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

细胞氧化还原状态的传感和信号传导允许生物体适应其环境的变化。这些信号帮助细胞区分未受干扰的氧化还原稳态和来自非生物或生物变化的氧化应激，并导致持续生长和应激反应程序之间的重大决定。在这里，硫酸盐同化/谷胱甘肽合成在植物叶绿体中的代谢途径已被选择来证明氧化还原开关的决策作用。基于体外证据，我们提出，APR和GCL控制这一途径的两个分支点的内在二硫桥在体内的氧化还原调节。对应激反应程序的决定后，感知氧化应激的指示流量的硫远离稳态功能，如蛋白质翻译和二级化合物的形成对使用半胱氨酸谷胱甘肽synthesization.The氧化还原调节的精确机制将被确定在APR中，启动硫酸还原和GCL催化谷胱甘肽合成的第一步。在GCL的情况下，谷胱甘肽对二聚体形成和反馈抑制调节的可能的双重作用需要解剖。基于这些发现，靶向氧化还原不敏感的APR和GCL突变体蛋白将被产生，并用于补充缺乏APR（apr 1，2，3）和GCL（gcl）活性的拟南芥突变体。这些riAPR 1：apr 1、2、3和riGCL：gcl线将交叉并与单线进行比较，以评估两个开关在两个分支点处的协调动作。在活细胞的功能背景下的氧化还原开关的行动的证据将通过比较野生型和氧化还原不敏感的突变株系在定义的氧化应激条件下（甲基紫精）。使用共聚焦成像，使用roGFP 2-Orp 1获得关于H2 O2的时间和细胞分布的详细信息，使用Grx 1-roGFP 2获得关于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相关代谢物和其他代谢物进行全面分析，并通过微阵列进行表达分析，将与标记代谢物的通量分析结合使用，以记录APR、GCL或两者中氧化还原开关修饰引起的代谢重定向。这将精确定位这些氧化还原开关在氧化应激时在细胞背景下进行决策的相关性。将通过氧化还原蛋白质组学评估转基因系中氧化还原感测修饰的全局读出，所述氧化还原蛋白质组学关注蛋白质中半胱氨酸残基的谷胱甘肽化和亚磺酰化。预计氧化处理后的不同基因型显示蛋白质巯基修饰的特征模式。时间序列将用于剖析可归因于应激处理的早期信号传导和后期时间点的保护或损伤的修饰。

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.