Rice Research Newton Fund: Exploiting a Cyanobacterial CO2 Concentrating Mechanism to Increase Photosynthesis and Yield in Rice

Lead Research Organisation: Lancaster University
Department Name: Lancaster Environment Centre

Abstract

Food security is internationally recognised as one of the major global challenges of the 21st century. By 2050, it is predicted that world food production will have to increase by 50% to meet demand. This is against the pressures of global climate change and resource limitations. Meeting this challenge is going to require the development of innovative strategies to make use of our unprecedented knowledge of modern bioscience in the post genomic era. Since rice is the most important human food, consumed by more than half of the world's population, developing new highly-productive rice varieties will be fundamental to meeting the 2050 goal.

Whilst substantial gains in rice yield have been obtained by traditional breeding, achievement of major increases in the future will require novel approaches. Improved photosynthesis has been identified as a major target for bioengineering for enhanced yield of C3 crop plants. Improving photosynthetic efficiency does not simply increase the amount of carbohydrate produced per hectare of land, but it also increases the amount produced per unit of water and per unit of nitrogen. This means not only environmental sustainability, but also higher income and better food security for small farmers.

The properties of the carbon-fixing enzyme Rubisco (ribulose-1:5-bisphosphate carboxylase/oxygenase) are known to limit the efficiency of photosynthesis in land plants. Rubisco catalyses the combination of RuBP (ribulose-1,5-bisphophate) with CO2, but also catalyses the reaction of RuBP with oxygen, leading to photorespiration, a process in which NH3 and previously fixed CO2 are lost. Some land plants (C4 plants) and bacteria have evolved mechanisms that concentrate CO2 near Rubisco and thereby both increase photosynthetic CO2 assimilation and decrease the competing wasteful reaction with oxygen. However, rice lacks this ability. As a result, rice utilizes a Rubisco enzyme that has higher CO2 affinity but is slower than Rubisco enzymes in plants with carbon-concentrating mechanisms such as maize. Consequently, the plants must devote considerable amounts of protein, and thereby, nitrogen, to allow Rubisco to carry out adequate carbon fixation, reducing yield and biomass production. Replacing endogenous Rubisco with a faster enzyme with less CO2 specificity, along with a carbon concentrating mechanism (CCM), could significantly improve CO2 fixation, according to published models.

We propose to engineer the nuclear genome of rice to express components of the cyanobacterial beta-carboxysome, including a faster Rubisco enzyme. We will identify transgenic lines of rice with the required levels of carboxysome shell proteins, internal proteins, and cyanobacterial Rubisco, which can then be crossed to express all of the novel proteins in the same line. , we will install a combination of bicarbonate transporters in order to supply CO2 to the engineered carboxysome. Finally we will also reduce the expression of the chloroplast stromal carbonic anhydrase and endogenous rice Rubisco through RNA silencing technology Based on promising results with the model plant tobacco, we believe it is time for forge ahead with a project that could bring the cyanobacterial CCM into rice for the future.

Planned Impact

Our proposed research project focuses on enhancing photosynthetic efficiency and capacity to substantially increase yield in rice. The proposed research is transformational; although high risk, it has the potential to deliver enormous gains in terms of increased rice yields using an approach that has not previously been utilised in breeding programmes. Modelling studies indicate that a functional CCM in a C3 plant such as rice could result in increase in yield of 36-60% (McGrath and Long, 2014a). The project builds on earlier (but now ended) investments by BBSRC.

Communications and Engagement
Outcomes of the project will be disseminated to the wider scientific community and beyond, through a number of means. Findings will be published in high ranking peer reviewed journals. To reach a broader scientific audience all partners regularly engage in publication of widely read review articles. In addition, the PIs and PDRAs will present their work at international conferences.
The project team will engage with a wide range of audiences (public, policy makers, farmers, and academics). These engagements will encompass wider social issues such as GM and food security as well as the details of the project. In addition, the applicants regularly engage in outreach activities such as visiting schools, organising public open days and providing materials and participation in summer schools.

