D-A-CH Project:Regulation of primary metabolism under oxidative stress

植物由于其固着的生活方式，其分布和表现受到许多非生物因素的显着影响。供水、温度和土壤质量对植物生长的影响最为相关，也具有相似的影响。在细胞水平上，这些应激源可以对膜和蛋白质造成损害，在许多情况下，这是由氧化应激引起的，氧化应激导致细胞内产生活性氧物种。以往的研究表明，模式植物拟南芥在逆境耐性上的显著差异与可溶性碳水化合物的积累和中心碳水化合物代谢酶活性的提高有关。然而，研究也表明有必要分析整个初级代谢的亚细胞组织，以便描绘涉及植物胁迫反应的复杂机制的代表性图景。在拟议的项目中，我们的目标是在亚细胞水平上剖析氧化胁迫对初级代谢的代谢和信号影响。我们的项目旨在阐明在受到非生物胁迫的干扰后，有助于氧化还原动态平衡重新调整的昼夜调节和隔室相互作用。已加入哥伦比亚-0基因的拟南芥植株以及缺失己糖激酶1和S177A己糖传感功能的gin-2突变体将暴露在光、热和冷胁迫条件下。对于初级代谢的亚细胞分析，非水分离(NAF)技术将与代谢组和蛋白质组的高通量测量相结合。拟议的NaF与鸟枪式蛋白质组学、代谢物图谱和改进的数据处理技术的新组合旨在更准确地呈现植物细胞作为协调其子系统的氧化还原和代谢活动的系统的图景。这将涉及对低分子ROS清除剂(如抗坏血酸)以及氧化损伤指示剂(如丙二醛)和保护性分子(如糖或脯氨酸)的特定隔室分析。叶片初级代谢的昼夜动态和亚细胞代谢相互作用的数学模型将产生有关代谢适应非生物胁迫的瓶颈的信息。除了生化分析和全面的植物-环境相互作用的系统生物学特征外，该项目旨在提供一种增强的方法学，使人们能够了解植物细胞内不同反应空间的相互作用。我们打算模拟整个细胞的氧化还原调节，从而证明或反驳细胞氧化还原状态的概念。反过来，这应该会打破旨在提高植物非生物胁迫耐受性的育种理念。

Due to their sessile lifestyle plants are significantly affected in their distribution and performance by many abiotic factors. Water supply, temperature and soil quality are most relevant and share similar effects on plant growth. At the cellular level, these stressors can cause damage to membranes and proteins, and in many cases this is entailed by oxidative stress, resulting from intracellular production of reactive oxygen species. Previous studies revealed significant differences in stress tolerance of the model plant Arabidopsis thaliana to be correlated with accumulation of soluble carbohydrates and increased enzyme activities of the central carbohydrate metabolism. Yet, studies also indicated the necessity of analysing subcellular organisation of the whole primary metabolism to allow drawing a representative picture of the complex mechanisms involved in plant stress response.In the proposed project, we aim to dissect metabolic and signalling effects on primary metabolism caused by oxidative stress at the subcellular level. Our project is set out to elucidate diurnal regulation and compartment interactions contributing to the re-adjustement of redox homeostasis after a disturbance by abiotic stresses. Plants of the Arabidopsis thaliana accession Columbia-0 as well as the gin-2 mutant defective in hexokinase 1 and the S177A complement of its hexose sensing function will be exposed to conditions of light, heat and cold stress. For subcellular analysis of primary metabolism, the non-aqueous fractionation (NAF) technique will be applied in combination with high-throughput measurement of the metabolome and proteome. The proposed novel combination of NAF with shotgun proteomics, metabolite profiling and refined data processing techniques are intended to present a more precise picture of the plant cell as a system co-ordinating redox and metabolic activities of its subsystems. This shall involve the compartment-specific analysis of low molecular weight ROS scavengers like ascorbate as well as indicators of oxidative damage, e.g. malone dialdehyde and protective molecules like sugars or proline. Mathematical modelling of both diurnal dynamics and subcellular metabolic interactions of leaf primary metabolism shall yield information about bottlenecks for metabolic adaptation to abiotic stress. Beyond biochemical analysis and comprehensive systems biological characterization of plant-environment interactions, this project intends to contribute an enhanced methodology that allows understanding of the interplay of the different reaction spaces within a plant cell. We intend to simulate whole cell redox adjustments, thus proving or disproving the concept of a cellular redox state. This should, in turn, burst breeding concepts aimed at improving plant abiotic stress tolerance.