Peroxidase assay kit

Contents of soluble sugar, starch, and sucrose were determined according to the method described in a previous study [40 (link)]. Content of soluble protein was determined according to the Total Protein Quantitative Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China); Peroxidase Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the POD activity.

The plant samples were firstly ground into powder in liquid nitrogen and then suspended in ice-cold phosphate buffer (0.1 M, pH = 7). The sample was vortexed at maximum speed for 1 min and centrifuged at 12,000 rpm for 15 min. The supernatants were then used for further determination of the hydrogen peroxide level and antioxidant enzyme activity. The hydrogen peroxide level of different samples was determined using the Hydrogen Peroxide Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China). The absorbance at 405 nm was determined by a Scandrop spectrophotometer (Analytikjena, Germany). The hydrogen peroxide level was calculated based on a previously described formula [56 (link)]. The MDA, proline, and total soluble sugar level were separately determined using the MDA Assay Kit, the Proline Assay Kit, and the Plant Soluble Sugar Content Test Kit (Jiancheng Bioengineering Institute, Nanjing, China), as previously described [24 (link)].
The antioxidant enzyme activities were also determined using the spectrophotometric method. SOD, POD, and CAT activities were separately determined using the Total Superoxide Dismutase (T-SOD) Assay Kit, Peroxidase Assay Kit, and Catalase (CAT) Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China), as previously described [56 (link)].

The oxidation and antioxidative activities were determined according to the manufacturer’s recommended protocol. Nitric oxide (NO) induction was measured using the nitrate reductase method and the nitric oxide assay kit (Nanjing Jiancheng Bioengineering Institute). Catalase (CAT) activity was measured using the ammonium molybdate method and the catalase assay kit (Nanjing Jiancheng Bioengineering Institute). Peroxidase (POD) activity was measured using the absorbance method and the peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute). Superoxide dismutase (SOD) activity was measured using the WST-1 method and the superoxide dismutase assay kit (Nanjing Jiancheng Bioengineering Institute).

Herein, 1 mg/mL Di-amino benzidine (DAB) and 1 mg/mL nitroblue tetrazolium (NBT) were used to stain the 5th to 8th rosette leaves from 7-week-old seedlings for H₂O₂ and O₂⁻, respectively [49 (link)], according to the previous method [16 (link)]. The measurements of H₂O₂, O₂⁻, and MDA were performed according to the previous studies [50 (link),51 (link)]. Briefly, leaf samples used for determination of H₂O₂ and O₂⁻ were extracted by 50 mmol/L phosphate buffer. Then, the supernatant was mixed with titanium sulphate and determined at 415 nm to calculate the concentration H₂O₂, while the supernatant was mixed with sulphanilic acid and α-naphthylamine and detected at the absorbance of 530 nm to determine the concentration of O₂⁻. MDA was determined by trichloroacetic acid and thiobarbituric acid, and the absorbance was determined at 532, 600, and 450 nm.
In addition, the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were detected using the Total Superoxide Dismutase Assay Kit (Nanjing Jiancheng, A001-1, Nanjing, China), the Catalase Assay Kit (Nanjing Jiancheng, A007-1, Nanjing, China), and the Peroxidase Assay Kit (Nanjing Jiancheng, A084-3, Nanjing, China), respectively.

To examine the physiological parameters of NT poplar and PeHKT1;1 transgenic lines, the three-year-old soil-grown poplar plants were treated with 0.8% (w/v) NaCl at different time points (0, 0.5, 1, 1.5, 2, and 6 h). The CAT, SOD, and POD activities were measured by leaves according to Li et al. [29 (link)] using the corresponding assay kits: total protein assay kit (BCA method, A045-2), catalase assay kit (visible light, A007-1), total superoxide dismutase (T-SOD) assay kit (hydroxylamine method, A001-1), and peroxidase assay kit (A084-3), according to the manufacturer’s respective manuals (Jiancheng Bioengineering Inc., Nanjing, China). The experiments were repeated at least five times. Statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Data were compared using one-way analysis of variance (ANOVA) followed by Duncan’s test.

Harvested P. armandii roots (0.1 g for each seedling) were first ground in liquid nitrogen with a pestle and the resultant paste soaked in 100 mM phosphate buffer (pH 7.5) containing 1 mM EDTA and 1% polyvinylpyrrolidone (w/v) at 4°C. The homogenate was centrifuged at 8000 g at 4°C for 10 min, and then, the supernatant was centrifuged at 12 000 g at 4°C for 20 min. This final supernatant was used to measure the enzyme activity.
SOD activity of root extracts was determined by the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) following the method of Fridovich (2011). Peroxidase (POD) activity was measured using a peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. Using a spectrophotometer (MODEL U‐3900/3900H; Hitachi High‐Technologies Corporation, Tokyo, Japan), one unit of enzyme activity (1 U) was defined as the change in 0.01 absorbance units per minute at 460 nm.

Cyanobacterial cells were pelleted by centrifugation at 12,000 × g and 4 °C for 20 min and washed twice with PBS (50 mM, pH 7.8). After the pellets had been resuspended in PBS, the cells were disrupted using an Ultrasonic Cell Disruption System (NingBo Scientiz Biotechnological Co., Ltd, China) (200 W, ultrasonic time: 2 s; rest time: 8 s, 30 times) in an ice-bath. The supernatant was then collected by centrifugation for 20 min at 12,000 × g and 4 °C, and used to investigate the physiological changes, including antioxidase activity, total protein and malondialdehyde (MDA, a byproduct of lipid peroxidation) content. Total protein content, concentration of MDA and the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were determined using Total Protein Quantitative assay kit (No. A045-2), Malondialdehyde assay kit (No. A003-1), Total Superoxide Dismutase (T-SOD) assay kit (No. A001-1), Catalase assay kit (No. A007-1), and Peroxidase assay kit (No. A084-3) purchased from Nanjing Jiancheng Institute, China, respectively, according to the manufacturer’s instructions.