*2.5. Quantification of Proline, Total Soluble Sugar, and Protein Contents*

The Bates method, with minor modifications, was used for proline determinations [51]. The fresh leaves (0.5 g) were homogenized in 5 mL of sulfosalicylic acid (3%) and incubated at 100 ◦C for 10 min. The supernatant (2 mL) was mixed with 2 mL of ninhydrin reagent and 2 mL of glacial acetic acid. The mixture was then allowed to cool to room temperature and centrifuged for 10 min at 3000 rpm. The mixture was incubated at 100 ◦C for 1 h before being cooled in an ice bath for 15 min. As a reaction reagent, 4 mL of toluene was added to the previous mixture, and the absorbance at 520 nm was measured. The proline content was determined using the standard curve and expressed as g g−<sup>1</sup> FW.

The total soluble sugars were determined using the phenol–sulfuric acid method [52]. The fresh samples (0.5 g) were homogenized in 10.0 mL of 80% ethanol and centrifuged for 20 min at 2000 rpm, then the supernatant was collected. The supernatant (0.1 mL) was mixed with 1.0 mL phenol (5%) and 5.0 mL of sulfuric acid (98%). The mixture was then allowed to stand in a 30 ◦C water bath for 20 min. Finally, the absorbance was measured at 490 nm. The amount of available soluble sugar was calculated using a glucose calibration curve (10–100 mg/mL) and expressed as mg/g FW.

The soluble protein was measured using the method used by Guy [53]. The fresh samples (0.5 g) were homogenized in a solution of 50 mM Tris-HCl (pH 7.5), 2 mM EDTA, and 0.04% (*v/v*) β-mercaptoethanol and centrifuged at 10,000× *g* for 15 min. After mixing 1 mL of supernatant with 1 mL of Coomassie Brilliant Blue, the absorbance was read at 595 nm.

#### *2.6. ROS Determination—Hydrogen Peroxide (H2O2) and Superoxide Anion (O2* •−*)*

The H2O2 content was determined using the method developed by Okuda et al. [54]. The fresh leaf samples (200 mg) were milled in 2 mL of 200 mM perchloric acid in an ice bath and then centrifuged for 10 min at 12,000× *g*. After centrifugation, 4 M KOH was used to neutralize the perchloric acid in the supernatant. The insoluble potassium perchlorate was then removed by centrifugation at 500× *g* for 3 min. The supernatants (1 mL) were combined with 400 μL of 3-(dimethylamino) benzoic acid (12.5 mM) in phosphate buffer (0.375 M, pH 6.5), 80 μL of 3-methyl-2-benzothiazoline hydrazone, and 20 μL of peroxidase (0.25 unit). The reaction was started by adding peroxidase at 25 ◦C, then the absorbance was measured at 590 nm.

The superoxide radical (O2 •−) content was determined using the techniques used by Bu et al. [55] and Lang et al. [56] with minor modifications. The fresh leaf samples (0.2 g) were treated for 1 h with 1 mL of hydroxylamine hydrochloride. The mixture was then incubated at 25 ◦C for 20 min with 1 mL each of α-naphthylamine and 2-aminobenzenesulfonic acid. The absorbance of the solution at 530 nm was measured. The O2 •− content was calculated using a NaNO2 calibration curve (10–100 mg/mL).
