Posttranslational Regulation of Pep-Carboxylase Activity in Higher Plants

本实验计划包括三个领域的研究，通过调节磷酸化和变构代谢物效应物在体内控制高等植物磷酸烯醇丙酮酸羧化酶（PEPC）的活性。具体的研究目标包括：(i)通过对完整C4和CAM植物的原位研究，使用综合生化、免疫学和/或分子遗传学方法，继续审查高度调控的pepc -激酶信号转导链，并通过对完整C4-叶肉细胞和原生质体的原位研究进一步阐明复杂的C4 pepc -激酶信号转导链。这些目标的结果可能会强烈影响我们对负变构效应物l -苹果酸调节PEPC活性的基本理解。相反，项目（ii）将重点研究正磷酸化效应物（如葡萄糖6-P、三糖- p）对C4/CAM PEPC活性的相反控制，方法是利用光亲和标记和定点诱变技术绘制该激活物结合域。最后，项目（iii）将通过体内（分离的叶片和根瘤）、原位（完整的叶片原生质体）和体外（分离的PEPC和PEPC激酶）综合分析，批判性地评估C3植物叶片和根瘤中非光合作用PEPC可能的调控磷酸化。这三个项目的研究结果不仅将揭示C4-和CAM-光合作用控制的重要细节，以及细胞质PEPC水平上植物一般C/N代谢的细节，同时也将为高等植物中可逆蛋白磷酸化的酶调节提供急需的见解。本项目主要研究高等植物中二氧化碳（CO2）固定酶磷酸烯醇丙酮酸羧化酶（PEPC）的翻译后调控。虽然这种酶在C4光合作用期间的二氧化碳固定和玉米、高粱、甘蔗和菠萝（CAM）等作物的天冬酸代谢（CAM）中发挥的作用最为人所知，但它也在一般植物的碳/氮代谢中发挥作用。除了这些重要的生理和功能考虑外，PEPC酶是高等植物中为数不多的由蛋白磷酸酶和蛋白丝氨酸激酶的相反作用控制的酶之一。因此，这种复杂的调控磷酸化循环形成了我们目前许多研究的中心焦点。了解这个循环是如何运作的，对二氧化碳同化和植物代谢的调节是很重要的。它可以构成植物生物技术工程的基础，以提高生长和生产力。本项目由MCB代谢生物化学项目和IBN植物综合生物学项目联合支持。＊＊＊

9315928 Chollet The experimental plan is composed of three areas of research on the control of higher plant phosphoenolpyruvate carboxylase (PEPC) activity in vivo by regulatory phosphorylation and allosteric metabolite effectors. Specific research objectives include (i) the continued scrutiny of the highly regulated PEPC-kinase signal- transduction chain by in situ studies with intact C4 and CAM plants using an integrated biochemical, immunological and/or molecular- genetic approach, and the further elucidation of the complex C4 PEPC-kinase signal-transduction chain by in situ studies with intact C4-mesophyll cells and protoplasts. The outcome of these objectives could strongly affect our basic understanding of the regulation of PEPC activity by L-malate, the negative allosteric effector. Conversely, project (ii) will focus on the opposing control of C4/CAM PEPC activity exerted by positive phosphorylated effectors (e.g., glucose 6-P, triose-P) by mapping this activator binding domain with photoaffinity-labeling and site-directed mutagenesis techniques. Finally, project (iii) will critically assess the possible regulatory phosphorylation of nonphotosynthetic PEPC in leaves and root-nodules from C3 plants by integrative in vivo (detached leaves and nodules), in situ (intact leaf protoplasts), and in vitro (isolated PEPC and PEPC-kinase) analysis. Results from these three projects will not only delineate important details of the control of C4- and CAM- photosynthesis, and details of general C/N metabolism in plants at the level of cytosolic PEPC, but at the same time will provide much needed insight into enzyme regulation in higher plants by reversible protein phosphorylation. %%% This project is focused on the posttranslational regulation of the carbon dioxide (CO2)-fixing enzyme phosphoenolpyruvate carboxylase (PEPC) in higher plants. While this enzyme is best known for its role in CO2 fixation during C4 photosynthesis and Crassulacean acid metabolism (CA M) in crops as maize, sorghum, sugarcane, and pineapple (CAM), it also functions in general plant carbon/nitrogen metabolism. Besides these important physiological and functional considerations, the PEPC enzyme represents one of the few well- known examples in higher plants of an enzyme that is controlled by the opposing action of a protein phosphatase and a protein-serine kinase. Consequently, this complex regulatory phosphorylation cycle forms the central focus of many of our present studies. Understanding about how the cycle operates is important in regulation of CO2 assimilation and in plant metabolism in general. It can form the basis for biotechnological engineering of plants to improve growth and productivity. This project is supported jointly by Metabolic Biochemistry Program, MCB and the Plant Integrative Biology Program, IBN. ***