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

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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.