## 3 Results and Discussion
### Effect of Adaptation to Cd Stress on Biomass of the Middle European Ecotype of S. nigrum
#### 3.3.5 Second Line Defence Antioxidant Activities  
In reducing Cd stress, a group of scavenging antioxidants also plays an important role. This second line defense antioxidants [41] scavenge and neutralize free radicals by donating electron to them, thus converting themselves into free radicals, but of lesser damaging effect. They, in turn, are easily neutralized by other antioxidants of this group. The prominent representatives of this group are ascorbate and glutathione enzymatic (APX, AsA, GR) and non-enzymatic compounds (GSH, GSSG). Besides scavenging ROS directly, they perform other functions that enhance antioxidant capacity and reduce oxidative stress [42]. Of these antioxidants, a very strong reaction to increasing Cd stress in \(S\). _nigrum_ showed ascorbate antioxidant compounds: ascorbate peroxidase APX and ascorbate AsA (Figure 6). APX occurs in cytosol and organelles (chloroplast, mitochondria and peroxisome). Ascorbate AsA is present also in nucleus and vacuoles of leaves cells [40]. With respect to APX in the leaves of the non-adapted N0 variety of \(S\). _nigrum_, increase of its activity (by 14% in T10 related to T0), typical for this ecotype occurred, indicating activation of its defense functions. However, it was followed by the breakdown of defense capacity and fast decline of APX activity up to its thorough extinction over the T10-T50 range of Cd treatments. An enzymatic co-factor ascorbate (AsA) was affected even more, showing sharp decrease (by 33% compared to T0) until complete extinction at T30. Enrichment of ascorbate compounds in the leaves of the adapted variety A50 appeared to be considerably bigger and showed much higher defense capacity. After an increase of APX at T10 by 28% compared to T0, its defense activity permanently exceeded control up to T30, while at T50, APX activity declined scarcely by 37% related to T0 (Figure 6a). Also, AsA contents in the adapted A50 variety appeared to be much less responsive to Cd stress than in N0 in the range T0-T30 and was extinct only at T50 (Figure 6b). Figure 5: Response of SOD isozyme (**a**) Cu/ZnSOD, (**b**) MnSOD (**c**) FeSOD activities 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 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 \(p<0.05\). Simultaneously, the levels of second line glutathione antioxidant compounds present in plant cells (GR, GSH) and the level of disulfide GSSG generation in both _S. nigrum_ varieties suggest much weaker response of these compounds to increasing Cd stress (Figure 6c-e). The level of glutathione reductase (GR) appeared to be similar in both varieties and did not show significant (\(p<0.05\)) changes over the entire T0-T50 range of Cd treatments (Figure 6c). In turn, the contents of non-enzymatic antioxidant glutathione (GSH) were also stable in both varieties. Differences in AsA (Figure 6b) and GSH trends (Figure 6d) in response to Cd stress suggest also other mechanisms besides regulation of Ascorbate-Glutathione (AsA-GSH) pathway [42] in detoxifying ROS in this Middle-European ecotype of _S. nigrum_. While AsA showed a fast decline, GSH contents were basically constant, in A50 about 30% bigger than in N0. GSH displayed a very slight general increasing trend at T10 followed by the same slight (above T0 level) decrease in the T10-T50 range in the N0 variety and no significant changes in the adapted A50 variety (Figure 6d). Inversely, the levels of oxidized GSH (GSSG) were in the non-adapted variety N0 somewhat higher (by 7-10%) than in the adapted one A50, at the similar slight decreasing trend (Figure 6e).