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

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The enzyme pyruvate,orthophosphate dikinase (PPDK) is most well known asan enzyme of the C4 photosynthetic pathway where it catalyzes theformation of PEP (phosphoenolpyruvate), the primary CO2 acceptormolecule, from pyruvate. Because it is a key enzyme in the C4 pathway,PPDK activity is highly regulated so the overall pathway can functionoptimally in CO2 assimilation. This regulation is conferred byreversible phosphorylation of a PPDK active-site threonine, with theenzyme being inactive in the phosphorylated state. A single bifunctionalprotein kinase/phosphatase enzyme, PPDK regulatory protein (RP),catalyzes this regulatory phosphorylation and dephosphorylation. PPDK isalso present in C3 plants although it does not participate inphotosynthesis. Further, it is present in all tissues of the plant butin very low amounts. Its function in C3 plants is essentially unknown,although it is proposed to participate in cellular metabolism andguard cell function. Recently, it was demonstrated that PPDK in leavesof C3 plants undergoes light/dark mediated regulatory phosphorylationsimilar to C4 leaf PPDK. The major aim of this project is to furthercharacterize C3 plant PPDK regulatory phosphorylation by identifyingorgan, tissue, cellular, and subcellular sites of regulatory PPDKphosphorylation. Further, the C3 RP or RP-like activity mediatingreversible phosphorylation of PPDK in C3 plant tissue will bescrutinized in vitro for understanding its biochemical nature. Finally,this project seeks to clone the gene for C4 PPDK RP from a maize cDNAlibrary. This will provide the "ultimate" tool for resolving theperplexing riddle of how this uniquely bifunctional enzyme is regulatedin C4 plants, as well as the requisite gene probe for understanding itsprecise expression in C3 and C4 plants. In summary, the experimentaloutcomes of this project will enable the hitherto unknown metabolicfunction(s) of PPDK in C3 plants to be elucidated.