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

丙酮酸正磷酸二激酶（PPDK）是C4光合途径中最著名的酶，它催化丙酮酸形成PEP（磷酸烯醇丙酮酸），PEP是主要的CO2受体分子。因为它是C4途径中的关键酶，所以PPDK活性受到高度调节，因此整个途径可以在CO2同化中发挥最佳作用。这种调节是由PPDK活性位点苏氨酸的可逆磷酸化赋予的，该酶在磷酸化状态下无活性。一种单一的双功能蛋白激酶/磷酸酶，PPDK调节蛋白（RP），催化这种调节磷酸化和去磷酸化。PPDK不参与光合作用，但也存在于C3植物中。此外，它存在于植物的所有组织中，但含量非常低。它在C3植物中的功能基本上是未知的，尽管它被提出参与细胞代谢和保卫细胞功能。最近的研究表明，C3植物叶片中的PPDK与C4植物叶片中的PPDK一样，也经历了光/暗介导的调节性磷酸化。 本项目的主要目的是通过鉴定器官、组织、细胞和亚细胞的PPDK磷酸化调节位点来进一步表征C3植物PPDK磷酸化调节。此外，C3 RP或RP样活性介导C3植物组织中PPDK的可逆磷酸化将在体外进行筛选，以了解其生化性质。最后，本项目试图从玉米cDNA文库中克隆C4 PPDK RP基因。这将为解决C4植物中这种独特的双功能酶是如何调节的这一令人困惑的谜团提供“终极”工具，以及了解其在C3和C4植物中精确表达所需的基因探针。总之，本项目的实验结果将使迄今未知的C3植物中PPDK的代谢功能得以阐明。

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.