RUI: Uncovering the Occurrence and Mechanism of Regulatory Phosphorylation of Non Photosynthetic Pyruvate,Orthophospate Dikinase in C3 Plants

Minnesota State University Moorhead, Moorhead MN

Investigators

Abstract

The enzyme pyruvate,orthophosphate dikinase (PPDK) is most well known as an enzyme of the C4 photosynthetic pathway where it catalyzes the formation of PEP (phosphoenolpyruvate), the primary CO2 acceptor molecule, from pyruvate. Because it is a key enzyme in the C4 pathway, PPDK activity is highly regulated so the overall pathway can function optimally in CO2 assimilation. This regulation is conferred by reversible phosphorylation of a PPDK active-site threonine, with the enzyme being inactive in the phosphorylated state. A single bifunctional protein kinase/phosphatase enzyme, PPDK regulatory protein (RP), catalyzes this regulatory phosphorylation and dephosphorylation. PPDK is also present in C3 plants although it does not participate in photosynthesis. Further, it is present in all tissues of the plant but in very low amounts. Its function in C3 plants is essentially unknown, although it is proposed to participate in cellular metabolism and guard cell function. Recently, it was demonstrated that PPDK in leaves of C3 plants undergoes light/dark mediated regulatory phosphorylation similar to C4 leaf PPDK. The major aim of this project is to further characterize C3 plant PPDK regulatory phosphorylation by identifying organ, tissue, cellular, and subcellular sites of regulatory PPDK phosphorylation. Further, the C3 RP or RP-like activity mediating reversible phosphorylation of PPDK in C3 plant tissue will be scrutinized in vitro for understanding its biochemical nature. Finally, this project seeks to clone the gene for C4 PPDK RP from a maize cDNA library. This will provide the "ultimate" tool for resolving the perplexing riddle of how this uniquely bifunctional enzyme is regulated in C4 plants, as well as the requisite gene probe for understanding its precise expression in C3 and C4 plants. In summary, the experimental outcomes of this project will enable the hitherto unknown metabolic function(s) of PPDK in C3 plants to be elucidated.