DIFFERENTIAL RESPONSES TO PARAQUAT-INDUCED OXIDATIVE INJURY IN A PEA (PISUM-SATIVUM) PROTOPLAST SYSTEM

Antioxidant responses to varying degrees of paraquat stress in Freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cu ltivars Progress and Nugget were studied. Leaves of comparable maturit y were used for protoplast isolation. Nugget protoplasts were more res istant to paraquat in the micromolar range under our conditions. In Nu gget, a non-bleaching paraquat concentration (10 mu M) inhibited CO2-d ependent O-2 evolution ca 50% during the first 40 min, remaining at th at rate (''coping behavior'') for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not e xhibit coping behavior. Antioxidant enzyme activities were unaltered t hroughout the time course of the experiment ir. treated protoplasts fr om Nugget and in chloroplasts isolated From them. Thus, the coping beh avior of Nugget protoplasts cannot be attributed to changes in activit ies of the three an;ioxidant enzymes tested. Paraquat treatment did no t affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated From them. When higher doses of paraquat were us ed (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapid ly bleached and total protein decreased to ca 30% of pre-stress levels . Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplas ts From both cultivars under the severe stress conditions in concert w ith declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time c ourse, increasing to ca 150% of original levels by the final, 12-h tim e point. The activity of the plastid Cu,Zn isoform, expressed as a per centage of total SOD activity, declined over the time course of the ex periment while th:dt of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement wi th some earlier reports and are compatible with the hypothesis that SO D activity levels increase in response to reactive oxygen species leve ls, even under conditions leading to cell death.