H2o2 test kit

H₂O₂ production was examined in 6–8 tomato leaves at 2, 4, and 6 h after treatment with 1 μM BcGs1 or Tris-HCl buffer. Leaves were excised and placed in water with 0.01% Triton-X-100 and 1 mg/mL nitro 3, 3′-diaminobenzidine (DAB); then, the solution was infiltrated with low vacuum pressure for 5 min, and the leaves were incubated overnight at room temperature. After that, the leaves cleared in alcoholic lactophenol (95% ethanol:lactic acid:phenol, 2:1:1) at room temperature until the leaves did not contain chlorophyll, and they were then rinsed with water. H₂O₂ can be visualized by a brownish-red precipitate that formed by polymerization with DAB.
H₂O₂ content was detected in the leaves using the H₂O₂ Test Kit (Jiancheng Biotechnology, Nanjing, China) at 0, 2, 4, 6, 12, and 24 h post-treatment. Samples of 150 mg were ground using a grinding machine and homogenized in extraction buffer (0.05 mM Phosphate buffer, pH 7.2). The supernatant was collected for H₂O₂ content detection after centrifugation at 10000 g for 10 min at 4 °C. The reagent provided by the kit was incubated with the initial solution following the manufacturer’s instructions. The H₂O₂ content was detected at 405 nm wavelength. Each experiment was repeated three times.

The activities of POD and SOD, H₂O₂ content, and GSH level in soybean hairy roots were measured as described previously [63 ,64 (link)]. In brief, total proteins were extracted using the vegetable protein extraction kit (KeyGene BioTECH, Nanjing, China) and quantified using a BCA protein assay kit (KeyGene BioTECH). The activities of POD and SOD, H₂O₂ content, and GSH level were measured using a peroxidase kit (Jiancheng BioTECH, Nanjing, China), superoxide dismutase kit (Keming BioTECH, Suzhou, China), H₂O₂ test kit (Jiancheng BioTECH, Nanjing, China), and GSH test kit (Jiancheng BioTECH, Nanjing, China) according to the instructions from the manufacturers and previously published methods [63 ,64 (link)]. All measurements were performed with four biological replications and three independent soybean hairy roots were used in each replication.

After being subjected to trimming treatment for 0, 1, 3, 6, 12, 24, 48 and 72 h, the sample was weighed at 0.1 g. Subsequently, nine times the volume of PBS (pH 7.0–7.4, 0.1 mol/L) was added following a weight (g) to volume (mL) ratio of 1:9. The supernatant homogenate was mechanically shaken under an ice-water bath and collected for determination using a hydrogen peroxide (H₂O₂) test kit (Jiancheng, Nanjing, China).