*2.5. Gas Exchange and Chlorophyll Fluorescence Measurements*

The infrared gas analyzer-based portable photosynthesis system (LI-6400T, Li-Cor Inc., Lincoln, NE, USA) was applied for measuring the net photosynthetic rate (*P*n) in plants under heat or controlled environment. The measurements were carried out at 1000 μmol m−<sup>2</sup> s−<sup>1</sup> photosynthetic photon flux density (PPFD), 400 μmol mol−<sup>1</sup> atmospheric carbon dioxide (CO2) concentrations, and 25 ◦C leaf temperature, respectively. Fluorescence measurements for chlorophyll were conducted using a MAXI Version of the Imaging-PAM M-Series fluorescence system (Heinz-Walz, Effeltrich, Germany). For 30 min prior to measurement, plants were kept in the dark. According to previous descriptions, the maximum quantum yield of PSII (*Fv/Fm*) was measured and calculated [39].

#### *2.6. Analysis of H2O2, O2* •− *and Malondialdehyde (MDA)*

In order to observe the accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2 •−) on leaves, the DAB and NBT staining were performed as previously described with minor modifications [40].

For O2 •− staining, leaf samples were stained with 0.5 mg mL−<sup>1</sup> NBT in 25 mM N-2 hydroxyethylpiperazine-N-ethane-sulphonic acid (HEPES) (pH 7.8) and incubated in the dark under 25 ◦C for 6 h. For H2O2 staining, leaf samples were stained with 1 mg mL−<sup>1</sup> DAB in 50 mM Tris-HCl (pH 3.8) and incubated at 25 ◦C for 12 h in the dark. In both cases, leaf samples were washed in 95% (v:v) ethanol for 10 min at 95 ◦C, kept in lactic acid/phenol/water (1:1:1; v:v:v), and photographed.

The H2O2 concentration in the leaves was quantified based on the method described previously with minor modifications [41]. In brief, a 0.3 g leaf sample was taken for analysis. After being ground with 3 mL 0.2 M HClO4 in liquid nitrogen, the material was centrifuged at 6000 g for 10 min at 4 ◦C. A total of 4 M KOH was used to neutralize the pH to about 6–7. 0.05 g activated carbon was added and the solution was centrifuged at 12,000× *g* for 5 min at 4 ◦C. The 0.22 μm filter membrane was used to filter the supernatant into a new centrifuge tube to obtain extracting solution. A total of 100 mM potassium acetate buffer (pH 4.4, containing 1 mM ABTS) was used as the reaction buffer. For the nonenzymatic tube reaction system, 1 mL H2O2 sample and 1 mL reaction buffer were mixed and the absorption peak at 412 nm was determined. For the enzyme tube reaction system, 1 mL H2O2 sample, 996 μL reaction buffer, and 4 μL horseradish peroxidase (POD) were mixed. Finally, the absorption peak at 412 nm was determined to measure the content of H2O2. The content of MDA in the leaves was measured according to a previous protocol [39]. Extracted leaves were heated at 95 ◦C for 25 min with trichloroacetic acid containing 0.65% 2-thiobarbituric acid (TBA). By subtracting the absorbance at 532 nm of a TBA-free solution containing the plant extract, non-MDA compounds were corrected.
