | A method to test for ubiquitination of a membrane protein by a membrane-bound E3 ligase |
Description | We developed an in vitro method to test for the ubiquitination of membrane proteins as mediated by a membane-bound E3 ligase. The method involves the use of radiolabelled, in vitro transcribed/translated substrate proteins, and the use of recombinant, bacterially-expressed E3 ligase lacking its transmembane regions (through replacement with a flexible linker sequence). |
Type Of Material | Technology assay or reagent |
Year Produced | 2012 |
Provided To Others? | Yes |
Impact | This resource supported a major publication (see below), and two successful grant applications (BB/K018442/1, BB/N006372/1). Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659. |
Title | Antibody against the SP2 protein |
Description | A domain of SP2 was expressed in bacteria, purified to homogeneity, and used to immunize rabbits (by Harlan Laboratories, Loughborough). The antiserum was affinity-purified and proven to be effective by ELISA analysis. |
Type Of Material | Biological samples |
Provided To Others? | No |
Impact | It has been used to support a PhD studentship and a pending BBSRC Responsive Mode application (BB/R00272X/1). |
Title | Clone encoding FLAG-tagged ubiquitin |
Description | This clone encodes Arabidopsis ubiquitin bearing an N-terminal FLAG epitope tag, and is useful for monitoring ubiquitination in planta |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Provided To Others? | No |
Impact | This resource supported a major publication (see below), and two successful grant applications (BB/K018442/1, BB/N006372/1). Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659. |
Title | Improved protoplast transfection system for the analysis of various membrane protein functions |
Description | We developed an optimized system for investigating the functions of membrane proteins in relation to localization, topology, protein-protein interactions, responses to pharmacological agents, and ubiquitination. The system is based on the analysis of transfected Arabidopsis protoplasts. |
Type Of Material | Technology assay or reagent |
Year Produced | 2012 |
Provided To Others? | Yes |
Impact | This resource supported a major publication (see below), and two successful grant applications (BB/K018442/1, BB/N006372/1). Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659. |
Title | Vectors and resulting transgenic plants (brassica, AG DH1012) that overexpress or are silenced for SP1 |
Description | Vectors and resulting transgenic plants (brassica, AG DH1012) that overexpress or are silenced for SP1. The transgenic brassica plants were generated at BRACT, John Innes Centre, Norwich, with support of Plant Biosciences Ltd. (PBL). |
Type Of Material | Biological samples |
Provided To Others? | No |
Impact | The plants are being used to provide proof-of-concept for the application of SP1 in crop improvement. The analysis is still in progress. |
Title | Vectors and resulting transgenic plants (rice, Kitaake) that overexpress or are silenced for SP1 |
Description | Vectors and resulting transgenic plants (rice, Kitaake) that overexpress or are silenced for SP1. |
Type Of Material | Biological samples |
Provided To Others? | No |
Impact | The plants are being used to provide proof-of-concept for the application of SP1 in crop improvement. The analysis is still in progress. |
Title | Vectors and resulting transgenic plants (tomato, Ailsa Craig) that overexpress or are silenced for SP1 and its homologue SPL2 |
Description | Vectors and resulting transgenic plants (tomato, Ailsa Craig) that overexpress or are silenced for SP1 and its homologue SPL2 |
Type Of Material | Biological samples |
Provided To Others? | No |
Impact | Furtherance of our BBSRC-funded research. This work is covered by a PCT stage patent application, and by a licensing agreement in place with PBL, Norwich. |
Title | Vectors and resulting transgenic plants (wheat, Fielder) that overexpress or are silenced for SP1 |
Description | Vectors and resulting transgenic plants (wheat, Fielder) that overexpress or are silenced for SP1. The transgenic wheat plants were generated at NIAB, Cambridge, as part of the BBSRC-funded Community Resource for Wheat Transformation. |
Type Of Material | Biological samples |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | Furtherance of our BBSRC-funded research. This work is covered by a PCT stage patent application, and by a licensing agreement in place with PBL, Norwich. |
Title | Vectors for the in vitro transcription/translation of SP1 and various TOC proteins (full-length and truncated forms) |
Description | pBluescript-based plasmid vectors containing coding sequences for SP1 and various TOC proteins (either full-length or truncated forms), under the control of the T7 promoter, to enable the synthesis of radiolabelled proteins by coupled in vitro transcription/translation. |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Provided To Others? | No |
