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

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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 H 2O 2, 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.