## 2 Materials and Methods
### Experimental Methods and Sample Analysis
#### 2.2.2 Impact of Cd on the Physiological Parameters of _S. nigrum_
The reaction mixture was illuminated (50 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\)) at 25 \({}^{\circ}\)C for 10 min and the absorbance measured at 560 nm. One unit of SOD activity was defined as the amount of the enzyme required to inhibit the reduction of NBT by 50% under the specified conditions. SOD activity of the extracts was expressed as U mg\({}^{-1}\) protein. Catalase (EC 1.11.1.6) activity was measured according to [PERSON] et al. [29]. The enzyme activity was monitored spectrophotometrically at 240 nm for 60 s using the following mixture: 50 mM potassium phosphate buffer (pH 7.0), 14.3 mM H\({}_{2}\)O\({}_{2}\) (both reagents from Merck, Darmstadt, Germany) and 100 \(\mu\)L of enzymatic extract. Purified CAT (Merck, Darmstadt, Germany) was used as a calibration standard. CAT activity was expressed as U mg\({}^{-1}\) protein. Data for both enzyme activities were expressed as means of independent biological replicates \(\pm\) SD. Glutathione reductase (EC 1.8.1.7) activity was analyzed as described by [PERSON] and [PERSON] [30] by following the rate of NADPH oxidation at 340 nm for 3 min. The as say mixture contained: 0.1 mM potassium phosphate buffer (pH 7.8), 0.5 mM NADPH, 10 mM oxidized glutathione (GSSG), 10 mM EDTA (all reagents from Merck, Darmstadt, Germany) and 100 \(\upmu\)L of enzyme extract. The GR activity was expressed as nmol NADPH min\({}^{-1}\) mg\({}^{-1}\) protein. Glutathione peroxidase (EC 1.11.9) activity was assessed as described by [PERSON] and [PERSON] [31] by following the rate of NADPH oxidation at 340 nm for 5 min. The reaction mixture contained: 0.5 M potassium phosphate buffer (pH 8.2), 10 mM EDTA, 1.14 M NaCl, 10 mM GSH, 2 mM NADPH, and 2.5 mM H\({}_{2}\)O\({}_{2}\) (all reagents from Merck, Darmstadt, Germany), and 100 \(\upmu\)L of enzyme extract. The reaction was started by adding 2 U of GR (Merck, Darmstadt, Germany). The GS activity was expressed as nmol NADPH min\({}^{-1}\) mg\({}^{-1}\) protein. The protein content in the enzymatic extracts was assayed by [PERSON]'s method [32], using bovine serum albumin (BSA) (Merck, Darmstadt, Germany) as a standard. Glutathione in the reduced (GSH) and oxidized (GSSG) form were assayed following procedure described by [PERSON] [33], which comprised grinding plant samples in liquid N\({}_{2}\), extraction in sulphosalicylic acid (1:10, \(w/v\)) (Merck, Darmstadt, Germany), centrifugation, and measuring absorbance values in the neutralized supernatant for total glutathione (GSH + GSSG) and in GSSG alone after GSH masking, in double-extracted and suitably incubated samples at 412 nm and 25 \({}^{\circ}\)C with the use of UV-Vis spectrophotometer (Thermo Fisher Scientific, Madison, USA). The results were expressed as molar concentrations of GSH (in nmol GSH g\({}^{-1}\) FW) and GSSG (in nmol GSSG g\({}^{-1}\) FW). The ascorbic acid (AsA) contents were determined as described by [PERSON] et al. [34] in supernatants of adequately prepared extracts from plant samples, by measuring absorbance values at 525 nm on UV-Vis spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA) and subsequent calculations of molar concentrations of AsA (mmol AsA g\({}^{-1}\) FW). Lipid peroxidation was estimated by determination of malondialdehydehyde (MDA) contents according to [PERSON] et al. [35], by measuring absorbance values at 532 and 600 nm with the use of extinction coefficient 155 mM\({}^{-1}\) cm\({}^{-1}\). Results were expressed as \(\upmu\)mol g\({}^{-1}\).