SP1 and ubiquitin-mediated control of chloroplast protein import

Lead Research Organisation: University of Leicester
Department Name: Biology

Abstract

Chloroplasts and mitochondria are normal components of many cells - they are sub-cellular structures called organelles. Interestingly, these two organelles evolved from bacteria that were engulfed by other cells more than a billion years ago, and in many ways they still resemble free-living bacteria. Chloroplasts are found in plant cells, contain the green pigment chlorophyll, and are exclusively responsible for the reactions of photosynthesis (the process that captures sunlight energy and uses it to power the activities of the cell). 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 essential roles in the biosynthesis of oils, proteins and starch. Although chloroplasts do contain DNA (a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and so are able to make some of their own proteins, over 90% of the 3000 proteins needed to build a fully functional chloroplast are encoded on DNA in the cell nucleus. Most chloroplast proteins are therefore made 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. 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 another in the inner envelope membrane called TIC. Each machine is made up of several different proteins which cooperate to ensure the efficiency of import. We work on a model plant called Arabidopsis that has many advantages for research, such as an availability of numerous mutants (each one with a mutation in a specific gene). One such mutant plant, ppi1, has a defect in a TOC gene such that chloroplast protein import does not work efficiently. Several years ago, we identified another mutation called sp1 (this stands for 'suppressor of ppi1'), which significantly improves protein import efficiency in ppi1. Very recently, we discovered that the defective gene in sp1 (the SP1 gene) encodes a type of regulatory protein called a 'ubiquitin E3 ligase'. These usually work by labelling-up unwanted proteins and targeting them for degradation. Because this control mechanism was not previously known to operate in chloroplasts, we believe that we have made an important breakthrough. We think that SP1 controls protein import efficiency by regulating the amount of the TOC machinery. In the sp1 mutant, this control mechanism is disturbed somewhat, allowing certain TOC proteins to accumulate to a higher level, thereby improving protein import efficiency. We will do experiments to test these theories. 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 without a functional chloroplast protein import machinery are unable to survive (in fact, they die at 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. Because chloroplasts play major roles in the synthesis of many economically important products (such as oils and starch), a more complete understanding of how these organelles develop will enable us to enhance the productivity of crop plants, or otherwise manipulate their products.

Technical Summary

We found that SUPPRESSOR OF PPI1 LOCUS 1 (SP1) encodes a RING finger (RNF) ubiquitin E3 ligase of the chloroplast envelope. We propose that SP1 controls chloroplast protein import by targeting the Toc75 translocon component for degradation. This is highly novel as no role for the ubiquitin proteasome system (UPS) at the chloroplast surface was reported before. We will elucidate the role of SP1 and UPS in chloroplast import. 1. To confirm that Toc75 is a substrate of SP1, we will test the Toc75-SP1 association by several methods: in vitro pull-down using recombinant and in vitro translated proteins; in vivo co-immunoprecipitation using either epitope-tagging or SP1 antibody; possibly also BiFC, to confirm the interaction in intact cells if necessary. 2. To confirm that SP1 regulates turnover of Toc75 via the UPS, we will do ubiquitination assays in vitro (using recombinant proteins) and in vivo (in WT and sp1 plants, to test the requirement for SP1). We will also assay Toc75 stability in WT and sp1 cells, via protoplast transfection. 3. We will do biochemical tests to confirm that SP1 is an integral protein of the outer envelope membrane and to elucidate its orientation. 4. We will analyse functions of three SP1 domains: two transmembrane domains and the RNF. We will mutate each one and then assess for effects on: localization in vivo; ubiquitination in vitro; functionality in planta (complementation). 5. We will assess whether SP1 and its homologues, SPL1 and SPL2, have redundant or divergent roles. To do this we will study double and triple mutants (in the ppi1 and PPI1 backgrounds), and overexpress each of the genes (in the ppi1 and sp1 ppi1 backgrounds). 6. We will use tandem affinity purification and proteomics to seek additional substrates of SP1. Collaborative arrangements have been established. 7. We will assess for involvement of SUMO in chloroplast import, and conduct a new screen for sp mutants (the original screen was not saturating).

