Changes in gluthatione reductase activity and protein content in wheat leaves and chloroplasts exposed to photooxidative stress

The effect of different extents of oxidative stress on total glutathione reductase (GR, EC 1.6.4.2) activity, isozymic pattern, and chloroplastic GR protein content were studied in wheat (Triticum aestivum L. cv. Oasis) leaves exposed to increasing doses of paraquat (PQ). Low concentrations of PQ increased total GR activity, peaking at 0.25 μM. In 0.75 to 2 μM PQ total GR activity remained around 30% lower than control, while at 3 μM PQ activity decreased to 54% of control. Two GR isoforms were detected in crude extracts, one chloroplastic and one extrachloroplastic. PQ, at 0.25 and 0.50 μM, increased chloroplastic GR; but, at higher concentrations, it markedly decreased the activity and protein content of this isoform. A photooxidative-induced loss of GR protein was also observed in chloroplasts isolated from PQ pre-treated leaves and subsequently exposed to light. Extrachloroplastic GR was less affected by treatments. The influence of active oxygens and plastidic protease(s) on photooxidative-induced chloroplastic GR degradation was studied. While purified GR was not affected by treatments with H2O2, when exposed to an ·OH-generating system, a dose-dependent inactivation and breakdown were observed. Chloroplastic GR showed to be hydrolysed by a sulphydryl- and metal-containing protease, active at acid pH in both stressed and non-stressed chloroplasts. However, this protease degraded ·OH-pretreated GR more rapidily than native GR. It is suggested that the rapid degradation of chloroplastic GR under strong photooxidative stress could be mainly due to direct fragmentation and/or increased susceptibility of the enzyme to protease attack, caused by higher levels of ·OH radicals.