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

Lead Research Organisation: Rothamsted Research
Department Name: Plant Biology & Crop Science

Abstract

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

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The photosynthetic efficiency of C3 plants suffers from the reaction of Rubisco with O2 instead of CO2, leading to the costly process of photorespiration. Increasing the concentration of CO2 around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO2-concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have have transiently expressed multiple ß-carboxysomal proteins in tobacco leaves with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the ß-carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17-amino-acid CcmN peptide to the shell proteins inside chloroplasts. In tobacco plants we also managed to replace the native tobacco Rubisco with that from cyanobacteria and recover transformed lines that were photosynthetically competent, supporting autotrophic growth, at near wild type rates at elevated CO2 concentrations but with much less Rubisco. These are important step towards improved photosynthesis in plants and establish the feasibility of introducing a functional cyanobaterial concentrating mechanism into chloroplasts of crop plants
Exploitation Route The micro-compartment may be used for other purposes such as nitrogen fixation or packaging of phramaceuticals
Sectors Agriculture, Food and Drink

 
Description Bilateral NSF/BIO-BBSRC Synthesis of Microcompartments in Plants for Enhanced Carbon Fixation
Amount £1,400,000 (GBP)
Funding ID BB/N016009/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2016 
End 11/2020
 
Description Rice Research Newton Fund: Exploiting a Cyanobacterial CO2 Concentrating Mechanism to Increase Photosynthesis and Yield in Rice
Amount £341,000 (GBP)
Funding ID BB/N013662/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2016 
 
Description Cornell University 
Organisation Cornell University
Department Department of Molecular Biology and Genetics
Country United States 
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
 
Title Production of Bacterial Microcompartments in Eukaryotic Cells 
Description Production of Bacterial Microcompartments in Eukaryotic Cells 
IP Reference Provisional applications filed: 61/820,871 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact Some media interest
 
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 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 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