A003 3

Each index was determined by an assay kit as follows. The malondialdehyde (MDA) contents were measured with an MDA assay kit (A003-3, Jiancheng, China). A test method based on thiobarbituric acid was used. The SOD enzymatic activity was measured by an assay kit (A001, Jiancheng, China). The POD enzymatic activity was measured by an assay kit (A084-3, Jiancheng, China). The CAT enzymatic activity was measured by an assay kit (A007, Jiancheng, China). Each assay was replicated at least three times per sample.

To detect changes in physiological indices under normal and drought stress conditions, leaves were collected from the controls and TaCIPK2-overexpressing transgenic lines and treated with 0.1 M phosphate-buffered saline (pH 7.4) on ice. The crude extract was centrifuged at 8,000 g for 10 min at 4°C and the supernatants were used to measure the physiological index changes. The malondialdehyde (MDA) and H₂O₂ contents and the antioxidant enzyme activities [catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)] were measured using the corresponding detection kits (A003-3, A064, A007-1, A001, and A084-3; Jiancheng, China). Ion leakage (IL) was measured under normal and drought stress conditions. Leaves of all lines were harvested and incubated in 15 ml distilled water overnight at 23°C before measuring the initial conductivity (C1). After boiling the samples for 30 min, the final conductivity (C2) was determined. The following formula was used to calculate IL (%): C1/C2×100.

Levels of total protein (TP), hydrogen peroxide (H₂O₂), anti-superoxide anions, and malondialdehyde (MDA), and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR) were measured using A045-2, A064-1, A052-1, A003-3, A001-1, A084-3, A007-1, and A062-1 assay kits (Nanjing Jiancheng Bioengineering Institute). The total proline (Pro) content was determined as described by Bates et al. (1973) (link).