Pod enzyme activity assay kit

ROS content and SOD, POD activities in tomato fruits of transient transformation with pK7LIC4.0 and pK7LIC4.0-BvEP was determined. The tomato fruits were ground into powder with liquid nitrogen, 0.1g powder was weighed for the above index determination. Three biological replicates were determined for each sample, 5 tomato fruits were used as one biological replicate.
The ROS detection method was described previously (Hu et al., 2022 (link)). The ROS content in the fruits was determined by adding 0.1 g of tomato fruit powder to phosphate buffered saline (pH 7.4) followed by a commercial plant ROS enzyme-linked immunosorbent assay (ELISA) kit (Chundubio).
Methods for SOD and POD activities assay were also described previously (He et al., 2019 (link)). 0.1g of tomato fruit powder was added to 1 mL of the extraction solution, centrifuged at 8000g for 10min at 4°C, and the supernatant was taken on ice for determination. SOD activity was measured with the SOD enzyme activity assay kit (Solarbio) by UV spectrophotometer with absorbance at the maximum absorption wavelength of 470 nm. POD activity was measured with the POD enzyme activity assay kit (Solarbio) by UV spectrophotometer with absorbance at the maximum absorption wavelength of 560 nm.

A total of 0.1 g fresh samples were grinded into powder in liquid nitrogen and dissolved into 1 mL extraction buffer (BC1190, Solarbio, China). The mixture was centrifuged at 4 °C at 12,000 r.p.m for 20 min. Then the supernatant was sucked out on ice and the GPX enzyme activity was detected by measuring the decrease of NADPH absorbance at 412 nm according to the instructions of GPX assay Kit (BC1190, Solarbio, China). Similarly, the POD enzyme activity was determined according to the instructions of the POD enzyme activity assay Kit (BC0090, Solarbio, China). POD catalyzes H₂O₂ oxidation of specific substrates and has characteristic light absorption at 470 nm. The experiments were repeated three biological replicates.