*2.7. Antioxidant and Enzyme Activity Assays*

For nonenzymatic antioxidant assays, approximately 100 mg of leaf sample was powdered in liquid nitrogen and extracted into 1 mL 0.2 M HCl. The solution was centrifugated by 12,000 g for 10 min under 4 ◦C and then 0.2 M NaOH was used to neutralize the mixed solution to pH 4–5 containing 500 μL supernatant of the last step and 100 μL 0.2 M phosphate buffer (pH 5.6). Finally, spectrophotometric assays were used to measure the

extracting solution for ascorbic acid (AsA)/dehydroascorbic acid (DHA), and reduced glutathione (GSH)/oxidized glutathione (GSSG) according to previous methods [42].

To measure antioxidant enzyme activity, 300 mg leaf sample was milled with 3 mL of ice-cold enzyme buffer containing 25 mM HEPES, 0.2 mM ethylene diamine tetraacetic acid (EDTA), 2 mM AsA, and 2% polyvinylpolypyrrolidone (w:v) (pH 7.8). The extracting solution was centrifugated at 12,000× *g* for 10 min under 4 ◦C and then the supernatants were kept for measurement. Subsequently, SHIMADZU UV-2410PC spectrophotometer (Shimadzu, Kyoto, Japan) was employed to detect enzyme activity. The activities of antioxidant enzymes catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) were analyzed according to the previous protocol with minor modifications [43]. For analyzing CAT activity, 100 μL of enzyme solution, 1700 μL of 25 mM phosphate buffer Solution (PBS) (PH 7.0, containing 0.1 mM EDTA), and 200 μL of 100 mM hydrogen peroxide were mixed. The kinetic changes of OD240 were determined according to the kinetic program, and the enzymatic reaction rate was calculated by taking the kinetic changes of 10 s. For analyzing APX activity, 100 μL of enzyme solution, 1700 μL of 25 mM PBS (pH 7.0, containing 0.1 mM EDTA), 100 μL of 20 mM H2O2, and 100 μL of 5 mM AsA were mixed together at 25 ◦C. The kinetic changes of OD290 were determined according to the kinetic program, and the enzymatic reaction rate was calculated by taking the kinetic changes of 10 s. The reaction rate without H2O2 was used as blank control. For analyzing GR activity, 100 μL of enzyme solution, 1700 μL of 25 mM PBS buffer (PH7.8, containing 0.2 mM EDTA), 100 μL of 10 mM GSSG, and 100 μL of 2.4 mM NADPH were mixed together at 25 ◦C. The kinetic changes of OD340 were measured, and the enzymatic reaction rate was calculated by taking the kinetic changes of 10 s. For analyzing DHAR activity, 100 μL of enzyme solution, 1700 μL of 25 mM PBS (pH 7.0, containing 0.1 mM EDTA), 100 μL of 70 mM GSH, and 100 μL of 8 mM DHA were mixed together. The kinetic changes of OD265 were measured, and the kinetic change of 10 s was taken to calculate the enzymatic reaction rate. The enzyme activities of superoxide dismutase (SOD) and peroxidase (POD) were detected according to the previous protocol with minor modifications [44]. For analyzing SOD activity, 50 μL of enzyme solution and 3 mL reaction solution (containing 50 mM PBS (pH 7.8), 15 mM methionine, 65 mM NBT, 2 μM riboflavin, 0.1 mM EDTA) were mixed. After 15 min illumination at 25 ◦C, 4000 lx, the absorbance was measured at 560 nm. For analyzing POD activity, 100 μL of enzyme solution, 1700 μL of 25 mM PBS (pH 7.0, containing 0.1 mM EDTA), 100 μL of 10 mM H2O2, and 100 μL of 1% guaiacol were mixed together at 25 ◦C. The kinetic changes of OD470 were determined according to the kinetic program, and the kinetic changes of 10 s were taken to calculate the enzymatic reaction rate.

#### *2.8. Immunoblotting Assay*

Following the manufacturer's instructions, the oxidized protein fractions extracted from the soluble protein were tested with an OxyBlot Protein Oxidation Detection Kit (Chemicon International, Temecula, CA, USA).

For immunoblotting assay, the protein extraction and Western blotting assay were modified by protocol described previously [45]. A 0.1 g leaf sample was grinded in liquid nitrogen and added with the extraction buffer (100 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulphonyl fluoride, and 0.2% β-mercaptoethanol). The Bio-Rad protein assay kit was used to measure the protein concentration and the total protein concentration of all samples were adjusted to 6 μg/μL. After denaturation by 95 ◦C for 10 min, the protein samples were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred to nitrocellulose membrane (GE Healthcare Biosciences, Piscataway, NJ, USA). Antibodies of cytosolic Hsp90 (AS08 346, Agrisera, Vännäs, Sweden), Hsp70 (PHY0034S, Phytoab, San Jose, CA, USA), Hsp101 (AS07 253, Agrisera, Vännäs, Sweden) and Hsp17.6 (PHY0149S, Phytoab, San Jose, CA, USA) were used to detect proteins. Afterwards, the

goat anti-rabbit horseradish peroxidase-linked antibody (7074, Cell Signaling Technology, Boston, MA, USA) was used as the secondary antibody for these analyses.
