Proline assay kit

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 21-day-old plant leaves that were pre-treated under normal and drought stress conditions were collected for proline, MDA, and H₂O₂ content analysis. Drought stress treatment experiments were repeated three times. The proline, malondialdehyde (MDA), and H₂O₂ contents were determined by the proline assay kit, MDA assay kit, and H₂O₂ assay kit (Jiancheng, China), according to the instructions, respectively.

Leaf tissue homogenate was prepared using the same method for CAT content determination, and the supernatant after centrifugation was detected using a proline assay kit (colorimetric) produced by Nanjing Jiancheng Bioengineering Institute.