*2.5. Estimation of the Contents of Proline, Glycine Betaine (GB), Trehalose and Soluble Sugars*

Proline content was determined by adopting the ninhydrin method [44]. Briefly, fresh leaf tissues (300 mg) were homogenized in 3 mL of 3% sulphosalicylic acid, and the homogenate filtrate was reacted with 1 mL each of acid ninhydrin and glacial acetic acid for 1 h in a test tube placed in a water bath at 100 ◦C. The mixture was extracted with toluene, and the absorbance was measured on a spectrophotometer at 520 nm using L-proline as a standard.

Glycine betaine was determined by estimating the betaine-periodite complex [45] in a sample from 500 mg dried leaf powder mechanically shaken for 24 h at 25 ◦C with 20 mL of deionized water. After filtering the samples, the filtrates were diluted (1:1) with2NH2SO4. A portion (0.5 mL) was taken and centrifuged before cooling in ice water for one hour. After adding 0.2 mL of cold KI-I2 reagent, the reactants were gently stirred. The tubes were kept at 4 ◦C for 16 h and were centrifuged at 10,000× *g* for 15 min at 0 ◦C. After carefully aspirating the supernatant, the absorbance at 365 nm was measured after two hours. In 2 N H2SO4, reference standards for GB (50–200 μg mL−1) were created. The trehalose content was determined following the protocol given by Trevelyan and Harrison [46]. Dried leaf powder (500 mg) was extracted in 80% ethanol, followed by centrifugation at 5000× *g* for 15 min at 4 ◦C. A 100 μL sample of the supernatant was combined with 4 mL of anthrone reagent and 2.0 mL of trichloroacetic acid (TCA). The absorbance was read at 620 nm. A standard curve was plotted using glucose.

The method developed by Xu et al. [47] was used to measure the amount of soluble sugars. A total of 100 mg of the dried sample powder was extracted with 10 mL of 80% ethanol and incubated at 80–85 ◦C for 30 min. Three additional extractions were performed after centrifuging the extract and transferring the supernatant to a 100 mL volumetric flask. At 80–85 ◦C, alcohol extract was evaporated over a water bath. Following the addition of 100 mL distilled water, all three supernatants were poured into the flask. An aliquot of the extract was used to measure the amount of soluble sugars using the anthrone reagent, and the reaction mixture's absorbance was observed at 630 nm using a spectrophotometer.
