## 2 Materials and Methods
### Experimental Methods and Sample Analysis
#### 2.2.2 Impact of Cd on the Physiological Parameters of _S. nigrum_  
To assess the mechanisms of plant defense and adaptability to Cd stress, the physiological parameters were determined in fresh leaves of all _S. nigrum_ samples from each pot, i.e., as the results of experiment conducted in three independent biological replicates. The analysis was performed at the Institute of Biology, University of Szczecin (Poland). In this research, a considerably broader scope of physiological parameters was investigated than that presented in other research on _Solanum nigrum_ L. as Cd hyperaccumulator(e.g., [5, 21, 22, 23]). Except photosynthetic pigments, the applied methods for determination of other physiological parameters differed in some significant details (mostly in the units used) with respect to these presented in the relevant publications. This did not allow direct comparison of results, but clearly presented trends. To avoid misinterpretation, methods used for assessment of physiological parameters are presented here in more detail. Concentrations of photosynthetic pigments were determined using Lichtenthaler and Buschmann protocol [24]. To estimate chlorophyll a, chlorophyll b and carotenoids, leaves were homogenized and extracted in 80% (\(v/v\)) acetone (Merck, Darmstadt, Germany) (leaves, FW:acetone, 1:10, \(w/v\)). After rotation and centrifugation, the supernatant was used for measurements of absorbance values at 470, 645, and 665 nm by UV-Vis spectrophotometer (ThermoFisher Scientific, Madison, WI, USA) and subsequent calculations of chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids. The results were expressed as mg g\({}^{-1}\) FW. Superoxide anion and hydrogen peroxide (ROS) content was assessed according to the procedure described by [PERSON] [25] using dihydroethidium (DHE) (ThermoFisher, Waltham, MA, USA) for O\({}^{2\bullet-}\), and CDCDHFDA-AM (6-carboxy-2,7\"-dichlorohydrofluorescein diacetate) (ThermoFisher, Waltham, MA, USA) for H\({}_{2}\)O\({}_{2}\) analysis. The labelled cells were analyzed using flow cytometer (Partec, Goerlitz, Germany) with an air-cooled 20 mV argon-ion laser. The relative O\({}^{2\bullet-}\) and H\({}_{2}\)O\({}_{2}\) level was expressed as the mean fluorescence intensity (percentage of the control). Enzyme extraction and western blot analysis were performed after fine grinding plant samples in liquid N\({}_{2}\) with the use of a Retsch MM200 (Retsch, Haan, Germany) laboratory ball mill, homogenization in the lysis buffer, and subsequent boiling and centrifugation. Samples containing 50 \(\mu\)g protein were loaded per line and separated on 12% SDS-PAGE gel [26]. After electrophoresis, the gels were electroblotted onto PVDF membranes (AmershamTM protranTM 0.2 \(\mu\)m NC, Amersham, UK). Following triple washing in TBST (Merck, Darmstadt, Germany), the blotting membranes were incubated in a blocking solution and probed with the polyclonal antibody (Agrisera, Vannas, Sweden): Cu/ZnSOD (AS10 652), MnSOD (AS09 524), FeSOD (AS06 125), CAT (AS09 501), APX (AS08 368), GR (AS06 181), GPX (AS06 183), RbCl (AS03 037), and RbCS (AS07 259). The membranes were then washed three times in TBST and probed with peroxidase conjugated secondary antibody (AS09 602 or AS09 603, Agrisera, Vannas, Sweden). The immunoblots were incubated with a detection solution containing acetate buffer, diaminobenzazoline (Merck, Darmstadt, Germany) and H\({}_{2}\)O\({}_{2}\) (Merck, Darmstadt, Germany). The data were as immunoblot band visualization and the band intensities were determined using the Fiji ImageJ software v2.9.0 (LOCI, University of Wisconsin, Madison, WI, USA) [27]. Superoxide dismutase (EC 1.15.1.1) activity was tested according to [PERSON] and [PERSON] [28] by the inhibition of NBT chloride photoreduction. The assay was carried out using the following reaction mixture: 0.1 M potassium phosphate buffer (pH 7.8), 1.3 \(\mu\)M riboflavin, 13 mM methionine), 63 \(\mu\)M NBT, 0.1 mM EDTA (all reagents from Merck, Darmstadt, Germany) and 100 \(\mu\)L of the enzymatic extract.