Determination of antioxidant capacity and the content of proline

Proline content was evaluated by the acid ninhydrin method [60]. The O²⁻ content was quantified by the hydroxylamine oxidation method [61]. 3,3′-diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) staining were performed to detect H₂O₂ and O²⁻ levels in tomato leaves. Specifically, tomato leaves were first immersed in a 0.5 mg/mL DAB staining solution (pH = 3.8) or 0.5 mg/ml NBT solution, and then placed in a dark, high-humidity plastic box for about 24 h. Next, the staining solutions were decanted and the leaf samples were incubated with an ethanol/lactic acid/glycerol solution (3:1:1, v:v:v) in a boiling water bath for 5 min [62]. The SOD activity was determined by the photochemical reduction method using nitroblue tetrazolium [63]. The CAT activity was determined by the ultraviolet absorption method and change in the optical density at 240 nm (OD240) of 0.1 within 1 min was defined as one unit of enzyme activity [64]. POD activity was measured by the guaiacol method at OD470, and one unit enzyme activity was defined as 0.1 decease in OD470 per minute [65]. SOD, POD, and CAT activities are shown as U·min^− 1·g^− 1 (FW).

The degree of damage to the cells was determined by measuring the relative conductivity of tomato leaves. Ten leaf disks (0.8 cm in diameter) from the WT or transgenic lines were submerged in 20 mL distilled water, vacuumed for 30 min, shaken for 3 h at room temperature, and measured for the initial electric conductivity (S1). Subsequently, these leaf samples were boiled for 30 min and cooled down to room temperature to measure the final electric conductivity (S2). The electric conductivity of pure distilled water was used as the blank (S0). The relative electronic conductance (REC) was calculated as REC=S1−S0S2−S0∗100. MDA content was determined by the thiobarbituric acid method [66]. Tomato leaves were stained with the Evans blue staining solution to assess cell viability [67].