*2.2. Germination Experiment*

Prior to the germination experiment, cold stratification was performed (4 ◦C, 30 days). Then, the seeds were sown on Petri dishes (Ø 7 cm) containing Whatman No. 2 filter paper (three replicates of 35 seeds in each salinity variant). We watered the seeds with four variants of a solution: control (0 mM NaCl), 200, 400, and 800 mM NaCl. NaCl concentrations were selected according to those observed in the field studies [23]. Piernik et al. [23] indicated the minimum (ca. 2 dS/m), maximum (ca. 100 dS/m), and optimum (ca. 30 dS/m) salinity for *T. pannonicum* growth. These correspond, respectively, to 20, 1000, and 300 mM NaCl. However, we did not obtain seedlings in the germination experiment at 1000 mM NaCl. Therefore, we established finally an upper limit of 800 mM NaCl. The Petri dishes with seeds were put into the growth chamber with day/night (25 ◦C), a humidity of 50–60%, a photon flux density of 1000 mmol m <sup>−</sup><sup>2</sup> s−1, and a photoperiod of 16/8 h (light/dark) (LED lights with white, full-spectrum light) [24]. The number of germinated seeds was determined daily (in the same part of the day) until the end of the 14th day after sowing.

The germination parameters were calculated based on the International Seed Testing Association (ISTA) method [25].

Germination percentage (GP):

$$\text{GP} = (\text{n/N}) \times 100 \,\text{\AA} \tag{1}$$

where n is the number of normally germinated seeds and N is the number of all seeds sown. Germination index (GI):

$$\text{GI} = \sum (\text{G}\_{\text{l}} / \text{T}\_{\text{l}}) \,\tag{2}$$

where Gt is the number of seeds germinated on day t, and Tt is the number of days. Mean germination time (MGT):

$$\text{MGT} = \sum (\text{T}\_{\text{i}} \times \text{N}\_{\text{i}}) / \sum \text{N}\_{\text{i}\prime} \tag{3}$$

where Ni is the number of newly germinated seeds at time Ti.

Germination energy (GE) was assessed on the fourth day by counting the number of typical seedlings according to the ISTA (2006) standard [25].

#### *2.3. Pot Experiments*

Following 14 days of germination, the seeds were transferred into individual pots (height: 5.3 cm, diameter: 5.5 cm) with a mixture of vermiculite and sand (1:1) as a substrate. Each pot was saturated to full capacity by solutions of 0, 200, 400, and 800 mM NaCl (ca 35 mL of solution for 1 pot with the substrate) to reflect the salinity of the soil in the field. For individual variants of salinity, we prepared six pots (total 6 pots × 4 variants of NaCl concentration). The pots were located on individual trays filled with 210 mL NaCl solution

(35 mL × 6 pots). Because in the first step we saturated the pots to the full capacity with NaCl solution, the NaCl was still present in the "soil solution" and the concentration of NaCl in this medium did not change during the experiment [24,26,27]. Seedlings were irrigated for three months with 210 mL of Hoagland's solution added to each tray every two days [22]. Following three months of plant development, the growth parameters and biochemical parameters were estimated per triplicate for each NaCl concentration (plants were randomly selected). We evaluated the salinity effect on six growth parameters: shoot length (SL), root length (RL), total fresh mass (FMT), shoot fresh mass (FMS), root fresh mass (FMR), and number of leaves in the rosette (No.LR); and on biochemical parameters: the activity of antioxidant enzymes (APX and POD) and salinity stress indicators (hydrogen peroxide and proline) in the roots, stems and leaves.

The phenotype photos after treatment with different salt concentrations were performed with a Sony digital camera and processed according to Cárdenas-Pérez et al. [24].

#### *2.4. Long- and Short-Term Effects of Salinity*

The three-month-old plants (growing without NaCl application) were stressed with 800 mM NaCl and the leaves were harvested after 1 h, 3 h, and 5 h (short-term salinity stress), and after 24 h, 48 h, 5 days, and 7 days (long-term salt stress) of NaCl addition. Then, the antioxidant enzyme activities (POD and APX) and salinity stress indicators (hydrogen peroxide and proline) were assessed.