Impact | These resources supported two major publications (see below), and two successful grant applications (BB/K018442/1, BB/N006372/1). Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659. Ling, Q. and Jarvis, P. (2015) Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants. Curr. Biol. 25: 2527-2534. |
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 | NIAB, Cambridge |
Organisation | National Institute of Agronomy and Botany (NIAB) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Generation of constructs, and analysis of transgenic wheat plants |
Collaborator Contribution | Transformation of wheat to generate transgenic plants with altered expression of the SP1 protein |
Impact | Transgenic plants with altered expression of the SP1 protein, and improved understanding of SP1 function and possible applications in crops |
Start Year | 2013 |
Title | ATSP1, AN E3 UBIQUITIN LIGASE, AND ITS USE |
Description | The invention relates to plants with improved phenotypes and related methods. These improved phenotypes are conferred by altering the expression of the SP1 gene which is involved in plastid development or altering the activity of the SP1 protein. |
IP Reference | 16/643507 |
Protection | Patent application published |
Year Protection Granted | 2015 |
Licensed | Yes |
Impact | A BBSRC Follow-on Funding Pathfinder grant was awarded in 2013 (BB/FOF/PF/15/12), which related to earlier patent filings of the same technology. A subsequent BBSRC Follow-on Funding Standard grant was applied for in 2017 (BB/R005591/1; application pending). |
Title | ATSP1, AN E3 UBIQUITIN LIGASE, AND ITS USE |
Description | The invention relates to plants with improved phenotypes and related methods. These improved phenotypes are conferred by altering the expression of the SP1 gene which is involved in plastid development or altering the activity of theSP1 protein. |
IP Reference | WO2014037735 |
Protection | Patent application published |
Year Protection Granted | 2014 |
Licensed | Yes |
Impact | The technology is currently in development. |
Description | 2nd International Photosynthesis Workshop, 2009, Munich, Germany |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Invited speaker International networking; inspiration for research; esteem no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
URL | http://www.hfsp.org/frontier-science/awardees-articles/new-theories-origin-cerebral-cortical-convolu... |
Description | BBSRC press release associated with Ling et al. 2012 Science paper on SP1 |
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 | Media (as a channel to the public) |
Results and Impact | A huge amount of media interest was generated as a result of this press release, which focused on the control of plastid biogenesis by the ubiquitin-proteasome system, published in November 2012 (Science 338: 655-659). I was interviewed for BBC television (East Midlands Today News; aired on November 2nd 2012), BBC Radio Leicester (live on November 2nd 2012), the Daily Telegraph (November 2nd issue), Australian National Radio (ABC Rural), and Scientific American (December issue). The story was also carried by the Daily Mail, and by numerous online news and media outlets. In addition, it featured on the front page of the BBSRC website, and was a headline story in the November 9th edition of the BBSRC newsletter. |
Year(s) Of Engagement Activity | 2012 |
URL | http://www.bbsrc.ac.uk/news/food-security/2012/121102-pr-chloroplast-hold-key-fruit-ripening.aspx |
Description | Plant Signaling and Behavior Meeting, 2009, Florence, Italy |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Invited speaker International networking; inspiration for research; esteem no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
Description | University press release associated with Ling et al. 2012 Science paper on SP1 |
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 | Media (as a channel to the public) |
Results and Impact | A huge amount of media interest was generated as a result of this press release, which focused on the control of plastid biogenesis by the ubiquitin-proteasome system, published in November 2012 (Science 338: 655-659). I was interviewed for BBC television (East Midlands Today News; aired on November 2nd 2012), BBC Radio Leicester (live on November 2nd 2012), the Daily Telegraph (November 2nd issue), Australian National Radio (ABC Rural), and Scientific American (December issue). The story was also carried by the Daily Mail, and by numerous online news and media outlets. In addition, it featured on the front page of the BBSRC website, and was a headline story in the November 9th edition of the BBSRC newsletter. |
Year(s) Of Engagement Activity | 2012 |
URL | http://www2.le.ac.uk/offices/press/press-releases/2012/november/could-chloroplast-breakthrough-unloc... |