*4.4. H2O2 Histological Analysis: 3,3-Diamino-Benzidine (DAB) Staining*

DAB-vascular uptake staining method was used to measure the H2O2 production. The cucumber leaves (1 cm sections) were immersed in a solution containing 1 mg/mL DAB that was dissolved in HCl-acidified (pH 3.8) distilled water, then incubated under light for 8 h, then the treated leaves were examined under a microscope [36].

#### *4.5. ROS Assay*

The H2O2 content in cucumber was measured according to the method described by Gong et al. using potassium iodide [37]. Superoxide anion (O2 •−) content was measured according to the method of Wang et al. [38].

### *4.6. Antioxidative Enzyme and Antioxidant Substances Analysis*

To assay antioxidative enzymes, 0.5 g of cucumber leaves were weighed and ground together with 6 mL of 200 mmol/L phosphate buffer (pH 7.8) which contains 1% (*w*/*v*) soluble polyvinyl pyrrolidone (PVP) under ice bath conditions. The homogenates were centrifuged at 11,000× g for 20 min at 4 ◦C and the supernatant were collected to measure the activities of SOD, CAT, POD and the contents of soluble protein and MDA [8]. For the assay of antioxidant substances, 1.0 g of cucumber leaves were homogenized in 5 mL of 1% (*w*/*v*) saline buffer (1% (*w*/*v*) PVP, 1 mmol/L ethylenediaminetetraacetic acid (EDTA)). Homogenates were centrifuged at 11,000 g for 15 min at 4 ◦C and the supernatants were collected to check the content of ASA and GSH [39].

#### *4.7. Total RNA Extraction and Antioxidant Enzyme Gene Expression Analysis*

Total RNA was isolated from cucumber leaves using Trizol total RNA Extraction Reagent (Bioer, Hangzhou, China). First-strand cDNA was synthesized by a HiFiScript cDNA Synthesis Ki (CoWin Biosciences, Beijing, China). The expression of selected genes was analyzed by real-time quantitative polymerase chain reaction (RTq-PCR). The corresponding primer sequences were selected from Zhao et al. [23]. The qPCR was performed with the iCycleriQTM 5 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) following the manufacturer s instructions. Gene expression levels were calculated on the basis of the 2−ΔΔCt method [40]. Three biological and technical replications were performed, respectively.
