A genomics-based analysis of chloroplast replication/recombination/repair pathways using RNAi proteomics and transplastomic plants
Lead Research Organisation:
University of Manchester
Department Name: Life Sciences
Abstract
Chloroplasts are microscopic compartments in plant cells and contain the green pigment chlorophyll. They harvest sunlight and capture carbon dioxide to produce living organic matter. These solar energy converters have considerable scope for bio-manufacturing useful products. Because the process requires little input and is energy-efficient and carbon-neutral it will allows sustainable production of bio-molecules important to maintain our quality of life. Chloroplasts are unique in containing their own blueprint comprised of a circle of ~100 genes. These genes were known to be very important but resisted our early attempts to study them using modern molecular tools. Transplastomics, is a new and exciting method, that allows the detailed study of chloroplast genes. Despite the importance of chloroplast genes to life on this planet and sustainable manufacture of high-value products, we know virtually nothing about the processes that maintain these genes in plants. This project will study these processes by identifying the proteins that interact with chloroplast genes. We will use advanced transplastomics to find out what effects these proteins have on chloroplast genes. This work will allow us to understand better how these genes are protected from the damaging effects of sunlight, and how they are maintained and inherited in plants. By studying the machinery responsible for maintaining chloroplast genes we will understand one of the key molecular processes required for chloroplasts to function and this underpins the healthy growth and development of plants.
Technical Summary
An important set of extra-nuclear genes are located in the plastids of plant cells. Plastid genes are conserved, present in many copies per cell, inherited from the female parent in most angiosperms, and are essential for photosynthesis and plant development. Transgenes integrate by homologous recombination and are highly expressed in plastids. These features drive academic and industrial research on plastid genes. Despite the importance of plastid genes our knowledge of DNA-replication/recombination/repair (DNA-RRR) pathways in plastids is extremely poor. Proteins involved in these pathways are encoded by the nucleus. We have identified plastid targeted DNA-RRR proteins using bioinformatics, genome databases and published proteomic studies. Reverse genetics will be used to study the roles of these genes. Because knockouts of plastid DNA-RRR genes can be lethal we will use RNAi-mediated down-regulation to reduce protein levels. We will target 5 key genes involved in homologous recombination, repair and replication of plastid DNA. We will examine the effects of reduced protein levels on plastid DNA-RRR pathways using transplastomic plants, enabling recombination rates to be measured and plastid inheritance to be followed. We will determine the effects of reduced DNA-RRR proteins on overall phenotype, tolerance to DNA damaging agents, plastid mutation rate and plastid DNA levels. We will purify and compare plastid fractions enriched in DNA-RRR proteins from wild type and down-regulated lines, using an established method, to study interactions between DNA-RRR proteins and to identify new proteins in the pathway. This first detailed study of plastid DNA-RRR proteins will provide the first experimental results to test genomics-based models of plastid DNA-RRR pathways and will have a major impact on transplastomic research.
People |
ORCID iD |
Anil Day (Principal Investigator) |
Publications
Day A
(2007)
Cell and Molecular Biology of Plastids
Day A
(2011)
The chloroplast transformation toolbox: selectable markers and marker removal.
in Plant biotechnology journal
Day A
(2007)
Cell and Molecular Biology of Plastids
Gisby MF
(2011)
A synthetic gene increases TGFß3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.
