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Seasonal changes in levels of reactive oxygen species (ROS), low-molecular weight antioxidants and activities of antioxidant enzymes were analyzed in relation to the freezing tolerance of 1-year-old needles from four populations of Norway spruce. Throughout the study period (from January until May), no significant changes were observed in the superoxide anion radical (O2-) or hydrogen peroxide (H2O2) levels in the needles. By contrast, a marked reduction was observed in concentrations of lowmolecular weight antioxidants, including flavonoids (FL), ascorbic acid (AsA) and slight glutathione (GSH), during deacclimation. The activities of superoxide dismutase (SOD) (EC. 1.15.1.1.) and guaiacol peroxidase (PO) (EC. 1.11.1.7.) also decreased significantly. The activity of catalase (CAT) (EC. 1.11.1.6.) did not change significantly. Levels of low-molecular weight antioxidants (AsA, FL and GSH) and SOD activity were correlated significantly with freezing tolerance in the studied populations. The reactions were similar in all populations. This suggests that the response of the antioxidant system depends more strongly on climatic conditions than on population origin. The ability of spruce trees to cope with active oxygen species is discussed as an aspect of defense and a factor associated with freezing tolerance.
The symptoms of oxidative stress and antioxidative response were investigated on Norway spruce seedlings subjected to freezing desiccation conditions. Three-year-old seedlings were exposed to freezing desiccation at -3oC and -10oC for 45 days in two acclimation stages: autumn (October) and winter (January). The stress enhanced the production of reactive oxygen species (ROS): superoxide radical anion (O2.-), and hydrogen peroxide (H2O2). Concentrations of low molecular antioxidants: glutathione (GSH), ascorbic acid (AsA) and a-tocopherol declined at both low temperatures and acclimation stages. The activity of superoxide dismutase (SOD) increased with ROS production, while guaiacol peroxidase (POX) activity decreased. The freeze-induced desiccation of needles was significantly correlated with the cold hardiness (LT50), the level of low-molecular antioxidants, and POX activity, but not with SOD activity. Under extreme freezing desiccation conditions, these reactions continued, leading to the degradation of membrane phospholipids and a strong decrease in cold hardiness. The results show that membranes are the primary site of injury induced by ROS, produced under the influence of low temperature combined with dehydration. The acclimation response of Norway spruce needles to the oxidative stress generated by long-term cold and/or freezing desiccation is discussed.
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