Collaborations
It is highly possible that direct industrial collaborations will occur during the timeframe of this project. We will identify outputs with potential impact by discussion of the research with industrial contacts. We will also fully engage (visits and correspondence) with national and international agencies working to increase rice yields.

Capability
The PI's have relevant experience and a track record in public dissemination of research findings. Any publicity activities will be coordinated through the Science Communication offices in both host organisations.

Exploitation and Application
Potential intellectual property will be identified and properly protected through the commercialisation offices of both universities. Non-patentable findings will be disseminated to the scientific community through conference presentations and peer-reviewed publications and to the wider public through the media, national and local events.
Material Transfer Agreements will be arranged between the partners in this proposal to safeguard ownership and rights of the research organisations involved, liabilities, and to cover any IP arising. This also covers other important aspects of working together such as acknowledgement of the source of the materials in any publication, provision of raw data, reports or publications or inventions relating to the materials and arising from the specific research programme. The agreement also requires subsequent discussion of any intellectual property arising, and negotiation on how it should be exploited, with what division of work related to its exploitation and the revenues between the partners. The supplier of materials will retain the right to use an Invention for non-commercial research purposes. The PIs have relevant experience and track record in filling patents and in public dissemination of research findings.

Training
The proposed research will provide advanced training in both China and the UK.

Publications

10 25 50
 
Description The minimal set of components required to build a functional carboxysome has been identified, and the gene sequences have been amplified or synthesised de novo. We have expressed the three key carboxysome shell proteins (CcmK2, CcmL and CcmO) in rice protoplasts and have observed fluorescent loci in the stroma, suggesting these proteins are interacting and have been successfully targeted. We have also built individual cassettes for the proteins inside the carboxysome (Rbcl, Rbcs, CcmM58, CcmM35, CcaA), and have demonstrated that these have been successfully targeted to the chloroplast stroma. We are currently assembling constructs to express combinations of the internal proteins, as-well-as constructs to express both shell and internal proteins by making use of the higher-order Golden Gate cloning levels.
We have also used the protoplast transient expression system to identify additional promoters that will be useful to the project. A large suite of available promoters is beneficial since we aim to eliminate the repetition of promoters in multigene expression cassettes in order to avoid undesirable recombination events. In addition, optimising the relative amounts of the expressed proteins in planta will be necessary for correct carboxysome assembly, and this will require an array of promoters of different strengths. We are using a ratiometric fluorescence method to assess the relative strengths of seventeen promoters as yet unused in this project - chloroplast-localised GFP driven by a promoter-of-interest is normalised to cytosolic DsRed driven by the constitutive 35S promoter. The majority of these new promoters are expressed in mesophyll protoplasts, and show a range of strengths, with some conferring very high levels of expression (above the level of the much-used p35S, pAct and pUbi monocot promoters).
As part of this project, it is necessary to silence expression of some rice genes in order to maximise the benefit conferred by installing the carboxysome. The native stromal carbonic anhydrases (CAs) and Rubisco must all be silenced, and we are using genome editing to achieve this. Multiplex CRISPR-Cas9 cassettes have been built, and used to stably transform rice plants. In one construct, we have a cassette that is able to direct editing in the CA and Rbcs genes at the desired target site in planta. Simultaneous editing of the four expressed Rbcs genes is unsurprisingly assumed to be lethal, as triple but no quadruple knock-outs have been recovered. Interestingly, plants with only one copy of one of the Rbcs genes appear to be able to grow as well as wild-type in ambient levels of carbon dioxide. We have also assembled CRISPR-Cas9 cassettes to edit three of the four Rbcs genes (four different combinations) in order to discover more about the roles of each individual protein on Rubisco catalysis.
As-well-as installing the carboxysome, it will also be necessary to localise cyanobacterial carbon transporters to the chloroplast envelope. We are currently building and testing transit peptide-transporter combinations using the transient expression system in order to successfully localise these proteins.
Exploitation Route The findings will be progressed in this and related projects
Sectors Agriculture, Food and Drink

