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导使生物体能够适应环境的变化。这些信号帮助细胞区分未受干扰的氧化还原平衡和氧化应激，氧化应激来源于非生物或生物变化，并导致持续生长和应激反应程序之间的主要决定。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通过氧化还原调节体内固有二硫桥来控制这一途径的两个分支点。在感知氧化应激后，对应激反应程序的决定将硫的通量从蛋白质翻译和二级化合物形成等稳态功能转向使用半胱氨酸来合成谷胱甘肽。氧化还原调控的精确机制将在APR中确定，APR启动硫酸盐还原，GCL催化谷胱甘肽合成的第一步。在GCL的情况下，谷胱甘肽可能对二聚体形成和反馈抑制调节的双重作用需要剖析。基于这些发现，将产生靶向的氧化还原不敏感的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。将这些riAPR1:apr1,2,3和riGCL:gcl线交叉并与单线进行比较，以评估两个分支点上两个开关的协调作用。氧化还原开关在活细胞功能背景下的作用证据将通过比较野生型和氧化还原不敏感突变系在确定的氧化应激条件下（甲基紫）获得。利用roGFP2-Orp1获得H2O2的时间和细胞分布的详细信息，利用共聚焦成像技术利用Grx1-roGFP2获得谷胱甘肽氧化还原状态。利用内部代谢组学核心设施和微阵列表达分析对硫相关和其他代谢物进行综合分析，并结合标记代谢物通量分析，记录由APR、GCL或两者中氧化还原开关的修改引起的代谢重定向。这将查明这些氧化还原开关在细胞氧化应激环境下决策的相关性。通过氧化还原蛋白质组学，我们将对转基因系中氧化还原传感修饰的全局解读进行评估，重点关注蛋白质中半胱氨酸残基的谷胱甘肽化和亚砜化。预计不同的基因型在氧化处理后会显示出蛋白质硫醇修饰的特征模式。时间序列将用于剖析可归因于早期信号和晚期应力处理时间点的保护或损害的变化。

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.