## 3 Results and Discussion
### Effect of Adaptation to Cd Stress on Biomass of the Middle European Ecotype of S. nigrum
#### 3.3.4 First Line Defense Antioxidant Activities  
Among antioxidant defense systems in plants, key antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) are considered the most important and efficient antioxidants that prevent or suppress the formation of free radicals and ROS in plant cells. Specifically, CAT degrades H\({}_{2}\)O\({}_{2}\) into H\({}_{2}\)O and O\({}_{2}\). SOD catalyzes the dismutation of two superoxide anions (O\({}_{2}\)\({}^{\bullet-}\)) and water into H\({}_{2}\)O\({}_{2}\) and O\({}_{2}\)[39; 40; 41].  
Activities of these first line antioxidant enzymes in the leaves of non-adapted N0 and adapted A50 varieties of the Middle-European occupy of S. _nigrum_ showed Cd stress-related changes following specific patterns. In N0 leaves, up-regulation of all key enzymes (CAT, SOD, GPX) indicating growing defense mechanism was observed up to T10 (with respect to SOD up to T20). This was followed by a down-regulation due to the weakening of the defense system (Figure 4). The strongest relative up-regulation trends were observed in SOD contents (up to 32% at T20), and the weakest in GPX activities (up to 12% at T10). The strongest relative down-regulation occurred in CAT activities (by 60% at T50 compared to T10 treatment).  
In contrast, in the adapted variety A50, the first line antioxidant activities, after reaching the highest level at T10 (by 24% for CAT, by 20% for SOD and by 15% for GPX), remained virtually unchanged, showing only a slight decrease above the initial level at T0 (CAT, GPX), while SOD activity kept a weak upward trend up to T50 (Figure 4).  
Worth mentioning that SOD activities were always bigger in N0 than in A50 at all Cd treatments, except that at T50, when decline of SOD in N0 reversed the relationship. Of three SOD isozymes distinguished by covalently linked metal ions and cellular locations (Cu/ZnSOD, MnSOD and FeSOD) [39], Cu/ZnSOD isozyme showed the same N0/A50 relations as SOD enzyme, indicating its predominance in both varieties (Figure 5a). In the adapted A50 variety, activities of all three isozymes showed increase at T10 (by \(\sim\)19%, 13% and 21%, respectively) and stable level (\(p<\) 0.05) in the Cd range T10-T50, for MnSOD and FeSOD considerably higher than in N0 (Figure 5a-c). Opposite, in the non-adapted variety N0 activities of MnSOD isozyme showed a slight increase at T10 (by 7% compared to T0),  
Figure 4: Response of the first-line defense antioxidant (**a**) CAT, (**b**) SOD and (**c**) GPX) activity in the leaves of non-adapted N0 and adapted A50 varieties of S. _nigrum_ to the growing Cd stress. (1) 0 to 50 means control and soil treatments with Cd (mg kg\({}^{-1}\)); (2) Data for the same treatments marked by the same capital letters over bars are not significantly different at \(p<\) 0.05. Data for different treatments marked by the same lowercase letters over bars are not significantly different at \(p<\) 0.05.  
followed by a decrease (by 32%) in the range T10-T30 and stabilization at T30-T50 (\(p<0.05\)) (Figure 5b). Activities of FeSOD in N0 remained at the same lower level compared to T0 over the entire Cd treatment range (by19%, \(p<0.05\)). Differential expression of antioxidant isozymes in the leaves of \(S\). _nigrum_ exposed to Cd stress indicated substantial differences in production superoxide anions in specific cellular locations. Since Cu/ZnSOD isozyme, which shows the biggest affinity to overall SOD pattern and expression dynamics, is found in cytoplasm and in chloroplasts, this indicates that superoxide anions requiring deactivation are generated mostly in these cellular locations. Lesser activity changes showed MnSOD located in mitochondria, while the most stable activity displayed FeSOD found in chloroplasts.