Dynamic analysis of metabolism under circumstances of altered photorespiratory flux

最近的研究表明，光呼吸途径以及光合作用代谢的一般网络都具有意想不到的复杂性。光呼吸已被清楚地显示出分为四个隔室，并与叶酸代谢、乙醇胺代谢和跨线粒体膜的质子耗散相互作用。澄清这种相互作用对于从根本上理解所涉及的途径和最大限度地利用生物技术方法操纵它们都是至关重要的。为此，将建立代谢组学和通量分析方法的组合，并将其应用于拟南芥材料，这些材料要么在适当的反应步骤中含有遗传损伤，要么受到环境条件的改变。在特定情况下，这将与非水分离技术一起进行，以解决代谢调节的亚细胞方面。预计所获得的结果将有助于加强我们对光呼吸及其密切相关的过程是如何被有条件地调控的理解，并进一步通过定义实施这一调控的反应步骤来提供信息。在第一个资助期，我们在这些试验目标中的几个方面取得了重大进展，并建立了必要的专门知识，以便能够开展进一步的试验，从而使我们能够充分解决悬而未决的问题。部分通过合作，我们整理了大量的多水平数据，包括在非光呼吸条件下和过渡到环境空气后的生长情况，以及转移到不同温度、不同光强以及二氧化碳和氧气分压的倒数变化。这些研究为光呼吸途径与相关代谢和转录过程的相互作用提供了丰富的信息。这项工作的各个方面将被提前到第二阶段，包括与植物能量信号转导方面的特征良好的突变体进行比较。同时，我们建立了一种新的13CO2喂养方法，包括在代谢水平和隔室水平上通过初级代谢对重标记物进行动力学示踪。作为第一种方法，该方法被用于表征环境空气中的新陈代谢。根据该模型计算的通量与由独立方法确定的主要通量进行了基准比较，例如光合作用和光呼吸的总速率以及蔗糖和淀粉的生物合成。在第二阶段，建议继续并完成将这种方法应用于光呼吸通量受到环境或遗传手段干扰的植物的实验。这些数据将与PROMICS的其他合作伙伴一起输入到一个关系数据库和一个计算模型中，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.