Seryl-Phosphorylation / Dephosphorylation of Plant Metabolic Enzymes in Leaves (PEPC) and Root Nodules (SuSy, PEPC)

这个更新项目的重点是该实验室长期以来对通过调节蛋白的磷酸化/去磷酸化周期控制植物初级代谢的兴趣。总体研究的两个相关领域特别涉及磷酸烯醇丙酮酸羧化酶（PEPC）和蔗糖合成酶（SuSy），这两种靶酶已知在完整叶片和固定n2的豆科根瘤中发生丝氨酸磷酸化。虽然胞质PEPC以其在催化C4光合作用和天冬酸代谢（CAM）过程中对大气CO2的固定作用而闻名，但这种无处不在的植物羧化酶也在大量非光合作用环境中发挥作用，包括一般的C/ n代谢（例如，在C3叶片中）以及更特殊的固氮根瘤，叶保护细胞和种子发芽。植物PEPC的多面性使其受到高度调控的Ca2+独立蛋白丝氨酸/苏氨酸激酶（PEPC-激酶）和相反蛋白磷酸酶- 2a的复杂控制，成为继续研究的极具吸引力的靶点。因此，本更新项目的三个具体研究目标中的两个集中在(1)C4和/或CAM PEPC-kinase，以及(2)其在叶片（C4, Ca， CAM）和根瘤中必要但看似多样化的信号通路。最近的研究表明，除了PEPC外，SuSy也是两种生理上不同的植物“库”组织（伸长的玉米叶片和大豆根瘤）中可溶性Ca2+依赖性蛋白激酶磷酸化的靶标。这些观察结果具有潜在的重要意义，因为人们对植物中SuSy活性的翻译后控制或该酶如何在质膜和细胞质溶胶之间进行物理分区一无所知。显然，其磷酸化状态的可逆变化是可能的，但尚未探索这些现象的机制。这些关于植物SuSy的新问题将在本更新项目的目标3中通过继续对该酶在大豆中的结节增强形式nodin -100的磷酸化/去磷酸化的生化和分子研究来解决。这个基础研究项目的主要意义在于它描述了在包括玉米、烟草和大豆在内的重要农学作物物种的各种组织中，二氧化碳同化（通过PEPC）和蔗糖裂解（通过SuSy）通过可逆蛋白磷酸化调控的重要基本细节。同时，研究结果可为通过生物技术对植物碳氮代谢及其调控机制进行建设性的调控提供必要的基础。必须强调的是，PEPC和SuSy代表了植物中蛋白激酶和蛋白磷酸酶靶向的植物代谢酶的少数文献例子中的两个。因此，该研究将为通过可逆蛋白磷酸化控制植物代谢提供急需的见解。

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.