 
Description Information on photosynthetic traits that contribute to CO2 ingress will be incorporated into rice breeding programmes. Data on promoter expression can be exploited in other projects and programmes.
First Year Of Impact 2018
Impact Types Societal

 
Title Transient expression system using rice mesophyll protoplasts 
Description We have developed a high-throughput transient expression system using rice mesophyll protoplasts for assessing the efficacy of expression cassettes prior to stable plant transformation. This, coupled to our extensive library of DNA parts and the highly efficient Golden Gate cloning system, is allowing us to screen large numbers of single and multigene expression cassettes. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact This method coupled to our extensive library of DNA parts and the highly efficient Golden Gate cloning system, is allowing us to screen large numbers of single and multigene expression cassettes and identify useful promoters. 
 
Description Cornell University 
Organisation Cornell University
Department Department of Molecular Biology and Genetics
Country United States of America 
Sector Academic/University 
PI Contribution Led the development of grant proposals building on our previous collaborative research work
Collaborator Contribution Assisted in the development of this grant proposal building on previous collaborative research work
Impact Multiple publications listed under awarded grants. Numerous talks at International events.
Start Year 2012
 
Description Food, Health and Environmental Security 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact New breeding approaches to increase the yield & quality of crops. Palma de Mallorca, February 20 - 22nd, 2018
Year(s) Of Engagement Activity 2018
 
Description 2nd Agriculture and Climate Change Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 2nd Agriculture and Climate Change Conference
Year(s) Of Engagement Activity 2017
 
Description 3rd Synthetic Biology Congress, 20th -21st October, 2016, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited presentation 'Synthesis of Microcompartments in Plants for Enhanced Carbon Fixation' at the 3rd Synthetic Biology Congress, 2016, London
Year(s) Of Engagement Activity 2016
 
Description CCM9 Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at the IXth International Symposium on Inorganic Carbon Utilization by Aquatic Photosynthetic Organisms held in Cambridge.
Year(s) Of Engagement Activity 2016,2017
 
Description DEFRA University Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This workshop brought together a group of universities with Defra policy teams / evidence specialists to explore the opportunities available through academic partnerships (including fellowships, studentships and other grant processes) and the benefits of engaging with external academic experts through developing contacts and networks.
Year(s) Of Engagement Activity 2018
 
Description Invited lecture 'Improving Rubisco' at Enhancing Photosynthesis in crop plants: Targets for improvement' at the Royal Society London December 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited lecture 'Improving Rubisco' at Enhancing Photosynthesis in crop plants: Targets for improvement' at the Royal Society London December 2016
Year(s) Of Engagement Activity 2016
 
Description Keynote for Keys Symposia 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Keynote presentation at Keygene Symposia
Year(s) Of Engagement Activity 2017
 
Description Newton Sustainable Rice Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Increasing photosynthesis and yield of rice through exploitation of a cyanobacterial CO2 concentrating mechanism
Year(s) Of Engagement Activity 2017
 
Description Waitrose Agronomy Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Plant breeding and genetics for enhanced yield
Year(s) Of Engagement Activity 2017
 
Description World Grain Forum 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Keynote lecture and breakfast discussion with minister of agriculture at the World Grain forum in Sochi, Russia
Year(s) Of Engagement Activity 2016
 
Description World Life Science Conference in Plant and Environment session, Beijing 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Keynote lecture 'Improving Photosynthesis the engine of Life to Increase Crop Yields' in Plant and Environment session at the World Life Science Conference, Beijing 2016
Year(s) Of Engagement Activity 2016
 
Description • 'Plenary Lecture at the 1st International Workshop on Food and Health Security, Lima Peru October 2016. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary talk on 'Discovery and creation of genetic variation to increase crop performance for current and future environments' at the 1st International Workshop on Food and Health Security, Lima Peru October 2016.
Year(s) Of Engagement Activity 2016