Hydrogen peroxide assay kit

H₂O₂ content was determined using the method described by Deniz et al. [51 (link)]. Fresh cotton leaves (0.1 g) were homogenized in 1 mL of cold acetone. Then, H₂O₂ content was determined using a hydrogen peroxide assay kit (Solarbio, Beijing, China) and its absorbance was measured at 415 nm. Data are represented as the amount of H₂O₂ per gram leaf (μmol/g). All analyses had three biological replicates.

The H₂O₂ content was measured using a hydrogen peroxide assay kit (Solarbio, Beijing, China) [42 (link)]. The content of H₂O₂ in rice leaves was detected by measuring the absorbance of titanium-peroxide complex formation at 415 nm.

H₂O₂ content and O₂^•− content were detected using the hydrogen peroxide assay kit (Beijing Solarbio Science and Technology Co. Ltd., Beijing, China) and superoxide anion assay kit (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China), respectively. The results were expressed as mmol·kg⁻¹.
DPPH scavenging ability detection followed Tang et al. [43 (link)]. An amount of 25 μL phenolic extract was mixed with 175 μL DPPH solution (350 μM in methanol), and placed in darkness at 25 °C for 4 h. The results were recorded by measuring absorbance at 517 nm and expressed as mmol Trolox equivalent kg⁻¹.
MDA content detection followed Jiang et al. [40 (link)]. An amount of 5 g powder was extracted with 5 mL trichloroacetic acid (100 g·L⁻¹) and centrifuged at 10,000× g, 4 °C for 10 min. The supernatant (2 mL) was mixed with 2 mL thiobarbituric acid (6.7 g·L⁻¹) and then incubated in boiling water for 20 min. After cooling to room temperature, the mixture’s absorbance was recorded at 450 nm, 532 nm and 600 nm. The result was expressed as µmol·kg⁻¹.

For NBT staining, the salt-treated leaves from NtNAC028-OE lines and WT were soaked in NBT staining buffer (10 mM potassium phosphate buffer, pH 7.2) for 12 h. After that, leaf chlorophyll was removed by a mixed solution (glycerol/acetic acid/ethanol, 1:1:3) and then stored in a solution consisting of ethanol/glycerol (4:1). The hydrogen peroxide content was determined using a hydrogen peroxide assay kit (Solarbio, China) according to the manufacturer’s instruction.

Biochemical indicators were measured using corresponding assay kits from Solarbio Life Sciences (Beijing, China) following the manufacturer’s protocols. In detail, the Starch Content Assay Kit (Solarbio, Cat#BC0700), α-Amylase Assay Kit (Solarbio, Cat#BC0615), Hydrogen Peroxide Assay Kit (Solarbio, Cat#BC3595), and Proline Content Assay Kit (Solarbio, Cat#BC0290) were obtained for subsequent spectrophotometric measurements.

For observation of the water-soaked/hypersensitive reaction, leaves of 5- to 6-week-old IR24 rice were infiltrated with different Xoo suspensions with an optical density at 600 nm (OD₆₀₀) of 0.5 using a needleless syringe, as previously described (Streubel et al. 2013 (link), Yang and Bogdanove 2013 (link)). The water-soaked symptoms were scored and photographed 3 dpi. For H₂O₂ accumulation assays, rice leaves infiltrated with different Xoo suspensions at the indicated time points (1 and 3 dpi) were examined by 3,3′-diaminobenzidene (DAB) staining following the methods described previously (Girija et al. 2017 (link), Sathe et al. 2019 (link)). Then, H₂O₂ accumulation was visualized by visible light microscopy using a 10× objective. To measure H₂O₂ levels in rice leaves, a Hydrogen Peroxide Assay Kit (Solarbio, Beijing, China) was used according to the instruction manual. All the rice inoculations with Xoo were biologically repeated at least three times, and each involved three replicates.