9727236 Chollet This renewal project is focused on this laboratory's long-standing interest in the control of plant primary metabolism by phosphorylation/ dephosphorylation cycles of regulatory proteins. The two related areas of overall investigation specifically concern phosphoenolpyruvate carboxylase (PEPC) and sucrose synthase (SuSy), two target enzymes known to undergo seryl-phosphorylation in intact leaves and N2-fixing legume nodules. While cytosolic PEPC is best known for its cardinal role in catalyzing the fixation of atmospheric CO2 during C4 photosynthesis and Crassulacean acid metabolism (CAM), this ubiquitous plant carboxylase also functions in a vast array of nonphotosynthetic contexts, including C/N-metabolism in general (e.g., in C3 leaves) and in the more specialized cases of N2-fixing root nodules, leaf guard cells, and seed germination. The multifaceted nature of plant PEPC makes its intricate control by a highly regulated, Ca2+-independent protein-Ser/Thr kinase (PEPC-kinase) and an opposing protein phosphatase-2A an extremely attractive target for continued study. Accordingly, two of the three specific research objectives of this renewal project focus on (1) C4 and/or CAM PEPC-kinase, and (2)its requisite but seemingly diverse signaling pathways in leaves (C4, Ca, CAM) and root nodules. Recent work has shown that SuSy, in addition to PEPC, is a target for seryl-phosphorylation by a soluble, Ca2+-dependent protein kinase in two physiologically distinct plant "sink" tissues: elongating maize leaves and soybean root nodules. These are potentially significant observations because virtually nothing is known of the posttranslational control of SuSy activity or of how the enzyme physically partitions between the plasma membrane and cytosol in plants. Clearly, reversible changes in its phosphorylation state are a possible but as yet unexplored mechanism for these phenomena. These newly emerged questions about plant SuSy will be addressed in Objective 3 of this renewal project by the continued biochemical and molecular investigation of the phosphorylation/dephosphorylation of nodulin-100, the nodule-enhanced form of this sucrose-cleaving enzyme in soybean. The major significance of this basic research project is its delineation of important fundamental details of the regulation of carbon-dioxide assimilation (by PEPC) and sucrose cleavage (by SuSy) by reversible protein phosphorylation in various tissues of agronomically important crop species, including maize, tobacco, and soybeans. At the same time, the results may provide the requisite underpinning for the constructive manipulation of plant carbon/nitrogen-metabolism and its control mechanisms through biotechnology. It must be emphasized that PEPC and SuSy represent two of the handful of documented examples of plant metabolic enzymes targeted by protein kinases and protein phosphatases in planta. Thus, the research will provide much-needed insight into the control of plant metabolism by reversible protein phosphorylation.
