Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures

Abstract

A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (CSH) synthesis in the presence of hydrogen peroxide. Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase. Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h. During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase. In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism. A 4-fold increase in the level of CSH, which was almost exclusively in the reduced form, was observed under the same treatment. To determine to what extent this increase in reduced CSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited CSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of y-glutamylcysteine synthetase. No significant oxidative injury was detected as a result of treatment with 50 WM BSO alone, and furthermore, this treatment had no effect on cell viability. However, addition of 2 mM aminotriazole to cells preincubated with 50 p~ BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation. Within 72 h, cell viability was lost completely. After removal of BSO from the growth medium, CSH levels recovered to normal over a period of 20 h. Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced CSH and the degree of protection against oxidative injury. These data strongly suggest that the induction of CSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress.

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Kinetics of Salicylate-Mediated Suppression of Jasmonate Signaling Reveal a Role for Redox Modulation

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