Creating a spatially defined, multidimensional, protein interactome of the eukaryotic algal CO2 concentrating mechanism

Photosynthesis harnesses energy from the sun to fix carbon dioxide (CO2) into sugars and the protein building blocks of life. To enhance photosynthesis some plants and nearly all algae have evolved mechanisms to increase the accumulation of CO2 from their surrounding environment, this process in known as a CO2 concentrating mechanism (CCM). It is predicted that the transfer of a CCM to crop plants, such as rice and wheat that have failed to evolve CCMs, could increase yields by up to 60%. A promising CCM donor candidate is a green alga called Chlamydomonas, which has a highly efficient CCM.

For the successful implementation of an algal CCM into crop plants it is essential that all the components are known, where they are found in the cell and how they function together. This project aims to rapidly identify all the proteins that make up the Chlamydomonas CCM and to determine how they interact with each other to form a functional unit that enhances CO2 uptake. To achieve this, we will determine where the different protein components of the CCM are located in the algal cell, we will then identify what proteins they are interacting with and how these interactions change when the CCM is switched on and off. This will allow us to understand the network of the CCM and how it is regulated. Finally, using mutants that lack individual proteins of the CCM, we will determine the functional importance of each protein component.

The localisation, interaction and protein function data will be combined to create a detailed 3D map of the CCM that can be easily explored through an open-access, online interactive viewing platform. It is anticipated that these data will facilitate the transfer of a CCM into crop plants to increase photosynthesis and yields.

The passive diffusion of CO2 from the surrounding environment to the active site of Rubisco can be limiting for photosynthesis. To overcome this nearly all algae and some plants have evolved CO2 concentrating mechanisms (CCMs) to concentrate CO2 in the proximity of Rubisco, saturating its active site, resulting in increased photosynthetic rates. CCMs are fascinating examples of highly coordinated protein networks. Elucidating protein-protein interactions, how they are regulated and how they change in response to environmental cues is one of the key steps in understanding biological processes. The proposed work aims to build a dynamic, spatially defined, protein-protein interaction network of the Chlamydomonas reinhardtii CCM.

To achieve a dynamic, spatial understanding of the CCM, 60 CCM proteins will be fluorescently tagged and imaged by time-lapse confocal microscopy during the induction of the CCM. In parallel, to understand changes in protein-protein interactions, affinity purification mass spectrometry will be performed in CCM on and CCM off conditions. Analysis of proteins for changes in post translational modifications (PTMs) during CCM induction will shed-light on the regulation of the CCM. To give further insight into the protein environment of CCM components we will apply a biotin labelling proximity assay. This data will allow us to identify weak interactions and proximally close proteins within the CCM network. In addition, we will perform a targeted mutant phenotype screen of all the protein-protein interaction network components. Mutants in components will be picked and growth rates quantified at high and low CO2 levels to identify mutants that have CCM defects.

The localisation, protein-protein interaction, protein proximity, PTM and phenotype data will be integrated to create a detailed, interactive 3D map of the CCM. It is anticipated that this dataset will guide the future transfer of CCM components into crop plants to improve yields.

In the short to medium term, UK biotechnology companies will benefit from technical advances made in the project. These include new algal synthetic biology tools and methods for the high-throughput expression and screening of foreign proteins in algae. I will actively build relations with companies to make them aware of technical advances. During the first year of the project I will approach UK based Algenuity (www.algenuity.com) and Sphere Fluidics (www.spherefluidics.com) about synthetic pathway assembly, high-throughput strain screening and foreign protein expression in Chlamydomonas using approaches developed within the project. Furthermore, I will attend and present work from my lab at leading UK and European algae biotechnology conferences.

In the long term, agricultural biotechnology companies such as Bayer CropScience and Syngenta may benefit from this research, as it will produce key targets for the improvement of photosynthesis in higher plants. These companies could use the full functional understanding of a CCM generated from the proposed research to transfer key components into crops. This would have the potential of increased yields and decreased water and nitrogen demands. If realised, these benefits would increase land use efficiency and crop drought resistance potentially providing multiple benefits to society. Companies will have access to the data through publications, online outlets including an interactive online viewing portal and through attendance and presentations at national and international meetings. In addition, charities that are already actively involved in funding photosynthesis research to improve crop yields, such as the Bill and Melinda Gates foundation, will be approached through links already established with the Centre for Novel Agricultural Products (CNAP) at York.

To ensure that the data generated from this project is freely available and easily accessible we will develop an online viewing website. To help in design and implementation the PDRA will work alongside York based website development companies. The PDRA will develop transferable skills in website design and implementation, and local companies will benefit from exposure through access to the website. The website and its development process will be of interest to other research groups that are involved in making large datasets easily available in an interactive manner.

Non-scientists will appreciate the understanding of how algae have evolved to become highly efficient at photosynthesis and the potential of this knowledge to improve future crop yields. They will have access to our findings through: 1) University of York Outreach via the University website and press releases, 2) communication through the Mackinder Lab website and Twitter, 3) talks by the PI at local events including the Pints of Science public debate and 4) interactions with secondary school students through the Future First network.

The project will give early career scientists training opportunities and lab experience. It is expected to provide short research placements for several undergraduate and high school students.