2.4. Measurement of Lipid Peroxidation Levels

Lipid peroxidation levels are reflected by MDA concentrations. Lipid peroxidation can produce MDA, which serves as a sensitive diagnostic index for plants under oxidative injury [25]. Two milliliters of 5% trichloroacetic acid (w/v) were added to fresh leaf samples, which were then ground, and the resulting homogenate was subsequently centrifuged at 300 g for 10 min. Afterward, 2 ml of 0.67% thiobarbituric acid (w/v) was added to 2 ml of the resultant supernatant, after which the mixture was heated at 100 °C for 30 min and then centrifuged at 300 g for 10 min. The absorbance of the supernatant was subsequently measured at 450 nm, 532 nm, and 600 nm. The MDA content was then calculated based on the following formula:

where A₄₅₀, A₅₃₂ and A₆₀₀ represent the absorbance value at a wavelength of 450, 532, and 600 nm, respectively [26].

The lipid peroxidation levels were expressed as micromoles of MDA per gram of fw.