Dynamic analysis of metabolism under circumstances of altered photorespiratory flux

最近的研究揭示了光呼吸途径以及光合代谢的一般网络中意想不到的复杂性。光呼吸已被清楚地显示为分支四个隔室，并与叶酸代谢、乙醇胺代谢和质子跨线粒体膜耗散相互作用。澄清这种相互作用将是至关重要的，无论是从根本上了解所涉及的途径，并在最大限度地利用生物技术的方法来操纵它们。为此，将建立代谢组学和通量分析方法的组合，并将其应用于在适当的反应步骤中具有遗传损伤或经受改变的环境条件的拟南芥材料。在特定情况下，这将与非水分级分离技术结合进行，以解决代谢调节的亚细胞方面。预计所获得的结果将是高度翔实的，在提高我们的理解，如何，光呼吸，与它密切相关的过程中，有条件地调节，并进一步通过定义的反应步骤，在该调节是强制执行的。在第一个资助期内，我们在其中几个实验目标方面取得了重大进展，并建立了必要的专业知识，以便能够进行进一步的实验，这将使我们能够充分解决悬而未决的问题。部分通过合作努力，我们整理了大量的多层次数据，用于在非光呼吸条件下和过渡到环境空气后的生长，以及转移到不同的温度，不同的光强度和二氧化碳和氧气分压的相互变化。这些研究提供了丰富的信息，光呼吸途径的相互作用，相关的代谢和转录过程。工作的方面将提前到第二阶段，包括与植物能量信号的多个方面的良好表征的突变体的比较。与此同时，我们建立了一种新的方法13CO2喂养，包括动力学示踪的重标记通过初级代谢和房室水平。作为第一种方法，该方法被应用于表征环境空气中的代谢。从这个模型计算出的通量基准对主要通量确定的独立方法，如光合作用和光呼吸和蔗糖和淀粉生物合成的总体速率。在第二阶段，建议继续和完成实验中，这种方法适用于植物的光呼吸通量已被环境或遗传手段扰动。这些数据将与其他Proximity合作伙伴一起输入到关系数据库和计算模型中，旨在改善并从而使我们能够影响光呼吸途径的代谢调节和相互作用。

Recent studies have revealed unexpected complexities both within the pathway of photorespiration as well as with respect to the general network of photosynthetic metabolism. Photorespiration has been clearly shown to branch four compartments and interacts with folate metabolism, ethanolamine metabolism and proton dissipation across the mitochondrial membrane. Clarification of such interactions will be crucial both for fundamental understanding of the pathways involved and in maximizing biotechnological approaches to manipulate them. For this purpose, a combination of metabolomics and flux profiling approaches will be established and applied to Arabidopsis material either harboring genetic lesions at appropriate reaction steps or subjected to altered environmental conditions. In specific instances, this will be carried out in conjuncture with the non-aqueous fractionation technique in order to resolve subcellular aspects of metabolic regulation. It is anticipated that results obtained will be highly informative in enhancing our understanding of how, photorespiration, and processes intimately related to it, is conditionally regulated and furthermore by defining the reaction steps at which this regulation is enforced. In the first funding period, we made significant progress with respect to several of these experimental goals as well as setting up the necessary expertise to enable the performance of further experiments which will allow us to fully address outstanding issues. Partially via collaborative efforts we collated large multi-level data for growth in non-photorespiratory conditions and after transition to ambient air, as well as transfer to different temperatures, different light intensities and reciprocal variations in carbon dioxide and oxygen partial pressures. These studies provided a wealth of information on the interaction of photorespiratory pathways to related metabolic and transcriptional processes. Aspects of the work will be brought forward into the second period including comparison with well characterized mutants of over aspects of plant energy signaling. In parallel we set up a novel method for 13CO2 feeding, including kinetic tracing of the heavy label through primary metabolism both at the metabolic and compartmental levels. As a first approach this method was applied to characterizing metabolism in ambient air. Fluxes calculated from this model were benchmarked against major fluxes determined by independent methods such as the overall rates of photosynthesis and photorespiration and sucrose and starch biosynthesis. In the second period it is proposed to continue and complete experiments in which this method is applied to plants in which photorespiratory fluxes have been perturbed by environmental or genetic means. Together with other Promics partners these data will be fed both into a relational database and a computational model aimed at improving and thereby allowing us to influence metabolic regulation and interaction of the photorespiratory pathway.