A064 1 1

The hydrogen peroxide (H₂O₂) and peroxide anion (O ${}_2^{-.}$ ) were detected following the manufactural instructions of a hydrogen assay kit (A064-1-1, Jiancheng, Nanjing, China) and method described by Xu et al. (2015 (link)). To further explore the ROS distribution in plant leaves, the visualization of H₂O₂ and O ${}_2^{-}$ was conducted using 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining according to the methods as described previously (He et al., 2019 (link)).

Cultured SWFs were collected and homogenized with PBS. Subsequently, centrifugation was performed at 800 rpm at 4 °C for 10 min, and the supernatant was aspirated to obtain a 10% tissue homogenate. The tissue homogenate was used to measure oxidation parameters and determine total protein concentration. The activity of CAT (A00171-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and T-SOD (A001-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), full antioxidant capacity (T-AOC) (A015-3-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the concentrations of MDA (A003-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), GSH (A006-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and hydrogen peroxide (H₂O₂) (A064-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and total protein concentration were determined using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Additionally, the cultured SWFs were homogenized in boiling water, heated in boiling water for 10 min, and then subjected to centrifugation. The ATP level was determined by an ATP assay kit (A095-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the instructions provided by the manufacturer.

The physiological indicators: PAL, H₂O₂, MDA, Pro, and POD were measured using detection assay kits (Cat. No. A137-1-1, A064-1-1, A003-3-1, A107-1-1 and A084-3-1 respectively; Nanjing Jiancheng Bioengineering Institute, China). All the tests were performed according to the manufacturer’s instructions (http://www.njjcbio.com/, accessed on 1 December 2022). A lignin test kit was obtained from COMIN company (Cat. No. MZS-1-G; Suzhou, China). The leaves were used for physiological detection. Expanded leaves were collected from L96 and NT plants after salt stress (0 d, 2 d and 5 d). Each sample was repeated 4 times. At the same time, root were collected, washed, and frozen in −80 °C for the following RNA extraction and plant hormone detection via HPLC (Agilent 1260, Santa Clara, CA, USA).