*2.3. Determination of the Soluble Amino Acid and Protein Contents*

The fresh plant material (roots and leaves) of the control and Tl treatments was homogenized (0.2 g mL−1) in 50 mM sodium phosphate buffer, pH 7.0, then filtered through muslin, centrifuged at 12,360× *g* for 15 min, and the supernatant was used for the protein and amino acid assays. For the protein assay, Bradford's method was used [67]. The results are expressed as mg g−<sup>1</sup> FW against an albumin standard curve. For the amino acid assay, the method of Yemm and Cocking [68] was used, with ninhydrin reagent. The results are expressed as mg g−<sup>1</sup> FW against a glycine standard curve.

#### *2.4. Determination of Photosynthetic Pigment Contents and Photosynthetic Efficiency*

The chlorophyll and carotenoid contents of the leaves were determined at the end of each trial. About 0.125 g of fresh leaves were incubated in 10 mL methanol for 24 h in the dark. The concentrations of chlorophylls and carotenoids were measured spectrophotometrically (Shimadzu UV1603) at A666, A653, and A470. The total chlorophyll and carotenoid content was calculated as described by Wellburn [69] and expressed as μg g−<sup>1</sup> FW.

For the determination of the photosynthetic parameters, the middle region of the fully expanded upper leaves at the end of each Tl treatment were adapted in the dark for 10 min, and then the minimal fluorescence (F0), the maximal chlorophyll fluorescence (Fm), and the maximum photosynthetic efficiency (Fv/Fm) were recorded with a handheld fluorometer (Chlorophyll Fluorometer, OS-30p, Opti-Sciences). The variable fluorescence (Fv) and the rate constants of photochemical and nonphotochemical deactivation of excited Chl molecules (Fv/F0) was calculated.

#### *2.5. Determination of Lipid Peroxidation and Reactive Oxygen Species (O2 .*<sup>−</sup> *and H2O2), NO, and H2S Contents, and SOD Activity*

To analyze oxidative stress, the formation of malondialdehyde (MDA) was determined using thiobarbituric acid (TBA). Briefly, 0.25 g of plant material (roots or leaves) was homogenized with 2.5 mL of solution containing 0.25% TBA and 10% trichloroacetic acid (TCA). The mixture was incubated at 95 ◦C for 30 min. The reaction was stopped by immersing the tubes in ice, then filtering, and centrifuging at 8800× *g* for 10 min. The MDA was determined spectrophotometrically in the supernatant at A532−A600 with ε = 155 mM−<sup>1</sup> cm−<sup>1</sup> and expressed as μmol MDA g−<sup>1</sup> FW [70]. The H2O2 content was analyzed in accordance with Velikova et al. [71]. Fresh material (roots or leaves) was homogenized (0.2 g mL<sup>−</sup>1) in 0.1% TCA. The homogenate was centrifuged at 12,000× *<sup>g</sup>* for 15 min. The reaction mixture contained 0.5 mL of supernatant, 0.5 mL 10 mM of potassium phosphate buffer (pH 7.0), and 1 mL of 1 M KI solution. The H2O2 concentration was estimated based on the reaction mixture absorbance at A390 using a standard curve of H2O2. The O2 .<sup>−</sup> generating and SOD activities were measured in an extract obtained from roots or leaves that was homogenized (0.5 g mL−1) at 4 ◦C in 50 mM phosphate buffer, pH 6.0, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM β-mercaptoethanol, and 1gL−<sup>1</sup> plyvinylpolypyrrolidone (PVPP). The homogenate was filtered and centrifuged at 39,000× *g* for 30 min at 4 ◦C, and the supernatant was collected as an enzyme extract. The O2 .<sup>−</sup> generation was assayed spectrophotometrically by measuring the oxidation of epinephrine to adrenochrome at A480 (ε = 4.020 mM−<sup>1</sup> cm<sup>−</sup>1) [72]. The reaction mixture

