Regul3P

Terrestrial life on Earth relies on plant photosynthesis to produce oxygen and to assimilate CO2 into organic matter. The most abundant enzyme in leaves is the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) that produces, by its carboxylase activity, the building blocks to make sugar phosphates and all other organic molecules within the plant. However, RuBisCO has also an oxygenase activity that produces 2-phosphoglycolate and other toxic metabolites that must be metabolized. This is achieved by the photorespiratory cycle. However, this important metabolic pathway has a cost since it uses energy and reducing power and it also leads to the liberation of assimilated carbon and nitrogen as CO2 and ammonia that is either re-assimilated (again at a certain energetic cost) or lost to the atmosphere. Therefore, the photorespiratory cycle has been described as “wasteful” to plant productivity and therefore it is a good target to manipulate with respect to improving plant yield. Indeed, Arabidopsis plants with a modified photorespiratory metabolism show an improved biomass compared to wild-type plants. Although we now have a good understanding of the eight core photorespiratory enzymes as well as certain by-pass pathways, the regulation of the photorespiratory cycle is poorly understood and little is known about how its interactions with other plant metabolic pathways and functions (photosynthesis, respiration, N-metabolism, C1 metabolism) are coordinated. Recent data show that all but one of the core photorespiratory enzymes can be phosphorylated. Generally protein phosphorylation leads to a modulation in protein function such as activity, sub-cellular localization, capacity to interact with other proteins, and stability. The overall aim of this project is to elucidate the role of protein phosphorylation in the control and regulation of the photorespiratory cycle. The will be achieved by (i) monitoring changes in photorespiratory enzyme phosphorylation state as a function of leaf photorespiratory activity by targeted phosphoproteomics, (ii) understanding the effect of phosphorylation on photorespiratory enzyme activity and kinetic properties by analyzing recombinant non-phosphorylatable or phosphorylation-mimic enzymes, (iii) evaluating the impact of photorespiratory enzyme phosphorylation/nonphosphorylation on plant physiology and metabolism by transforming mutants to express mutated photorespiratory enzymes and (iv) identifying protein kinases responsible for photorespiratory enzyme phosphorylation in peroxisomes. Our project will bring together the actors and the expertise necessary to achieve a better understanding of how each phosphorylation event affects photorespiratory enzyme function, and how this impacts on the photorespiratory cycle and interacting metabolic pathways. To attain our goals we will use complementary approaches including proteomics, phosphoproteomics, recombinant protein technology, site-directed mutagenesis, affinity chromatography, and targeted (using LC-MS, HPLC methods) and non-targeted (GC-TOF-MS) metabolite analyses. The novel data generated will increase our fundamental knowledge concerning this key metabolic pathway and its interaction with neighboring metabolisms. The new tools and results will indicate the plant metabolic pathways that can/should be modified to improve plant fitness in a changing environment (e.g. increased CO2 levels and temperature), with the aim of maintaining yield (biomass) using lower input amounts (e.g. fertilizers). Thus, we anticipate that the project will contribute to defining components and processes that can subsequently be validated in agriculturally important crop species as part of the ongoing effort to understand and promote appropriate strategies to improve yield in such species.