A major factor limiting photosynthetic efficiency in many crop plants is oxygen-induced inhibition of CO2 fixation by the enzyme RuBisCO, a first step in a process known as photorespiration. Photorespiration leads to CO2 release rather than fixation. Overall, photorespiration reduces the efficiency of photosynthesis by as much as 30%. To date, attempts to engineer reduced photorespiration have largely been unsuccessful. Several groups of organisms including, cyanobacteria, eukaryotic microalgae, and C4 plants have evolved mechanisms to concentrate CO2 near the active site of RuBisCO, reducing photorespiration. Based on knowledge gained from investigations on the biochemistry and cell biology of carbon concentrating mechanisms in algae, this project will engineer the C3 model plant, Arabidopsis thaliana, to concentrate CO2 near the active site of RuBisCO so as to favor CO2 fixation. The project will additionally quantify the expected changes in plant productivity at the biochemical and systems levels (isotopic labeling and metabolic flux analysis of leaves). The research strategies include: 1) enhancing photosynthetic carbon fixation by engineering the overexpression of bicarbonate transporters and other inorganic carbon metabolizing enzymes in leaves of Arabidopsis thaliana, and 2) developing leaf isotopic labeling and flux analysis methods to quantify photosynthetic metabolism and respiration in wild-type and mutant leaves. Results from flux analyses will be used to identify metabolic bottlenecks, assess the overall CO2 metabolism, and establish a platform to enable further metabolic engineering strategies for improving overall photosynthetic efficiency.
Broader Impacts. The scope of the research is highly interdisciplinary. Therefore, it is anticipated that the project will attract the interest of individuals from diverse backgrounds and education. The work will involve undergraduates, secondary science students, teachers, and scientists. The offering of summer internships to teachers and undergraduates, as well as professional development workshops for teachers, and an on-line mentoring program (eScience) involving student and scientist partners will result in a greater appreciation and understanding of science and stress the relevance of the research to food production and green energy that are aspects of everyday life. The transgenic lines and molecular tools, along with flux analyses and computational methods, will add to the growing body of information on leaf photosynthetic metabolism and engineering. Results will be disseminated through traditional journals and meetings, and the software and isotope data produced will be publicly available via the Web to serve as a valuable resource for research and teaching. From a societal context, increases in plant biomass serve to meet growing nutritional and chemical feedstock needs without a reliance on petroleum-based approaches.
Our overall objectives were to enhance photosynthetic carbon fixation in C3 plants by combining a metabolomics guided approach with engineering transgenic plants overexpressing the minimal number of genes from the algal carbon concentrating mechanism (CCM) in C3 plants to elevate chloroplast CO2 levels and enhance carbon fixation efficiency. As part of our objectives we developed comprehensive metabolic flux programs (INCA) and models to describe the path of carbon in C3 plants under atmospheric and elevated CO2 levels. Significantly, we demonstrated by isotopic and mass spec analyses, that C3 plants make insufficient ATP to support the energy-dependent bicarbonate uptake, a prerequisite for a functional CCM. This deficiency in ATP production could be complemented, however, by coexpression of transgenes that enhance cyclic photophosphorylation. In addition, we have identified a novel bacterial carbonic anhydrase (BCA) that enhances photosynthetic carbon fixation by 25%. Surprisingly, 13CO2 labeling and gas exchange studies independently indicate that the carboxylation efficiency in the BCA transgenics is not enhanced but that total carbon flux through the Calvin-Benson Cycle and the photorespiratory pathway is elevated. These results suggest that the effect of BCA on photosynthetic efficiency is not only due to direct effects on CO2 availability. The minimal suite of CCM genes to reconsitute a functional CCM is currently being expressed in engineered C3 plants.