contained 1 mM epinephrine in acetate buffer 25 mM, pH 5.0. The SOD activity was determined in 50 mM phosphate buffer, pH 7.8, 0.1 mM ethylene diamine tetra-acetic acid (EDTA), 1.3 μM riboflavin, 13 mM methionine, and 63 μM nitro blue tetrazolium (NBT) [73]. The reaction mixture was maintained in the dark at 25 ◦C, and the reaction was started by the addition of the riboflavin and enzyme extract and was illuminated for 2 min. The changes in absorbance A560 were measured. A unit of SOD is defined as the amount of enzyme required to cause 50% inhibition of NBT reduction. The NO content was determined using the method described by Zhou et al. [74]. Roots or leaves (0.5 g) were homogenized in 3 mL of 50 mM cool acetic acid buffer (pH 3.6, containing 4% zinc diacetate). The homogenates were centrifuged at 10,000× *g* for 15 min at 4 ◦C. The supernatant was collected. The pellet was washed in 1 mL of acetic acid buffer and centrifuged as before. The two supernatants were combined, and charcoal was added, followed by vortexing and filtration. A mixture of filtrate and the Griess reagent (1:1) was incubated at room temperature for 30 min. The absorbance was determined at A540. The NO content was calculated by comparison against a standard curve of NaNO2 and expressed as nmol NaNO2 g−<sup>1</sup> FW. The H2S concentration was determined using Li's method [75]. Roots or leaves were ground in liquid nitrogen, homogenized (0.5 g mL−1) in 20 mM Tris-HCl buffer, pH 8.0, containing 10 mM EDTA and 20 mM Zn(OAc)2, and centrifuged at 15,000× *g* for 15 min at 4 ◦C. The supernatant was combined with 30 mM FeCl3 (in 1.2 M HCl) and 20 mM DMPD (in 7.2 M HCl) (1:1:1). The mixture was incubated at room temperature for 15 min and the A670 was determined. The H2S content was calculated by comparison against a standard curve of NaHS and expressed as nmol H2S g−<sup>1</sup> FW.

### *2.6. Determination of the Components and Antioxidant Enzymes of the AsA–GSH Cycle*

To determine the total ascorbate and glutathione, fresh roots or leaves (1 g mL<sup>−</sup>1) were homogenized at 4 ◦C in 5% metaphosphoric acid. The homogenate was centrifuged at 20,000× *g* for 20 min at 4 ◦C, and the supernatant was collected for the determination of ascorbate and glutathione. The total ascorbate pool (AsA + DHA) and total glutathione pool (GSH + GSSG) were determined in accordance with De Pinto et al. [76]. Total ascorbate was determined by the reduction of DHA to AsA, and the concentration of DHA was estimated from the difference between the total ascorbate pool and the AsA. The ascorbate pool was determined at A525. The glutathione pool was determined from the change in absorbance at A412 over 1 min. GSH was estimated as the difference between the amount of total glutathione pool and that of GSSG.

To determine the activities of the enzymes involved in the AsA–GSH cycle—APX, MDHAR, DHAR, and GR—the roots or leaves (0.5 g L−1) were homogenized at 4 ◦C in 50 mM phosphate buffer, pH 7.5, 0.5 mM PMSF, 1 mM β-mercaptoethanol, 1 g L−<sup>1</sup> PVPP, and 5 mM AsA for the APX activity. The homogenate was filtered and centrifuged at 39,000× *g* for 30 min at 4 ◦C, and the supernatant was used for the enzyme determinations. The APX activity was determined spectrophotometrically by measuring the oxidation of ascorbate at A290 for 2 min (ε = 2.8 mM−<sup>1</sup> cm−1) [77]. The reaction mixture contained 0.5 mM ascorbate, 0.2 mM H2O2, and the enzyme extract, at 25 ◦C, in 0.1 M phosphate buffer, pH 7.5, and EDTA 0.5 mM, expressing the result as μmol ascorbate min−<sup>1</sup> mg−<sup>1</sup> protein. The DHAR activity was determined from the oxidation of DHA at A265 for 1 min (ε = 14 mM−<sup>1</sup> cm<sup>−</sup>1) [78] in a medium containing 0.1 M phosphate buffer (pH 6.5), 0.5 mM EDTA, 2.5 mM GSH, 0.5 mM DHA, and the enzyme extract. The DHAR activity is expressed as nmol ascorbate min−<sup>1</sup> mg−<sup>1</sup> protein. The MDHAR activity was determined from the oxidation of NADH at A340 for 1 min (ε = 6.22 mM−<sup>1</sup> cm−1) [79] in a medium containing 50 mM Tris-HCl buffer (pH 7.8), 10 mM AsA, 0.2 mM NADPH, 0.5 units of ascorbate oxidase, and the enzyme extract, expressing the result as μmol NADH min−<sup>1</sup> mg−<sup>1</sup> protein. The GR activity was determined at A340 from the oxidation of NADPH for 3 min (ε = 6.22 mM−<sup>1</sup> cm<sup>−</sup>1) [78] in a medium containing 0.1 M phosphate buffer (pH 7.5), 0.5 mM EDTA, 0.5 mM GSSG, 0.2 mM NADPH, and the enzyme extract, expressing the result as nmol NADPH min−<sup>1</sup> mg−<sup>1</sup> protein.

#### *2.7. Statistical Analyses*

The data to be presented are the means ± SE of at least 10 replicates obtained from three independent experiments. For each measurement, a Shapiro–Wilk normality test was performed (since *n* < 50) to verify if they had a normal distribution. Later, we applied a parametric test of one-way ANOVA. Those values where there are significant differences are marked with different letters, that is, where *p* ≤ 0.05. All statistical analyses were performed with Microsoft Excel 365 (Microsoft Inc., Alburquerque, NM, USA) and the SPSS v. 24 package (SPSS Inc., Chicago, IL, USA).

#### **3. Results**
