*2.8. Determination of Proline, Malondialdehyde, and Antioxidant Enzyme Activities on the Sixth Day after Treatment*

The determination of the free proline content was performed according to Bates et al. [24]. Leaf samples (0.5 g) from each group were homogenized in 3% (*w*/*v*) sulfosalicylic acid, and the homogenate was filtered through filter paper. After the addition of acid ninhydrin and glacial acetic acid, the resulting mixture was heated at 100 ◦C for 1 h in a water bath. The reaction was then stopped using ice bath. The mixture was extracted with toluene, and the absorbance of the fraction with toluene aspired from the liquid phase was read at 520 nm. The proline concentration was determined using a calibration curve and expressed as μmol proline g−<sup>1</sup> FW.

Lipid peroxidation can be measured by determining the malondialdehyde (MDA) content. The MDA determination followed the method described by Zhao [25] with minor modifications. Briefly, we took 1 mL of the sample supernatant extracted above and added 2 mL of 0.67% thiobarbituric acid (TBA). We used a sample containing only 1 mL of water as a negative control. Next, we placed the samples in a boiling water bath for 15 min, rapidly cooled them by immersion in cold water, and poured them into 10 mL centrifuge tubes. We then centrifuged all tubes at 4000 rpm for 20 min and determined the absorbance of all samples at 600 nm, 532 nm, and 450 nm using a spectrophotometer. The MDA content was determined using the following formula: MDA (μmol·<sup>g</sup> <sup>−</sup>1) = [6.452 (A532 − A600) − 0.56 × A450] × VT/(V0 × W), where VT = the total volume of the extract; V0 = the assay volume; and W = plant fresh weight.

All enzymatic activities were determined at 25 ◦C and expressed as U g−<sup>1</sup> protein. The CAT activity was measured by monitoring the decrease in H2O2 at 240 nm for 1 min

at 25 ◦C. The 3 mL reaction mixture contained 100 μL of enzyme extract and 2.9 mL of sodium phosphate buffer (50 mM, pH 6.0) containing 10 mM H2O2. The CAT activity was calculated as the amount of enzyme that caused a reduction in the absorbance at 240 nm of 0.01 per minute [25]. The peroxidase (POD) activity was determined by a colorimetric method [25] in a reaction mixture containing guaiacol as the substrate. The POD activity was determined based on the change in absorbance at 470 nm due to the oxidation of guaiacol to tetraguaiacol. The POD activity was defined as the amount of enzyme that caused an increase in absorbance at 470 nm of 0.001 per minute. The superoxide dismutase (SOD) activity was determined based on the ability to inhibit the photochemical reduction in nitroblue tetrazolium (NBT) [25]. The 3 mL reaction mixture was initiated by illumination for 2 min at 25 ◦C, and the absorbance of blue formazan was measured with a spectrophotometer at 560 nm. One unit of SOD activity (U) was defined as the amount of enzyme that caused a 50% inhibition of the NBT reduction. The APX activity (ascorbate peroxidase) was determined by measuring the oxidation rate of ascorbate at 290 nm according to Zhao [25]. The decrease in the AsA concentration was followed as a decline in the optical density at 290 nm, and the activity was calculated using the extinction coefficient (2.8 mM−<sup>1</sup> cm−<sup>1</sup> at 290 nm) for AsA. One unit of APX was defined as the amount of enzyme that breaks down 1 μmol AsA min−1. The guaiacol peroxidase (GPX) activity was measured using a modification of the procedure by Zhao [25]. The reaction mixture in a total volume of 2 mL contained 25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM EDTA, 0.05% guaiacol (2-ethoxyphenol), 1.0 mM H2O2, and 100 μL of enzyme extract. The increase in absorbance due to the oxidation of guaiacol was measured at 470 nm (E = 26.6 mM−<sup>1</sup> cm<sup>−</sup>1). The dehydroascorbate reductase (DHAR) activity was determined according to Zhao [25]. The reaction mixture in a total volume of 2 mL contained 25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM EDTA, 3.5 mM GSH, 0.4 mM dehydroascorbate (DHAR), and 100 μL of enzyme extract. The DHAR activity was measured by the formation of ascorbate at 265 nm (E = 14 mM−<sup>1</sup> cm−1). The enzyme activity was expressed as unit g−<sup>1</sup> min−<sup>1</sup> FW.

#### *2.9. Total RNA Extraction and Quantitative PCR Analysis on the Sixth Day after Treatment*

Genes of the antioxidant system were identified from the NCBI database (https:// www.ncbi.nlm.nih.gov/, accessed on 14 November 2022). RNA of leaves was extracted using the RNA Extraction Kit (Kangwei Century Technology Co., Ltd, Beijing, China) and reverse-transcribed to cDNA (Takara). The primers used in this study are shown in Supplementary Table S1. Quantitative PCR was performed to determine the expression of related genes. The reactions were performed using an Ultra SYBR Mixture (Kangwei, Beijing, China) Kit. Three biological replicates and three technical repeats were used in all qPCR reactions. The relative gene expression levels were calculated using the 2 –ΔΔCT method [26].

#### *2.10. Determination of TTC Assay and Electrolyte Leakage on the Sixth Day after Treatment*

The root viability was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) reduction method and expressed as the amount of TTC reduced by per gram of root [25].

The root electrolyte leakage was determined according to a previously described method [27]. Approximately 0.5 g of a root was taken in a test tube, mixed with 20 mL of distilled water, and the vacuum was evacuated three times for 20 min each time. The initial conductivity (S1) of the mixture was measured with a conductivity meter (DDS-11A, Shanghai Kanglu Instrument Equipment Co., Ltd., Shanghai, China). The mixture was then sealed and incubated in a boiling water bath for 10 min. The conductivity was measured after cooling (S2). The conductivity of the distilled water was also measured (S0). Then, the percentage of electrolyte leakage was calculated as follows: electrolyte leakage (%) = [(S1 − S0)/(S2 − S0)] × 100.

#### *2.11. Determination of Nitrogen Content and Nitrogen-Metabolism-Related Enzyme Activities in Leaves*

On the sixth day after the treatment, the nitrate–nitrogen (NO3-N) content of the leaves was determined by salicylic acid colorimetry [25]. Under an acidic condition, the leaf extract was mixed with salicylic acid to form nitrosalicylic acid, whose absorbance was read at 410 nm.

The ammonium–nitrogen (NH4 +-N) content was determined by Nessler's spectrophotometric method [28]. The leaf extract was mixed with hypochlorite and phenol under a strong alkaline condition to form a water-soluble blue indophenol whose absorbance was measured at 625 nm.

The nitrate reductase (NR) activity in the leaves was determined by sulfanilamide colorimetry [25]. The leaf extract was mixed with sulfanilamide in hydrochloric acid and N-1-naphtyl-ethylenediamine to form a red azo dye whose absorbance was measured at 520 nm.

We determined the glutamine synthetase (GS) activity using the plant GS enzyme activity assay kit and determined the glutamic acid synthetase (GOGAT) activity using the plant GOGAT enzyme activity assay kit (Jiangsu enzyme label Biotechnology Co., Ltd., Jiangsu, China), following the manufacturer's instructions.

#### *2.12. Data Processing and Analysis*

We collected three biological replicates for each treatment. Origin version 9.8 was used to conduct all statistical analyses. Duncan multiple range tests, which are included in SPSS version 20.0, were performed to detect any statistically significant differences in the mean values (IBM SPSS, Chicago, IL, USA). The threshold of statistical significance used for all tests was *p* < 0.05.

#### **3. Results**
