*2.10. Determination of Proline and Soluble Sugars*

Proline was calculated by using an assay kit (A107-1-1). Fresh leaf samples were homogenized with buffer available in the kit and tested at 520 nm following the company's protocol. Approximately 50 mg of fresh leaves were homogenized in 0.45 mL ddH2O for the analysis of soluble sugars. The homogenate was boiled at 95 ◦C for 15 min and then centrifuged at 7500× *g* for 15 min. After that, the supernatant was collected and diluted with ddH2O at 1:9. Using a test kit (A145-1-1), the soluble sugar content of the diluted extracts was determined at 620 nm [17,32].

#### *2.11. Determination of Total Polyphenols and Flavonoid Content*

A fresh leaf sample of 1 g was homogenized with 60% ethanol. After that, 1.25 mL of 10% Folin–Ciocalteu reagent was added to 250 μL of extract and 1 mL of sodium carbonate (0.75 g/mL). After incubating for 15 min at 45 ◦C, the mixture was allowed to remain at room temperature for 30 min. As a final step, the absorbance was recorded at 765 nm, and the results were presented as Gallic acid equivalents per gram (GAE/g) to quantify total polyphenols [16,34].

Approximately 0.25 mL of NaNO2 solution (0.5 g/mL) and 2 mL ddH2O were mixed with 0.5 mL of extract to measure flavonoids. After being retained at 25–28 ◦C for 5 min, 150 μL of aluminium chloride (1 g/mL), 1 mL of NaOH (1 M), and 1.2 mL of ddH2O were added simultaneously. As a final step, its absorbance was measured at 510 nm with Catechin (CAE) used as a standard, and its results were presented as CAE/g [16,34].

#### *2.12. Scanning Electron Microscopy (SEM)*

To observe the stomatal morphology, we used a published protocol [30]. To remove any debris, leaves were acetylated in 80% ethanol for two to three min. The tiny sections of leaf were prepared using s-cutting, and after that platinum was used to fix the abaxial and adaxial surfaces and sputtered using Leica Mikrosystem GmbH (ACE600) for 25 min, and finally examined under a SEM (Thermo Scientific, Verios G4 UC, Waltham, MA, USA).

#### *2.13. Statistical Analysis*

Three individual replications were used to obtain phenotypic, physiological, and biochemical indices. Significant differences (*p* ≤ 0.05) between means were determined using SPSS 25.0 software, and Duncan tests were applied for the means comparison, while ± represents a standard error (S.E). Figures were plotted with GraphPad Prism 7. The "ggplot2" package in R (version 3.3.4, https://CRAN.R-project.org/package=ggplot2 (accessed on 24 August 2022)) was used for principal component analysis (PCA) and Pearson correlation analysis.
