14-PSIL Combining Algal and Plant Photosynthesis (CAPP2)

In most plants, growth rate is limited by the rate at which carbon dioxide from the atmosphere is taken up and converted to sugars in the process of photosynthesis. The enzyme responsible for the first step in this process, Rubisco, does not work at its potential maximum efficiency at the current levels of carbon dioxide present in the atmosphere. If levels were much higher, photosynthesis would be increased and plant productivity would be higher. There is an immediate requirement for increased crop productivity to provide food for the rising population of the planet. Our project addresses this problem. We are studying a mechanism present in unicellular green algae that results in high concentrations of carbon dioxide inside their photosynthesising cells (called a Carbon Concentrating Mechanism, or CCM), enabling Rubisco to work at maximum efficiency. During the initial CAPP1 programme, we discovered important new information about this mechanism, and using new and rapid methods we have identified novel algal genes and additional regulatory components which allow the CCM to operate in association with a specific micro-compartment called a pyrenoid. We have also successfully introduced some of these components into a model higher plant, Arabidopsis, and also successfully introduced a modified form of Rubisco which may facilitate aggregation into the pyrenoid. The ambitious goals of the CAPP2 extension will be to combine the expression of the CCM and pyrenoid in the Advanced Plant. Firstly, we will continue to identify genes required by the algae to achieve high concentrations of carbon dioxide inside the cells, and develop new markers and sensors to reveal the location and activity of these genes when expressed in the higher plant. Secondly, we will identify additional regulatory elements needed to form a pyrenoid, as well as exploring the impact on Rubisco enzyme efficiency and light utilisation. Thirdly, we will continue to introduce successive components into our model Advanced Plant so as to "stack" up the activities of CCM components and examine the extent of pyrenoid formation and enhanced productivity associated with the CCM. This work will provide new insights into how plants and algae acquire and use carbon dioxide from the atmosphere, of great importance in predicting and coping with the current rapid changes in the atmosphere and hence in climate. The work will also contribute to strategies to increase global food security, because it will indicate new ways in which crop productivity can be increased.

The CAPP2 programme will continue to discover and characterise new components of the Chlamydomonas CCM, in addition to known bicarbonate pump(s) and carbonic anhydrases, and identify regulatory elements, protein modifications, linkers and chaperones needed to aggregate Rubisco into a pyrenoid and progressively incorporate key components into Arabidopsis. Our approaches include:
1) Identify and characterize components of the pyrenoid in Chlamydomonas, including growth phenotyping, complementation to identify the mutant locus and fluorescence tagging to identify prenoid-associated proteins. Pyrenoid defects will be screened from microscopy, Rubisco kinetics, CCM physiology and light use.
2) Pyrenoid structure, assembly and regulation will be defined using block-face scanning EM in Chlamydomonas and Advanced Plant chloroplasts. Chaperones and linkers will be identified using AUC and BN-PAGE, and other post-translational modifications including phosphorylation, ubiquitination and S-S bond forma tion by LC-MS/MS.
3) To assemble a pyrenoid in the Advanced Plant gene cassettes will be assembled and/or stacked into delivery vectors for transformation. Additional "known" CCM components will be introduced and location tested by fluorescent tags (e.g. GFP), and levels of untagged components by polyclonal antibodies. Repression of all native Rubisco in Arabidopsis double mutants, and CCM characteristics will be combined in a single plant.
4) The advanced plant lines will be characterized physiologically to determine effectiveness of introduced CCM.
5) A FRET nanosensor will be developed to allow bicarbonate pools accumulated through the CCM to be visualized.
6) Modelling of inorganic carbon accumulation and effectiveness of pyrenoid operation will be undertaken in collaboration with colleagues at UIUC Illinois.
7) A central repository of genetic tools and materials will be developed for open access to the scientific and commercial communities.

1. Academics and researchers in all fields of plant research.
2. Annotators in genomics and metabolomics, database and germplasm curators.
3. UK, US and international science base.
4.Agro-industry including biotechnologists and plant breeders seeking to increase plant productivity and/or harvest index; metabolic engineers and metabolic modellers.
5. Agricultural community and advisors.
6. Postdoctoral researchers employed on the project.
7. Public
8. The next generation: school children and undergraduate students
9. Multinational and Government Agencies
How will they benefit from this research?
1. Researchers will receive comprehensive new information about the CCM of algae, requirements for CO2 concentration in higher-plant chloroplasts, and mechanisms of assembly of supra-molecular complexes.
2. Researchers will have access at the point of publication to new genome annotation in Chlamydomonas, novel Arabidopsis material with altered primary carbon assimilation and models describing the relationship between the spatial distributions of inorganic carbon substrates and enzymes and the process of CO2 assimilation in chloroplasts.
3. The research will have a major impact on understanding of photosynthetic CO2 assimilation and its relationship to inorganic carbon concentrations in the chloroplast.
4. Agro-industry will receive information to underpin rational approaches to increase plant productivity, and relevant new genes and modelling methodologies.
5. The agricultural community will benefit in the longer term from sustainable crop improvements enabled by our research.
6. The PDRAs will receive a wide training in plant integrative biology, professional skills and wider training courses, and the opportunity to interact closely with researchers on an international scale. They will also receive training in transferable skills such as presentation and dissemination of results, and grant-writing. Enhanced career trajectory is already evident from support for the move of Dr McCormick to Edinburgh.
7. Our research findings relate to issues of public interest including sustainable crop production, global food security and atmospheric and climate change.
8. Our research has wide educational value, at all levels through schools and Universities
What will be done to ensure they benefit from this research?
1. Publish results in high-impact journals in a timely fashion, with open access where possible. Present research results at international meetings and institutions
2. Submit data and models to relevant international depositories. Notify new/corrected gene and enzyme annotations to community databases
3. Exploit extensive existing contacts of the PIs with other academics with relevant research interests as soon as any exploitable results/materials are generated.
4. Make informal contacts with biotechnologists as soon as exploitable results are generated; recognise and protect PI to ensure wise and fruitful exploitation. Collectively we have vibrant contacts with relevant industries.
6. Provide information and mentoring to ensure uptake of postdoctoral training schemes, including regular progress reviews and career development plans. Encourage participation in the dissemination of results, and understanding of the wider implications and applications of the research.
7. Use results as part of our regular engagement with non-academic audiences, e.g. local interest groups, schools, local and national shows, science showcases, media.
8. Involve school children and undergraduate students in a practical sense (visits, websites providing teaching resources, blogging and laboratory summer secondments for high school students and undergraduates).
9. Seek opportunities to inform the work of UN agencies and DFID (UK) in the developing world, and the CGIAR international network of germplasm repositories and strategic regional research.