Redox-sensitive switches in the core S-assimilation/GSH-biosynthetic pathway of plants

细胞氧化还原状态的感知和信号传递使生物体能够适应环境的变化。这些信号帮助细胞区分来自非生物或生物变化的未受干扰的氧化还原动态平衡和氧化应激，并导致在持续生长和应激反应计划之间做出重大决定。本文选择植物叶绿体中硫酸盐同化/谷胱甘肽合成的代谢途径来论证氧化还原开关的决策作用。根据体外证据，我们认为APR和GCL在体内通过氧化还原调节固有的二硫键来控制这一途径的两个分支点。在感觉到氧化应激后，对应激反应程序的决定将硫磺的流量从蛋白质翻译和次生化合物形成等动态平衡功能转向用于谷胱甘肽合成的半胱氨酸。氧化还原调节的精确机制将在启动硫酸盐还原的APR和催化谷胱甘肽合成第一步的GCL中确定。在GCL的情况下，需要分析谷胱甘肽对二聚体形成和反馈抑制调节的双重作用。基于这些发现，将产生靶向氧化还原不敏感的APR和GCL突变蛋白，并用于补充缺乏APR(apr1，2，3)和GCL(GCL)活性的拟南芥突变体。这些riAPR1：apr1，2，3和riGCL：GCL将被交叉，并与单一的线进行比较，以评估两个开关在两个分支点的协调作用。通过比较氧化应激条件下的野生型和氧化还原不敏感突变株(甲基紫精)，将获得氧化还原开关在活细胞功能环境中作用的证据。使用roGFP2-Orp1将获得有关过氧化氢的时间和细胞分布的详细信息，使用Grx1-roGFP2将使用共聚焦成像获得谷胱甘肽氧化还原状态的详细信息。使用内部代谢组学核心设施对硫磺相关和其他代谢物进行全面的分析和微阵列的表达分析将与标记代谢物的流量分析结合使用，以证明APR和/或GCL中氧化还原开关的修改导致的代谢重定向。这将明确这些氧化还原开关在细胞内对氧化应激做出决策的相关性。氧化还原蛋白质组学将评估转基因品系中氧化还原感觉修饰的整体读出，重点是蛋白质中半胱氨酸残基的谷胱甘肽化和磺化。预计氧化处理后不同的基因类型会表现出蛋白质硫醇修饰的特征模式。时间序列将被用来剖析可归因于在应激处理的早期信号和后期保护或损害的修改。

Sensing and signaling of the cellular redox status allows organisms to adapt to changes in their environment. These signals help cells to distinguish between undisturbed redox homoeostasis and oxidative stress that is derived from abiotic or biotic changes and lead to the major decision between continued growth and stress response programs. Here the metabolic pathway of sulfate assimilation/glutathione synthesis in plant chloroplasts has been selected to demonstrate the decision making role of redox switches. Based on in vitro evidence we propose that APR and GCL control the two branching points of this pathway by redox regulation of intrinsic disulfide bridges in vivo. The decision towards stress response program after sensing of oxidative stress directs the flux of sulfur away from homeostatic functions such as protein translation and secondary compound formation towards use of cysteine for glutathione synthesis.The precise mechanisms of redox regulation will be determined in APR that initiates sulfate reduction and in GCL that catalyzes the first step of glutathione synthesis. In case of GCL the possible dual effect of glutathione on dimer formation and feedback inhibition for regulation needs to be dissected.Based on these findings, targeted redox-insensitive APR and GCL mutant proteins will be generated and used to complement Arabidopsis mutants lacking APR (apr1,2,3) and GCL (gcl) activity. These riAPR1:apr1,2,3 and riGCL:gcl lines will be crossed and compared with the single lines to assess the coordinated action of both switches at the two branching points. Evidence for the action of the redox switches in the functional context of living cells will be obtained by comparing wild type and redox insensitive mutant lines under defined oxidative stress conditions (methyl viologen). Detailed information about the timing and cellular distribution of H2O2 will be obtained using the roGFP2-Orp1 and of the glutathione redox status using Grx1-roGFP2 using confocal imaging. Comprehensive profiling of sulfur-related and other metabolites using the in house metabolomics core facility and expression analysis by microarrays will be used in combination with flux analysis by labeled metabolites to document metabolic re-direction caused by the modification of redox-switches in APR, GCL or both. This will pinpoint the relevance of these redox switches for decision making within the cellular context upon oxidative stress. The global read-out of the modification of redox-sensing in the transgenic lines will be assessed by redox proteomics focusing on glutathionylation and sulfenylation of cysteine residues in proteins. It is expected that different genotypes after oxidative treatment show characteristic patterns of protein thiol modifications. A time series will be used to dissect modifications that can be attributed to signaling at early and protection or damage at late time points of stress treatment.