A007 1

The level of intracellular ROS in spermatozoa was measured using the fluorescent probe redox-sensitive-fluoroprobe-2′,7′-dichlorofluorescein-diacetate (DCFDA; D6883, Sigma-Aldrich, St. Louis, USA). Briefly, 1 × 10⁶ spermatozoa were washed in PBS and were incubated with 100 mM DCFDA at 37 °C for 30 min. The fluorescence of DCF was measured on a flow cytometer at a wavelength of 485/535 nm.
Mitochondrial protein was isolated from liquid-stored spermatozoa using a Mitochondria Isolation Kit (89801, Thermo Scientific, Rockford, USA) according to the manufacturer’s recommendations. The concentration of mitochondrial protein was determined using a BCA protein assay kits (23225, Thermo Fisher Scientific, Rockford, USA). The activities of mitochondrial SOD, CAT, and GSH-Px were measured using commercial assay kits (A001-3, A007-1, A005; Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. The SOD, CAT, and GSH-Px activities were determined using a microplate reader (Multiskan Spectrum, Thermo) at 450 nm, 405 nm, and 412 nm, respectively.

Plant extracts were prepared from tomato leaves after 45 days and antioxidant enzymes were assayed as described by Ahmad et al. (2015) (link). Briefly, fresh tomato leaves were ground using liquid nitrogen and the ground leaf samples were stored at –80 ^◦C. The ground leaf samples (approximately ∼1 g) were homogenized on ice using 10 mL of 50 mM phosphate buffer (pH 7.8) and then incubated for 10 min at 4 ^◦C. Subsequently, the homogenate was filtered using Advantech Qualitative Filter Papers (110 mm) and centrifuged at 4,000 × g for 15 min at 4 ^◦C. The supernatant was used for the determination of enzyme activities. The activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), and peroxidase (POD, EC 1.11.1.7), were measured using assay kits (kit Numbers. A001-1, A007-1, A084-3, respectively; Nanjing Jiancheng Bioengineering Institute, China), following the manufacturer’s instructions (http://elder.njjcbio.com/index_en.php) (Liang et al., 2017 (link)). This experiment was conducted in triplicate.

The catalase (CAT) activity, peroxidase (POD) activity, and superoxide dismutase (SOD) activity were measured using commercial CAT, POD, and SOD assay kits (A007-1, A084-3, A001-1, Jiancheng Bioengineering Institute, Nanjing, China), respectively.

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.

Longissimus thoracic samples (1 g) were grounded with a grinder and homogenized with ice‐cold saline (0.85%, 4.5 ml) and centrifuged (4,000 × g, 10 min, 4°C). The supernatants were used to assess catalase (CAT), superoxide dismutase (SOD), and glutathione (GPx) activities.
Catalase activity was determined using a hydrogen peroxidase assay kit (A007‐1; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instruction. Absorbance readings were performed at 550 nm (Persee TU‐1810; Persee Co. Ltd.). One unit of CAT activity was defined as 1 mg tissue protein decomposed with 1 μmol H₂O₂ per second at 37°C. SOD activity was assessed by measuring the content of hydroxylamine using a total superoxide dismutase assay kit according to the manufacturer's instructions (A001‐1; Nanjing Jiancheng Bioengineering Institute). Absorbance readings were performed at 550 nm. One unit of SOD activity was defined as the amount of enzyme in 1 ml of the reaction solution at 50% SOD inhibition at 37°C. GPx activity was measured colorimetrically using a glutathione peroxidase assay kit according to the manufacturer's instructions (A005; Nanjing Jiancheng Bioengineering Institute). Absorbance readings were performed at 412 nm. One unit of GPx activity was defined as the amount of enzyme capable of decomposing 1 μM glutathione per minute at 37°C.