*2.2. Determination of Photosynthetic Pigment Contents*

About 0.1 g fresh leaf samples were homogenized with 10 mL (4.5:4.5:1) mixed solution of ethanol, acetone, and distilled water until the green-colored leaf sample turned white. After that, absorbance readings were recorded at 645, 663, and 470 nm, and concentration (mg/g FW) of chlorophyll *a* (Chl *a*), Chlorophyll *b* (Chl *b*), total chlorophyll (Chl t), and carotenoid were calculated by the formula [20].

Chlorophyll *a* (mg/g leaf fresh weight) = [12.7 (OD663) − 2.69 (OD645)] × V/1000 × W (1)

Chlorophyll *b* (mg/g leaf fresh weight) = [22.9(OD645) − 4.68 (OD663)] × V/1000 × W (2)

Total chlorophyll (mg/g leaf fresh weight) = [20.2 (OD645) + 8.02 (OD663)] × V/1000 × W (3)

Carotenoid (mg/g leaf fresh weight) = [OD470 + (0.114 ∗ OD663) - (0.638 ∗ OD645)] × V/1000 × W (4)

According to Sairam et al., the chlorophyll stability index (CSI) was developed [21]. It is calculated as follows: CSI = (Total Chl under stress/Total Chl under control) × 100.

*2.3. Determination of Osmolyte Contents*

The proline concentrations were determined using a slightly modified method reported by Bates, 1973 [22]. The proline content of fresh leaf samples (0.1 g) was assayed

using aqueous 3% sulfosalicylic acid (5 mL) in a water bath for 10 min. Following this, the mixture was allowed to cool to ambient temperature, and then the resulting extract was filtered using filter paper. The supernatant from the second step was then combined with ninhydrin and acetic acid to make a 2.0 mL solution. The combination was then maintained in a boiling water bath for 30 min, and the reactions were terminated in an ice bath. After adding 5 mL of toluene, the mixture was left in the dark for 5 h. The absorbance of colored toluene at 520 nm was measured using the UV/Vis spectrophotometer (UV 2700, Shimadzu, Japan), with toluene serving as a blank. The amount of proline was tested using a standard curve constructed with L-proline common solution.

The amount of total soluble sugar (TSS) was determined using the anthrone method proposed by Yemn and Willis [23]. About 0.1 g fresh samples were placed in 10 mL cuvette, and after adding 10 mL distilled water, samples were heated at 100 ◦C for 1 h and then filtered into 25 mL volumetric flasks. The volumetric flask was filled up to mark by distilled water. Following that, 0.5 mL extracts, 0.5 mL mixed reagents (1 g anthrone + 50 mL ethyl acetate), 5 mL H2SO4 (98%), and 1.5 mL distilled water were added. After heating the mixture to 100 ◦C for a minute, the 630 nm absorbance was measured. Sucrose solution was used as a standard sample. The concentration of soluble sugar was measured using glucose as a standard solution.
