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Periodic Report Summary 3 - 3TO4 (3to4: Converting C3 to C4 photosynthesis for sustainable agriculture)

Project Context and Objectives:
Most plants use the C3 pathway of photosynthesis that is compromised by gross inefficiencies in CO2 fixation. However, some plants use a super-charged photosynthetic mechanism called C4 photosynthesis. The C4 pathway is used by the most productive vegetation and crops on Earth. In addition to faster photosynthesis, C4 plants demand less water and less nitrogen. Overall, our aim is to introduce the characteristics of C4 into C3 crops. This would increase yield, reduce land area needed for cultivation, decrease irrigation, and limit fertiliser applications. If current C3 crops could be converted to use C4 photosynthesis, large economic and environmental benefits would ensue from both their increased productivity and the reduced inputs associated with the C4 pathway. It is important to note that the huge advances in agricultural production associated with the Green Revolution were not associated with increases in photosynthesis, and so its manipulation remains an unexplored target for crop improvement both for food and biomass. Even partial long-term success would have significant economic and environmental benefits. Efficient C4 photosynthesis would be achieved by alterations to leaf development, cell biology and biochemistry. Although initially we will be using model species such as rice and Arabidopsis we envisage rapid transfer of technological advances into mainstream EU crops, such as wheat and rape, that are used both for food and fuel. We will build capacity for C4 research in Europe in this area by the training of future generations of researchers. To achieve this aim we need to increase our understanding of the basic biology underlying the C4 pathway. Our specific objectives will therefore address fundamental aspects of
C4 biology that are needed for a full understanding the pathway. In addition, to complement this long-term work we will also increase photosynthesis by introducing a synthetic bypass for photorespiration into European crops. We will also build capacity for C4 research in Europe by the training of future generations of researchers.
Specifically we aim:
(i) to adapt, via metabolic engineering and appropriate phenotypic screening techniques, photosynthetic CO2 assimilation mechanisms in C3-type terrestrial plants towards more efficient C4 architecture and biochemical mechanisms in crop species of economic relevance.
(ii) to increase biomass yields (e.g. by reducing photorespiration).
(iii) to utilise appropriate training opportunities (e.g. summer schools) aimed at early-career stage researchers and to build European research capacity in this area.
(iv) to investigate the response of the modified C3→C4-type plants to abiotic stress, in particular drought.

Project Results:
WP 1/2. In DUS The reporter gene construct GLDPA-Ft::LUC68 was stably transformed into Arabidopsis thaliana (Columbia). DUS expects to identify the first BSOM genes obtained by EMS mutagenesis in late 2014 and will start immediately on their verification by RNAi-driven knockout experiments and by their over-expression in the bundle-sheath cells of Arabidopsis.

WP 3/4. The sampling of C3 and C4 transcriptomes from dicots is complete. Work suggests that C4 cycle genes are not recruited from the same expression domain in C3. DUS has obtained libraries and sequenced transcriptomes from mesophyll and bundle sheath cells of C4 plants, detecting transcripts that accumulated preferentially in the M and BS respectively of C4 Cleome gynandra. This included transcription factors in these cells. The transcriptome of the PEPC mutant Amaranthus edulis was sequenced and quantified.

WP 5. The identification of the histone methyltransferase responsible for cell-type specific promoter modifications in maize identified five candidate maize genes. Together, combined approaches using RNAi lines, mutant resources and in vivo binding studies already revealed two candidates for methyltransferases that control C4 genes. A cDNA expression library has been constructed ready to be used for the yeast one hybrid (Y1H) screenings. These screenings allowed identification of three rice TFs binding to ZmPEPC1 promoter.

WP 6. In iBET and USFD studies are proceeding on post-translational modification (including phosphorylation) of C4 enzymes. 8 new putative phosphorylation sites were detected for PEPC , 3 new putative sites for PPDK, and a site for NADP-ME. More antibodies have been raised at USFD.

