2.5. Measurement of Leaves Biochemical Traits

Fully expanded leaves of carob seedlings grown under WW, DS, and REC conditions were harvested at the end of the light period, flash-frozen, and ground to fine powder in liquid N. The determination of total soluble sugars (TSS) was carried out on powdered frozen leaves (0.1 g) mixed with 4 mL ethanol (80% v:v) [46]. The obtained supernatant (0.2 mL) was added to 0.25 mL phenol and mixed with 1.25 mL sulfuric acid. The OD of the mixture was measured after 15 min at 485 nm.

Total soluble protein content and antioxidant enzyme activities were assessed by homogenizing 0.25 g of frozen leaf powder with 1 M phosphate buffer (pH 7) containing 5% polyvinylpolypyrrolidone. The resulting mixture was then centrifuged at 18,000× g for 15 min at 4 °C. The obtained supernatant was used to determine protein content and antioxidant enzyme activity [47]. Total soluble protein was determined by the Bradford method [48], using bovine serum albumin (BSA) as a standard.

The activity of superoxide dismutase (SOD) was evaluated according to the method described by Beyer and Fridovich [49]. A unit of SOD activity has been defined as the ability to inhibit 50% of the photochemical reduction of p-nitroblue-tetrazolium (NBT) at 25 °C. SOD activity was expressed in unit min⁻¹ mg protein⁻¹. Peroxidase (POX) activity was measured using the method described by Tejera García et al. [47]. The reaction mixture consisted of K₂HPO₄/KH₂PO₄ buffer (100 mM), guaiacol (40 mM), H₂O₂ (10 mM), and enzyme extract (0.1 mL). Measured activity was expressed in unit mg protein⁻¹. Measurement of polyphenoloxidase (PPO) activity was assessed by monitoring catechol oxidation at 410 nm, as described by Gauillard et al. [50]. The used solution consisted of K₂HPO₄/KH₂PO₄ buffer (100 mM, pH 6), catechol (50 mM), and enzyme extract (0.1 mL). PPO activity was expressed in mg protein⁻¹ units.

Malondialdehyde (MDA) concentration was determined by the method of Savicka and Škute [51]. Lipid peroxides were extracted from 0.25 g of frozen powder subsamples and mixed with 10 mL of 0.1% (w/v) trichloroacetic acid (TCA). After centrifuging the extract at 18,000× g for 20 min, 1 mL supernatant mixture was added to 2.5 mL thiobarbituric acid (TBA), resulting in chromogen formation. The supernatant was then incubated at 95 °C for 30 min and the reaction stopped by placing the tubes in an ice bath. The OD was measured at 450, 532, and 600 nm. MDA content was calculated as follows:

The amount of hydrogen peroxide (H₂O₂) was measured using the method described by Velikova et al. [52]. Frozen crushed leaves (0.25 g) were homogenized in 5 mL TCA 10% (w/v). The mixture was then centrifuged at 15,000× g for 15 min. The supernatant (0.5 mL) was mixed with 0.5 mL of potassium phosphate buffer (10 mM, pH 7) with the addition of 1 mL potassium iodate (1 M). After 1 h incubation in the dark, the OD was read at 390 nm and H₂O₂ concentrations were determined using a standard H₂O₂ curve.