Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis

The present study introduces metabolic modeling as a new tool to analyze th e network of redox reactions composing the superoxide dismutase-ascorbate ( Asc)-glutathione (GSH) cycle. Based on previously determined concentrations of antioxidants and defense enzymes in chloroplasts, kinetic properties of antioxidatiue enzymes, and nonenzymatic rate constants of antioxidants wit h reactive oxygen, models were constructed to simulate oxidative stress and calculate changes in concentrations and fluxes of oxidants and antioxidant s. Simulated oxidative stress in chloroplasts did not result in a significa nt accumulation of O-2(.-) and H2O2 when the supply with reductant was suff icient. Model results suggest that the coupling between Asc- and GSH-relate d redox systems was weak because monodehydroascorbate radical reductase pre vented dehydroascorbate (DHA) formation efficiently. DHA reductase activity was dispensable. Glutathione reductase was mainly required for the recycli ng of GSH oxidized in nonenzymatic reactions. In the absence of monodehydro ascorbate radical reductase and DHA reductase, glutathione reductase and GS H were capable to maintain the Asc pool more than 99% reduced. This suggest s that measured DHA/Asc ratios do not reflect a redox balance related to th e Asc-GSH-cycle. Decreases in Asc peroxidase resulted in marked H2O2 accumu lation without significant effects on the redox balance of Asc/DHA or GSH/G SSG. Simulated loss of SOD resulted in higher H2O2 production rates, thereb y affecting all subsequent steps of the Asc-GSH-cycle. In conclusion, model ing approaches contribute to the theoretical understanding of the functioni ng of antioxidant systems by pointing out questions that need to be validat ed and provide additional information that is useful to develop breeding st rategies for higher stress resistance in plants.