WP 7. SIBS has developed a kinetic systems model of Kranz-type C4 photosynthesis that includes a detailed description of not only the Calvin-Benson cycle, starch synthesis, sucrose synthesis, C4 shuttle and CO2 leakage, but also photorespiration and intercellular metabolite transport. Assessment of enzyme activities and fluxes by 13C labelling is proceeding at MPG using a combination of GC-MS and LC-MS/MS to quantify each isotopomer in metabolites including compounds from the Calvin-Benson cycle, organic and amino acids. Non-aqueous fractionation is being developed on both cell types to identify the subcellular distribution (cytosol, plastid and vacuole) of the labelled metabolites.

WP 8. The first round of oilseed transformation at NIAB generated unviable transformed plants. Two new constructs were designed and the rape plants transformed with the photorespiratory by-pass were generated at NIAB/CAM in summer 2013. However, the evidence suggests that plants transformed with the by-pass do not show a sufficiently strong phenotype to warrant an extensive work programme. A decision was taken to redefine WP8 using the natural photorespiratory by-pass in Moricandia. This work is now in progress.

WP 9. All constructs test candidate regulators of Kranz anatomy in rice have been generated and are now in the transgenic pipeline. CAM has sent constructs to IRRI to allow ribosomes to be tagged and then isolated in mesophyll or bundle sheath cells of rice. These plants are currently being tested. Crossing has taken place to generate double transformants for five of the C4 cycle transgenics to construct the C4 pathway in rice.

WP 10. Cels was unable to accede to the Grant Agreement leading to a 6-month delay in the start of the project. The proteomics work was taken over by iBET. Full consortium meetings were held in February 2012, June 2012, January 2013, July 2013, January 2014, June 2014, January 2015 and July 2015. Communication has been fully maintained with the C4 Rice programme at IRRI. A Risk register has been maintained. Quarterly on-line meetings have been held within the four pillars and progress reports have been posted onto the 3to4 website.

WP 11. The first summer training school on C4 photosynthesis was associated with the Annual Meeting that took place in Salzburg in June 2012. The second training school on Bioinformatics took place at Nebion in Zurich in August 2013. A number of secondments have taken place. The third training course was held at Potsdam in July 2014 and the fourth at Florence in July 2015.

WP 12. Meetings in Salzburg 2012, Potsdam 2014 and in Florence in 2015 were recorded and posted on the 3to4 website. Publications are available on the website and a large number of public dissemination activities by consortium members have taken place.

Potential Impact:
The UN Food and Agricultural Organization predict that crop yields need to increase 70% over the next 35 years to keep pace with projected population growth and dietary change. Increasing the productivity of the major food crops is a major challenge in maintaining food security and in countering the current large rises in the costs of these basic commodities. Increasing photosynthetic efficiency is seen as a key factor in meeting this challenge and helping to reduce the yield gap. At the time of writing the final scientific results cannot be foreseen as the project has effectively only been running for 12 months. However, the work is proceeding in tandem with a considerable effort worldwide to introduce C4 photosynthesis into C3 crops and to improve the efficiency of photosynthetic carbon fixation. Indeed elsewhere the Calvin cycle has already been manipulated to increase photosynthesis and production, and it is now possible to envisage the manipulation of Rubisco, carbon concentrating mechanisms and C4 photosynthesis on a scale that was not even imaginable even a few years ago. Nevertheless, attempts at crop improvement by manipulating photosynthesis remain in their very early stages, partly because food security has only recently emerged on the political agenda together with the realization of the importance of manipulating photosynthetic carbon fixation as an emerging route to increasing crop productivity, when other routes appear to be reaching their biological limits. The process of identifying what to manipulate, and how, continues and is a fundamental part of the 3to4 programme. The results from 3to4 will be directly exploited for use in the development of novel C4 crops in this and other consortia.
Effective collaborations are already being forged between partner institutions. Together, the participating researchers and the co-operating institutions will establish a close network, which will form a fertile source of potential future collaborations and research initiatives. Clearly the potential impact on both the ability to increase biomass, to reduce poverty by producing more food, and generate more material for biofuels is enormous. Even partial long-term success would have significant economic and environmental benefits. The first training programmes have already been successful at forming an esprit de corps among the researchers and this can be expected to increase as more secondments occur. Thus, a direct benefit of 3to4 will be establishment of a durable and effective European network built on a strong foundation of exceptional expertise that will be necessary for the duration of research in this area, which is likely to last for the next 15-20 years.

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Record Number: 188173 / Last updated on: 2016-08-24