Collaborative Research: Exploiting prokaryotic proteins to improve plant photosynthetic efficiency (EPP)

The efficiency of light interception and of partitioning of photoassimilate into the harvested components of the major food crops appears to have been fully optimised. Nevertheless, there is still very significant scope for increasing crop productivity by increasing the conversion efficiency of photosynthesis by decreasing the proportion of assimilated carbon that is lost through simultaneous metabolic processes. One such process, photorespiration, is in principle completely expendable and yet can account for assimilate losses of 25-50%. The objective of this proposal is to introduce a cyanobacterial carboxysome-based CO2 concentrating mechanism into the chloroplasts of a model C3 plant (tobacco) in order to reduce, or eliminate, photorespiration. This objective will be addressed using a multidisciplinary approach, through the collaboration of several research groups, each contributing complementary expertise in the essential areas of carboxysome genetics and assembly, plant transformation, photosynthetic systems modelling and physiological and biochemical characterisation of whole plant and component photosynthetic processes. The progress and outcomes from the project will be made available to breeders and to the scientific community, with data being presented on the project website, at scientific meetings, at stakeholder events and in peer reviewed publications

There is very significant scope for increasing crop productivity by increasing the conversion efficiency of photosynthesis by decreasing the proportion of assimilated carbon that is lost through simultaneous metabolic processes. One such process, photorespiration, is in principle completely expendable and yet can account for assimilate losses of 25-50%. The objective of this proposal is to introduce a cyanobacterial carboxysome-based CO2 concentrating mechanism into the chloroplasts of a model C3 plant (tobacco) in order to reduce, or eliminate, photorespiration. The incorporation of higher capacity, cyanobacterial forms of Rubisco into such carboxysomes will also be pursued, having the potential to further enhance photosynthetic capacity and nitrogen use efficiency. These objectives will be addressed using a multidisciplinary approach, through the collaboration of several research groups, each contributing complementary expertise in the essential areas of carboxysome genetics and assembly, plant transformation, photosynthetic systems modelling and physiological and biochemical characterisation of whole plant and component photosynthetic processes. The genetic elements governing the level of expression of cyanobacterial genes in the plant nucleus and chloroplast and the carboxysome proteins essential for the assembly of stable carboxysomes will be determined. We will develop systems modelling algorithms to predict (a) the most effective biochemical and anatomical features to maximise the value of carboxysomes in higher plant chloroplasts; and (b) the optimal investment of resources to maximise nitrogen use efficiency in such plants. To increase our understanding of the operation carboxysomes in chloroplasts, through iterative, high-resolution transmission electron microscopy and immunochemical characterization of carboxysome composition, together with parallel physiological and biochemical characterization of photosynthetic performance, in transformed plants.

This project will address the fundamental issue of the ability to express key prokaryotic proteins in higher plants, while addressing a key societal issue, an ability to keep pace with increasing global demand for grain production. To our knowledge this would be the first expression of a protein-based prokaryotic organelle (carboxysome) in a eukaryote, which in turn would be a prototype for a potential anaerobic compartment for new enzymatic activities, e.g. nitrogen fixation. It would allow the use of a Rubisco that is 3x faster than current C3 plant Rubiscos, allowing a huge reduction in N use. A crop plant model will be the experimental system. It will also deliver a theoretical framework and computational workbench to guide engineering and optimization of components of the CO2 concentrating mechanism. The project will also form a truly cross-disciplinary and trans-national team combining labs which specialize in: a) transformation of plastid and nuclear genomes and engineering genes for appropriate expression (Hanson); b) carboxysome and CA structural, functional and genetic diversity, and their adaptive evolution (Kerfeld); c) metabolic, systems and scaling models of photosynthesis (Long); and d) Rubisco, regulation, molecular physiology and phenotype characterization.(Parry). All collaborators will train postdoctoral associates, who will gain unusually broad training through exposure to the disparate expertise of the PIs and their labs. Depending on their home lab, trainees will receive either intensive training or exposure to cyanobacterial and chloroplast molecular genetics, structural biology, physiology of carbon assimilation, metabolic and systems modeling of photosynthesis, and fluorescence and electron microscopy. Exchanges between labs and international conferences will allow postdoctorals to learn about these diverse research areas and to network with leading researchers from around the world. All collaborating labs will offer undergraduate summer research experiences during the academic year and summer and all will participate in programs that encourage underrepresented minority participation. PI Long's Lab has trained 3-5 McNair US Minority undergraduates through summer research experiences as part of NSF supported research for the past 7 summers. Two of these students have won the annual award for the best research presentation, and 18 of the students have continued into Ph.D. programs in biological sciences at a range of Universities. The McNair movement formally recognized the consistent contribution PI Long had made in 2008. The McNair Scholars Summer Research Institute will assist in identifying appropriate candidates, provide research methods training, a 3-4 day visit to another research school, graduate school application support, and GRE training and support. PI Kerfeld's group will develop a new module for the IMG Annotation Collaboration Tool (IMG-ACT) which is widely used to teach bioinformatics and genomics to undergraduate students in the US23. The module will focus on prospecting for homologs to plant genes in microbial genomes. Subsequent comparative analysis will include sequence similarity, domain identification, protein structure comparison, active site identification and the construction of a phylogenetic tree.