2.3.4. Chlorophyll Stability Index (CSI)

The CSI was determined by heating fresh leaf samples (0.25 g) in 20 mL of distilled water at 56 ◦C in a water bath for 30 min [22]. Normal and heated leaf samples were homogenized in 80% acetone, then the total chlorophylls content was computed as above; the CSI was determined by using the following formula:

CSI (%) = (Total chlorophyll without heating − Total chlorophyll after heating) × 100

2.3.5. Electrolyte Leakage (EL) and Membrane Stability Index (MSI)

Electrolyte leakage was determined to evaluate membrane permeability, following the procedure of Guo et al. [23] with some modifications. Ten leaf discs of randomly selected plants per replicate were taken from the youngest fully expanded leaf. Then, the discs were placed in a 50 mL falcon tube and washed three times with distilled water to remove surface contamination. Next, the discs were placed in a 50 mL falcon tube containing 20 mL of deionized water (Aquinity2 P10, MembraPure GmbH, Hennigsdorf, Germany) and incubated, at room temperature, for 24 h. The bathing solution's (EC1) electrical conductivity (EC) was read after incubation using an electrical conductivity meter (BALRAMA, Digital EC Meter, New Delhi, India). Afterward, the same samples were placed into a boiling water bath for 20 min, and a second reading (EC2) was carried out

after cooling the solution to room temperature. The EL was expressed as a percent value using the following formula:

$$\text{EL (\%)} = \text{(EC1/EC2)} \times 100$$

The membrane stability index (MSI) was computed based on electrolyte leakage data and expressed as a percent value using the following formula:

$$\text{MSI} \text{ (\%)} = \text{[1 - (EC1/EC2)]} \times 100$$

#### 2.3.6. Total Sugar Content

The total sugar content was determined utilizing the phenol–sulfuric acid method, according to Dubois et al. [24]. To this aim, 0.5 g of fresh leaf was homogenized in 20 mL of 80% ethanol (*v*/*v*). One mL ethanolic solution was mixed with 1 mL of 5% phenol dissolved in water (*v*/*v*), followed by the addition of 5 mL of concentrated sulfuric acid. The absorbance was read at 490 nm by a spectrophotometer (JENWAY 6300, Staffordshire, UK). A standard curve was generated, employing a pure glucose solution, and the total sugar content was expressed in mg glucose equivalent g−<sup>1</sup> of fresh weight.
