Bc0200

Evans blue staining was used to identify dead cells. Deeper staining of more sites indicates less cell activity in the root or leaves. The staining approach was modified based on the methods proposed by Baker (1994) [71 ] and Chalivendra (2017) [72 (link)]. Three seedlings were harvested from both the control and treated-groups at 0 and 4 d. They were washed with tap water, distilled water, and deionized water. The washed-root tips and leaves of wheat seedings were placed in 0.25% Evans blue staining liquid (Solarbio Life Sciences, Cat#: G1810, China) for 8 min and 4 h, respectively in the dark. Then the stained roots and leaves of wheat seedlings were washed and photographed under a stereoscopic microscope (Nikon C-fled2, Nikon, Tokyo, Japan).
The in situ accumulation of H₂O₂ and O₂•^— in the roots and leaves of wheat seedlings was detected by histochemical staining with 3,3-diaminobenzidine (DAB, Sigma, USA) and nitro blue tetrazolium (NBT, Sigma, USA), respectively [29 (link)]. The contents of H₂O₂ and O₂•^— were determined using detection kits manufactured by Solarbio Life Sciences (Cat#: BC3595 and BC1290, China).
The activities of antioxidant enzymes including SOD, POD, and CAT were determined using commercial detection kits according to the manufacturer’s instructions (Solarbio Lifesciences, Cat#: BC0170, BC0090 and BC0200, China).

Fresh leaf samples (0.5 g) were ground to powder using liquid nitrogen and homogenized with 5 mL phosphate buffer (10 mM, pH 7.4). The homogenate was then centrifuged at 8,000 ×g for 15 min at 4 °C, and the supernatant was used to measure the activity of various antioxidant enzymes. The catalase (CAT, BC0200), superoxide dismutase (SOD, BC0170), ascorbate peroxidase (APX, BC0220), and glutathione reductase (GR, BC1165) activities were measured using ELISA kits from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China) following the manufacturer’s instructions.

The antioxidant enzymes SOD and CAT activities were measured using the detection kits (BC0170 and BC0200, respectively, Solarbio, Beijing, China). Briefly, Arabidopsis leaves were ground and homogenized in specific extraction buffers. After centrifuge, the resulting supernatants were used for enzyme activity assays. Total SOD and CAT activities were assayed by detecting changes in absorbance at 560 and 240 nm, respectively, following the kits’ protocols. The enzyme activities were expressed as U/g fresh weight, as described by us [39 (link)].