Dynamic analysis of metabolism under circumstances of altered photorespiratory flux

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Recent studies have revealed unexpected complexities both within the pathway of photorespiration as well as with respect to the general network of photosynthetic metabolism. Photorespiration has been clearly shown to branch four compartments and interacts with folate metabolism, ethanolamine metabolism and proton dissipation across the mitochondrial membrane. Clarification of such interactions will be crucial both for fundamental understanding of the pathways involved and in maximizing biotechnological approaches to manipulate them. For this purpose, a combination of metabolomics and flux profiling approaches will be established and applied to Arabidopsis material either harboring genetic lesions at appropriate reaction steps or subjected to altered environmental conditions. In specific instances, this will be carried out in conjuncture with the non-aqueous fractionation technique in order to resolve subcellular aspects of metabolic regulation. It is anticipated that results obtained will be highly informative in enhancing our understanding of how, photorespiration, and processes intimately related to it, is conditionally regulated and furthermore by defining the reaction steps at which this regulation is enforced. In the first funding period, we made significant progress with respect to several of these experimental goals as well as setting up the necessary expertise to enable the performance of further experiments which will allow us to fully address outstanding issues. Partially via collaborative efforts we collated large multi-level data for growth in non-photorespiratory conditions and after transition to ambient air, as well as transfer to different temperatures, different light intensities and reciprocal variations in carbon dioxide and oxygen partial pressures. These studies provided a wealth of information on the interaction of photorespiratory pathways to related metabolic and transcriptional processes. Aspects of the work will be brought forward into the second period including comparison with well characterized mutants of over aspects of plant energy signaling. In parallel we set up a novel method for 13CO2 feeding, including kinetic tracing of the heavy label through primary metabolism both at the metabolic and compartmental levels. As a first approach this method was applied to characterizing metabolism in ambient air. Fluxes calculated from this model were benchmarked against major fluxes determined by independent methods such as the overall rates of photosynthesis and photorespiration and sucrose and starch biosynthesis. In the second period it is proposed to continue and complete experiments in which this method is applied to plants in which photorespiratory fluxes have been perturbed by environmental or genetic means. Together with other Promics partners these data will be fed both into a relational database and a computational model aimed at improving and thereby allowing us to influence metabolic regulation and interaction of the photorespiratory pathway.