#### *2.5. Biochemical Analysis*

For the determination of the activity of antioxidant enzymes (POD and APX), the leaves, roots, and stems were homogenized in 50 mM potassium phosphate buffer pH 7.0, including 0.1 mM EDTA on ice in a mortar. Then, the homogenate was centrifuged at 15,000× *g* for 15 min at 4 ◦C. The obtained supernatant was used for the determination of the antioxidant enzyme activities and protein content. The peroxidase activity (POD) was examined according to Maehly and Chance [28]. The enzymatic reaction was initiated by adding 100 μL of supernatant to the mixture of 50 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol, and 40 mM H2O2. Changes in the absorbance of the reaction solution at 470 nm were read every minute. One unit of the enzyme activity was defined as the amount of enzyme causing a 0.001 change in absorbance per minute. The enzyme activity was presented as U·mg−1. The protein concentration was determined by the Bradford method [29]. The absorbance of the protein solution was measured at 595 nm with bovine serum albumin (BSA) as a standard. The ascorbate peroxidase activity (APX) was followed by the method of [30]. The assay mixture contained 0.1 mL of supernatant with enzyme, 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H2O2, and 1 mL of potassium phosphate buffer (pH 7.0). The decrease in the absorbance of ascorbate at 290 nm was measured and one unit of the enzyme activity was defined as the amount of enzyme causing a 0.001 change in absorbance per minute. The activity of APX was expressed as U·mg<sup>−</sup>1.

Hydrogen peroxide in plant organs was examined according to Sergiev et al. [31] with modifications described by Velikova et al. [32]. Plant tissues (500 mg) were homogenized in an ice bath with 5 mL 0.1% TCA. Then, the homogenate was centrifuged at 12,000× *g* for 15 min (4 ◦C), and 0.5 mL of the supernatant was added to 0.5 mL 10 mM potassium phosphate buffer (pH 7.0) and 1 mL 1 M KI. The solution was incubated in the dark for one hour and the absorbance of the supernatant was read at 390 nm. The final H2O2 concentration was expressed as μM.

The proline level was assessed according to the methodology of Abrahám et al. [33] with a small modification. Fresh plant material (500 mg) was homogenized on ice in a mortar with 3% aqueous sulfosalicylic acid solution (5 μL of solution per one mg of plant material). The homogenate was centrifuged (18,000× *g*, 10 min, 4 ◦C), and the supernatant was collected. The reaction mixture was composed of 2 mL of glacial acetic acid, 2 mL of acidic ninhydrin reagent, and 2 mL of supernatant. An acidic ninhydrin reagent was prepared according to Bates et al. [34]. The reaction mixture was shaken and incubated at 100 ◦C for 30 min. The reaction was inhibited by placing the samples on ice. To extract the chromophore, 4 mL of toluene was added and quantified spectrophotometrically at 520 nm. Proline concentrations were presented in μg/mL.

#### *2.6. Detection of Catalase Activity by Non-Denaturing Polyacrylamide Gel Electrophoresis (PAGE) in Roots, Shoots, and Leaves*

Leaves, roots, and stems were homogenized in 50 mM potassium phosphate buffer pH 7.0, including 0.1 mM EDTA on ice in a mortar. Then, the homogenate was centrifuged at 15,000× *g* for 15 min at 4 ◦C, and 10 μg of protein per sample was loaded on 6% resolving gel solution [35]. The electrophoresis was run at 15 mA at 4 ◦C for 2 h. Following electrophoresis, the gel was soaked in distilled water for 5 min at room temperature. Then, the gel was incubated with 100 mL of a solution with 4 mM H2O2 (10 min, RT) and washed with 100 mL of distilled water. Following this step, the gel was moved to 100 mL of a solution (1% (*w*/*v*) ferric chloride and 1% (*w*/*v*) potassium ferricyanide). When the gel turned dark green, the ferric chloride/potassium ferricyanide solution was removed and washed with distilled water. Bands of catalase activity were marked as clear bands and their intensity corresponded with the activity of CAT [36].