Planned Impact

We propose basic research on the Arabidopsis model plant in an area of profound biological importance with great potential to yield results of economical or societal significance. Non-academic impact of the research is discussed below. Chloroplasts are the site of photosynthesis in plants, algae and some protists, and so mediate much of the world's primary productivity. As photosynthesis is the only significant mechanism of energy-input into the biosphere, chloroplasts are essential for plants and animals alike; thus, agriculture is wholly dependent on chloroplast biogenesis. Chloroplasts or other plastids also mediate many biosyntheses (starch, amino acids, fatty acids). Many of these products are vital in mammalian diets, and knowledge on plastid biogenesis may enable improvements in their quantity or quality. Since plastids are so integral to cellular metabolism, plastid biogenesis defects can cause plants to die during (pre)embryonic development. Chloroplasts contain ~3000 proteins but only ~100 are encoded by the plastome. Thus >90% of plastid proteins are nucleus-encoded and cytosolically-synthesized, so that chloroplast biogenesis is dependent on efficient operation of the TOC/TIC import machinery. Because of chloroplasts' uniquely important role in the biosphere, we are all dependent upon proper chloroplast development (including protein import) for survival. This emphasizes the importance of knowledge on chloroplast protein import. Plastids offer many opportunities for agricultural or industrial exploitation. Depletion of fossil fuels and environmental effects of their use demand that renewable materials are used by the chemical and fuel industries. Biofuels have attracted much attention recently, and will likely become more significant as cost and efficiency issues are resolved. As raw materials for biofuel production are derived largely via chloroplast processes, improved understanding of plastid biogenesis will aid development of this important technology. As chloroplasts may contain >50% of total leaf protein, foreign proteins can be expressed to extremely high levels in plastids. Plastid manipulation may also enable accumulation of foreign proteins that would be harmful elsewhere in the cell. Plastids are inherited maternally and so (in relation to transplastomics) the possibility for transgene out-crossing is minimized. Knowledge on plastids may have medical or veterinary applications, as apicomplexan parasites (malaria, toxoplasmosis, coccidiosis) contain a relict plastid. Moreover, our work on SP1 may shed light on related processes in mitochondrial biogenesis; significantly, mitochondria and the ubiquitin proteasome system have both been implicated in ageing and neurodegenerative diseases. Short-term commercialization is not currently envisaged, but relevant mechanisms are nonetheless in place. For example, we will liaise with Leicester's Biobator which promotes commercial activities resulting from University research, and the Enterprise and Business Development Office which advises on intellectual property, its protection and commercialization. We will engage the public and broader community in various ways. We will continue to accept visitors into our lab via different schemes; e.g., sixth-formers funded by the Nuffield Foundation, undergraduates funded by the Genetics or Biochemical Societies, and ESF-funded trainee technicians. The Nuffield scheme gives school students an insight into scientific research and promotes outreach and science education. The Department is invited to present at local schools and colleges, and we will actively support this role. Further engagement will be through our lab web pages and our participation in undergraduate teaching. Our research directly influences undergraduate teaching, as research developments are presented in tutorials and lectures. We will engage the media via the University Press Office, with press releases on new publications and discoveries.

Publications

10 25 50
 
Description The project has led to several major discoveries in relation to the regulation of chloroplasts and other plastids by the regulatory protein SP1; these include, but are not limited to, the following:
1. We showed that the regulation of chloroplast import is an important component of the mechanisms by which plants tolerate stress, and that such regulation is dependent on SP1. We linked this role of SP1 to the down-regulation of photosynthesis under stress due to the degradation of the TOC protein import machinery. Mutant plants lacking SP1 show reduced tolerance to several stress conditions, while SP1 overexpressors show enhanced stress tolerance. These findings may be applied as a new way to increase the stress tolerance of crop plants.
2. We revealed how regulation of protein import into chloroplasts by SP1 is important for the way in which plants respond to, and tolerate, environmental stresses such as high salinity. In fact, plants that over-produce the SP1 protein display much enhanced stress tolerance, which suggests a novel way in which to make more hardy crop varieties.
3. The SP1 protein was identified in previous work, but it was not known whether SP1 required the activity of other proteins in order to fulfil its role. In this project, we have identified a chaperone protein that cooperates with SP1 by helping to mediate the removal of SP1 substrates from chloroplast membranes.
4. We also revealed an import new role for SP1 in the bulk removal of many unwanted chloroplast proteins, which is different from the previously described role of SP1 that deals only with individual proteins on a one-by-one basis.
Exploitation Route We and others may wish to explore the way in which SP1 can be used to modify chloroplast functions, for example in crop plants with a view to improving stress tolerance. This technology is covered by a patent application, and is being promoted commercially by PBL, Norwich.
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 a patent application and is being promoted by PBL, Norwich.
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
Amount £10,722 (GBP)
Funding ID BB/FOF/PF/15/12 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 03/2013
 
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 £374,416 (GBP)
Funding ID BB/K018442/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description BBSRC iCASE Studentship
Amount £95,042 (GBP)
Funding ID BB/M015165/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
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 04/2021
 
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 09/2021
 
Title A method to analyse chloroplast protein import using chloroplasts isolated from stressed plants 
Description We developed a method to analyse chloroplast protein import in vitro using chloroplasts isolated from stressed plants. The method enables the assessment of stress treatments on chloroplast protein import. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact These resources supported two publications (see below), and a successful BBSRC grant application (BB/N006372/1). 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. Ling, Q. and Jarvis, P. (2016) Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants. J. Vis. Exp. (117), e54717, doi:10.3791/54717. 
 
