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

植物核心 S-同化/GSH-生物合成途径中的氧化还原敏感开关

• 批准号: 251965288
• 负责人: Professor Dr. Rüdiger Hell
• 金额: --
• 依托单位: Forschungsgruppe Molekulare Physiologie der Pflanzen
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251965288?language=en
• 关键词: Redox; sensitive; switches; core; assimilation

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.

细胞氧化还原状态的传感和信号传导使生物可以适应其环境的变化。这些信号可帮助细胞区分未受干扰的氧化还原同源性和氧化应激，这些应激来自非生物或生物变化，并导致持续生长和压力反应计划之间的主要决策。这里选择了植物叶绿体中硫酸盐同化/谷胱甘肽合成的代谢途径，以证明氧化还原开关的决策作用。基于体外证据，我们建议APR和GCL通过体内内在的二硫键桥的氧化还原调节来控制该途径的两个分支点。感应氧化应激后，对应力反应计划的决策将硫的通量从稳态功能（例如蛋白质翻译和次级化合物形成）降低到使用半胱氨酸进行谷胱甘肽合成。氧化还原调控的精确机制将在APR中确定，以启动硫酸盐启动硫酸盐的启动硫酸盐。还原和在GCL中催化谷胱甘肽合成的第一步。在GCL时，2,3）和GCL（GCL）活性。这些RIAPR1：APR1,2,3和RIGCL：GCL线将被跨越并与单线线进行比较，以评估两个分支点上两个开关的协调作用。通过比较定义的氧化应激条件（甲基Viologen），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和谷胱甘肽氧化还原状态，使用GRX1-ROGFP2使用共共聚焦成像获得有关H2O2的时间和细胞分布的详细信息。使用内部代谢组学核心设施和微阵列的表达分析对硫相关和其他代谢产物的全面分析将与标记的代谢物结合使用通量分析，以记录由APR，GCL的Redox-Switches修饰引起的代谢重新指导或两者兼而有之。这将指出这些氧化还原开关在氧化应激下在细胞环境中决策的相关性。转基因线中氧化还原敏感性修饰的全局读数将通过重点蛋白质组学评估，该氧化还原蛋白质组学的重点是蛋白质中半胱氨酸残基的谷胱甘肽化和磺酰化。预计氧化治疗后的不同基因型显示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的修改，以及在压力治疗的晚期保护或损坏。