in Plant biotechnology journal
Madesis P
(2010)
A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera
in Journal of Biotechnology
Description | Chloroplasts are microscopic compartments in plant cells and contain the green pigment chlorophyll. They harvest sunlight and capture carbon dioxide to produce living organic matter. These solar energy converters have considerable scope for bio-manufacturing useful products. Because the process requires little input and is energy-efficient and carbon-neutral it allows sustainable production of bio-molecules important to maintain our quality of life. Chloroplasts are unique in containing their own DNA blueprint comprised of a circle of ~100 genes. These genes were known to be very important but resisted our early attempts to study them using modern molecular tools. Transplastomics is a new and exciting method, that allows the detailed study of chloroplast genes. Despite the importance of chloroplast genes to life on this planet and sustainable manufacture of high-value products, we know virtually nothing about the processes that maintain these chloroplast genes in plants. Maintenance of chloroplast genes is carried out by proteins that are encoded by the nucleus (nuclear genes). This project identified important nuclear genes involved in maintaining chloroplast DNA. We showed this by first identifying and then reducing the amounts of proteins made by these nuclear genes. This was followed by monitoring the impact this had on the chloroplast genes. Important nuclear genes involved in chloroplast gene maintenance had a large effect on the maintenance of chloroplast DNA and in turn this affected the growth of the plant. This demonstrated the importance of the chloroplast gene maintenance pathways we had discovered for growth and development of plants. This better scientific understanding provides us with new knowledge to monitor and ultimately improve a key molecular process required for the healthy growth of crops. In addition to significant new knowledge the project also led to: new skills and research methods to study the maintenance of chloroplast genes and a potential method to influence the inheritance of chloroplast genes in the vegetative parts of the plants. It also opened up new research opportunities for using our new found knowledge to improve transplastomic technologies in crops. The project enabled networks and collaborations with other research groups and the knowledge and skills needed to support the specialist training of three PhD graduate students. They in turn increased our research capability over the period of the grant. |
Exploitation Route | The findings provide new information on the key genes required for chloroplast gene maintenance. The project will be of interest to academic researchers and the agri-tech sector (green biotechnology) interested in improving chloroplast genes, which encode proteins that are essential for the growth and productivity of crops. Plants can be used for the sustainable manufacture of recombinant proteins for white and red biotechnology and chloroplast expression can boost protein yields. Understanding the key proteins that are responsible for maintaining chloroplast genes opens up new opportunities for improving transplastomic technologies in a wider range of crops for applications in green, white and red biotechnology. |
Sectors | Agriculture Food and Drink Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | The research project has had positive impacts throughout the research life cycle. The findings have provided new knowledge on a key set of genes that are required for the viability of the crops. We depend on crops performing well for our food security and this new and shared knowledge will contribute to our knowledge-based economy to enhance the quality of life. The project has the added value of training highly skilled scientists who have entered the private consultancy sector and secondary education to improve the economic competitiveness of the UK and effectiveness of public services. In addition, the project has provided networking opportunities outside academia to promote the research expertise developed during the project. |
First Year Of Impact | 2008 |
Sector | Agriculture, Food and Drink,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Cultural Societal Economic |
Description | BBSRC IAA |
Amount | £19,902 (GBP) |
Funding ID | BB/S506692/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2018 |
End | 03/2019 |
Description | Manipulating plastid DNA replication/recombination/repair pathways to study plastid genome maintenance & improve transplastomic technologies in crops. |
Amount | £359,268 (GBP) |
Funding ID | BB/I011552/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2012 |
End | 07/2014 |
Description | Proof of principle project |
Amount | £89,534 (GBP) |
Funding ID | G127199 |
Organisation | UMIP Premier Fund Ltd |
Sector | Private |
Country | United Kingdom |
Start | 01/2010 |
End | 12/2010 |
Description | TGAC Capacity and Capability Challenge Programme round1: Resources for sequencing and bioinformatic analyses: Analysis of chloroplast genomes using high throughput sequencing methods |
Amount | £6,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2010 |
End | 04/2013 |
Title | Clean gene transformation |
Description | Technology allowing precise manipulation of plant DNA to exclude marker genes. Understanding of the DNA maintenance machinery enables a better understanding of the parameters that influence the process. |
Type Of Material | Technology assay or reagent |
Provided To Others? | Yes |
Impact | The method is mentioned in work from consumer groups, for example: http://uspirg.org/sites/pirg/files/reports/Antibiotic_Resistance_USPIRG.pdf |
URL | http://www.isb.vt.edu/news/2001/news01.feb.html |
Description | Binding and glutathione conjgation of porphyrinogens by plant glutathione transferases |
Organisation | Durham University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Isolation of transplastomics plants expressing GSTs were provided to Durham. Provision of our plastid transformation expertise allowed project completion. |
Collaborator Contribution | Analyses of the GSTs in transplastomic plants |
Impact | Publication in a scientific journal |
Description | DNA Escape from Plastid to the Nucleus |
Organisation | University of Adelaide |