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 Additional sp mutants identified 
Description A new screen for suppressor of ppi1 (sp) mutants of Arabidopsis thaliana has been conducted, with the identification of >200 new mutants 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The mutants are still under characterization. Some of them have now been shown to be new alleles of sp1 and sp2. Others define novel loci. 
 
Title Antibody against the SP1 protein 
Description A domain of SP1 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 and immunoblot analysis. 
Type Of Material Antibody 
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 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 Identification of a set of new sp1 enhancer mutants 
Description We have screened for and identified a large number of new sp1 enhancer mutants in Arabidopsis, which was achieved by searching for plants with enhanced greening following EMS mutagenesis of sp1 ppi1 double mutant plants. These new mutants are likely define a series of new genes involved in chloroplast biogenesis regulation by SP1 and ubiquitin-proteasome system. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
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 Modification of p2GW7 and pH2GW7 vectors to enable addition of C-terminal HA tag to protein of interest 
Description These vectors (Trends Plant Sci., 2005, 10:103-105) were modified so that they now allow addition of a C-terminal HA epitope tag; they can be used for protoplast transfection (p2GW7-HA) and stable plant transformation (pH2GW7-HA) 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These resources are supporting several ongoing lines of investigation in the laboratory. 
 
Title Vectors and resulting transgenic plants (Arabidopsis) that over/underexpress a cytosolic Hsp100-type chaperone 
Description Vectors and resulting transgenic plants (Arabidopsis) that over/underexpress a cytosolic Hsp100-type chaperone. This chaperone is of interest because we hypothesize that it acts on the SP1 pathway for chloroplast protein degradation. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
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 encoding site-directed mutant forms of the SP1 protein, such that it lacks ubiquitination sites, for bacterial and plant expression, and resulting transgenic plants (Arabidopsis) 
Description Vectors encoding site-directed mutant forms of the SP1 protein, such that it lacks ubiquitination sites, for bacterial and plant expression, and resulting transgenic plants (Arabidopsis) 
Type Of Material Biological samples 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
Title Vectors for the analysis of the interaction of a cytosolic Hsp100-type chaperone with chloroplast proteins in vivo by BiFC 
Description Vectors for the analysis of the interaction of a cytosolic Hsp100-type chaperone with chloroplast proteins in vivo by bimolecular fluorescence complementation (BiFC). This chaperone is of interest because we hypothesize that it acts on the SP1 pathway for chloroplast protein degradation. Also included in this set of vectors are equivalent BiFC vectors for a range of control proteins. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Furtherance of our BBSRC-funded research 
 
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 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 NIAB, Cambridge 
Organisation National Institute of Agronomy and Botany (NIAB)
Country United Kingdom 
Sector Charity/Non Profit 
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 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 CONTROL OF PLASTID ASSOCIATED PROTEIN DEGRADATION II 
Description The patent application covers the possibility of manipulating PUX10 (CDC48) 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 GB1803834.9 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact Too early for impact to be assessed.
 
Title Transgenic Plants. 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 WO2014037735 
Protection Patent application published
Year Protection Granted 2014
Licensed Yes
Impact The technology is currently in development.
 
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 Technology New/Improved Technique/Technology 
Impact No actual Impacts realised to date 
 
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 Technology New/Improved Technique/Technology 
Impact No actual Impacts realised to date 
 
Description 10th International Congress on Plant Molecular Biology (IPMB), 2012, Jeju South Korea 
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 2012
 
Description 12th EMBL International PhD Symposium, 2010, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
URL http://www.icrs2012.com/Downloads/ICRS2012_Book_of_Abstracts.pdf
 
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 Chloroplast Biogenesis Session, Society for Experimental Biology (SEB) Annual Main Meeting, 2012, Salzburg, Austria 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Session co-organizer International networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Gordon Research Conference (GRC), Protein Transport Across Cell Membranes, 2010, Texas, USA 
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 2010
 
Description Molecular and Cellular Biosciences Theme Research Day, 2012, University of Leicester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited speaker Local networking; inspiration for research; esteem

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description The Rank Prize Funds Mini-Symposium on Photosynthesis, Grasmere, UK (October 2012; delivered by Dr. Qihua Ling) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach National
Primary Audience Other audiences
Results and Impact Dr. Qihua Ling delivered a presentation at the conference.

Promoted awareness and outcomes of our BBSRC-funded work, and made contact with researchers in related fields leading possible future collaboration.
Year(s) Of Engagement Activity 2012
URL http://www.rankprize.org/index.php/symposia/nutrition
 
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...