*2.7. H2O2 Content, Lipid Peroxidation, and Antioxidant Enzymes*

According to the method of Ding et al. [23], the lipid peroxidation of cell membrane was evaluated by the determination of malondialdehyde (MDA). The amount of MDA-TBA (thiobarbituric acid) complex was calculated from the coefficient of absorbance (155 mM−<sup>1</sup> cm<sup>−</sup>1). The content of H2O2 was estimated by monitoring the absorbance of the titanium peroxide complex at 415 nm. Next, 0.2 g of plant material were homogenized in 1.5 mL 50 mM phosphate buffer solution (PBS) (pH 7.5) containing 0.2 mM ethylenediamine tetra acetic acid (EDTA) and 2% polyvinylpyrrolidone (PVP). The homogenate was centrifuged in a refrigerated centrifuge at 13,000× *g* for 20 min and the supernatants were used for protein determination and enzyme assay. All the steps were carried out at 4 ◦C. superoxide dismutase (SOD) activity was measured by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm. The catalase (CAT) activity was determined by measuring the decrease in absorption of H2O2 at 240 nm.

#### *2.8. RNA Isolation and qRT-PCR*

Total RNA was extracted with an RNA Isolater Total RNA Extraction Reagent (Vazyme Biotech Co., Ltd., Nanjing, China). Reverse transcription was performed using the HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). The primer pairs used in this study are all listed in Table S1. The qRT-PCR was performed on an ABI7500 instrument (Applied Biosystems, Foster, CA, USA) using SYBR Green qPCR kits (TaKaRa, Kusatsu, Japan). The PCR amplification conditions included an initial heat-denaturing step at 95 ◦C for 3 min, followed by 40 cycles of 30 s at 95 ◦C, 30 s at 58 ◦C, and 1 min at 72 ◦C. Relative mRNA levels were normalized to that of the internal reference genes. Each sample was divided into 3 biological replicates. The data were processed on the basis of the 2−ΔΔCT method.