Country | Australia |
Sector | Academic/University |
PI Contribution | Isolation of transplastomic plants |
Collaborator Contribution | Analysis of transplastomic plants |
Impact | Publications in scientific journals |
Description | Expression of a therapeutic protein in chloroplasts |
Organisation | Renovo PLC |
Country | United Kingdom |
Sector | Private |
PI Contribution | Chloroplast biotechnology |
Collaborator Contribution | Expertise in the analysis, purification and activity of TGFbeta3 |
Impact | Research article in scientific journal |
Description | Screening human sera for HCV infection |
Organisation | Hellenic Pasteur Institute |
Country | Greece |
Sector | Academic/University |
PI Contribution | Strips containing chloroplast expressed HCV core protein were used to screen sera from patients to evaluate a low cost diagnostic method |
Collaborator Contribution | Expertise in chloroplast biotechnology combined with detection of HCV core antibodies in infected sera |
Impact | Scientific Research Article |
Start Year | 2008 |
Description | Syngenta University Innovation Centre |
Organisation | University of Manchester |
Department | School of Electrical and Electronic Engineering |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provision of growth facilities and expertise in plant sciences |
Collaborator Contribution | Expertise in electrical imaging in real time |
Impact | Multidisciplinary project with experimental results |
Start Year | 2008 |
Title | EXPRESSION OF TGF-BETA IN PLASTIDS |
Description | Provided is a method for the expression of a TGF-ß in a plant. A chimeric nucleic acid sequence comprising: (1) a first nucleic acid sequence capable of regulating the transcription in a plant cell of (2) a second nucleic acid sequence, encoding a TGF-ß, and adapted for expression in the plant cell; and (3) a third nucleic acid sequence encoding a termination region functional in said plant cell is introduced into a plant cell and the plant cell grown to produce TGF-ß. The nucleic acid sequence may preferably be adapted for expression in a plant chloroplast. It is preferred that the TGF-ß is TGF-ß3, whether full length or in the form of an active fragment. |
IP Reference | WO2008032035 |
Protection | Patent application published |
Year Protection Granted | 2008 |
Licensed | No |
Impact | The method provides a clear example of a route to increasing yields of therapeutic proteins |
Title | TRANSGENIC PLANTS |
Description | The invention provides method for producing a transgenic plant comprising a recombinant plastid genome containing an exogenous gene in the absence of a selectable marker gene introduced with the exogenous gene by using direct repeat sequences, nucleic acid constructs containing direct repeat sequences which may be used in the method and transgenic plants produced by the method. |
IP Reference | WO0181600 |
Protection | Patent application published |
Year Protection Granted | 2001 |
Licensed | Commercial In Confidence |
Impact | The technology has led to other patent applications in this area |
Description | Analysis of the recombination pathway responsible for marker excision and DNA maintenance in plastids. Presentation by Research Associate |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Participants in your research and patient groups |
Results and Impact | Role of the key RecA protein in plastid DNA recombination: Annual UK conference for early stage researchers guided by experienced research staff Specialist scientific knowledge no actual impacts realised to date |
Year(s) Of Engagement Activity | 2008 |
Description | Hosted school pupil for year 10 work experience |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Work experience placement in a lab Knowledge of research methods and material in biology. Written report by pupil for the school no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
Description | Public Understanding of Science |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Carnivorous Plants Exhibition A PhD student from my research team acted as a guide and demonstrator for this event. 'To celebrate Darwin's birthday, visitors to The Manchester Museum examined living carnivorous plants, using microscopes to look at the ways these plants have evolved to catch insects and maybe identify the plants' last meal! Importantly, scientists from our Faculty were on-hand to answer questions. Latest research on the mechanism of movement and gland secretions was illustrated in displays and leaflets to show how far the science has come since Darwin.' Photographs of carnivorous plants' no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
URL | http://www.ls.manchester.ac.uk/schoolsandcommunity/communityeventhighlights/gruesomecarnivorousplant... |
Description | Public Understanding of Science |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | A PhD student from the lab demonstrated at the 'Alien Plant Invasion' one-day event at Manchester Museum for the general public. It focused on how plants have evolved to be incredibly diverse in all aspects of their lives to enable them to survive in a wide range of conditions raising awareness of evolution by demonstrating how interesting,varied and useful plants are. The impact of humans on plants was covered to show how we have effectively hijacked evolution in the commercialisation of plant species for our own purposes. There was a range of interactive events divided over two education rooms for families to take part in, including a drawing competition. Materials and drawing competition no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
URL | http://www.ls.manchester.ac.uk/schoolsandcommunity/communityeventhighlights/alienplantinvasion/ |
Description | School outreach event |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | The presentation by PhD students (MEH and ZX) on the unique DNA in chloroplasts and hands on examination of plants growing in vitro stimulated questions on extra nuclear DNA. This included their origin from ancient bacteria. The yr 11 and 12 scholars particularly enjoyed talking to researchers active in the field who were able to answer their questions in depth. The enthusiasm demonstrated by the Yr11 and 12 pupils, which was apparent from the questions led to an increase in understanding to influence future decisions |
Year(s) Of Engagement Activity | 2014 |