Proline standard

Proline content was determined using 1% ninhydrin as we reported earlier [10 (link)]. For extraction, we used 30 mg of the freeze-dried tissue and 70% ethanol. Then, 100 μL of the obtained extract were mixed with 1 mL of reaction mixture containing 1% ninhydrin, 60% acetic acid, and 20% ethanol. Mixtures were heated at 95 °C for 20 min, cooled, and proline levels were measured at 520 nm using a UV–VIS spectrophotometer (Shimadzu BioSpec-1601, Kyoto, Japan). For the calibration curve proline standard (Sigma–Aldrich, Saint Louis, MO, USA) was used (0–1.6 mM, y = 1.4331x, R² = 0.999) and results are expressed as μmol g⁻¹ dw.

0.05 g ground, frozen leaf tissue was extracted in 5 ml of 3 % sulfosalicylic acid (Panreac, Barcelona, Spain) by sonication for 30 min. After centrifugation at 4000 g for 20 min at 4 °C, extracts were assayed for proline as described by Bates and others [31 (link)] with slight modifications. Briefly, 1 ml of the supernatant was mixed with 1 ml of glacial acetic acid and ninhydrin reagent (Panreac) in a 1:1 (v:v) ratio. The reaction mixture was incubated in a water bath at 100 °C for 1 h. After centrifuging at 2000 g for 5 min at 4 °C, absorbance was read at 520 nm. A standard curve was performed with standard proline (Sigma-Aldrich, St. Louis, MO, USA).

Cai et al. [24 (link)] procedure was employed for the analysis of phenolics in soybean, sunflower, and fungal filtrate. Different concentrations (100, 200, 300, 500, 600, 700, and 900 mg/ml) of gallic acid (Sigma Aldrich) were used to construct a standard curve. For the determination of proline, the method of Bates et al. [25 (link)] was followed, but after some modifications. A standard curve was plotted against different concentrations (2, 4, 6, 8, and 10 μg/ml) of standard proline (Sigma Aldrich) and the OD was taken at 520 nm. For the analysis of total flavonoids, the method of Mervat et al. [26 ] was used. A standard curve was constructed by using different concentrations of pure quercetin (15, 30, 60, 120, 240, and 480 μg/ml, Sigma Aldrich) and the OD were taken at 415 nm.

Fifty mg of ground material, frozen leaves, or roots was extracted in 5 ml of 3% sulfo salicylic acid (Panreac) by sonication for 30 min. After centrifugation at 4,000 rpm for 20 min at 4°C, extracts were processed as described in Bates et al. (1973) with slight modifications. Briefly, 1 ml of the supernatant was mixed with 1 ml of glacial acetic acid and ninhydrin reagent in a 1:1 (v:v) ratio. The reaction mixture was incubated in a water bath at 100°C for 1 hr. After centrifugation at 2,000 rpm for 5 min at 4°C, absorbance was read at 520 nm. A standard curve was performed with standard proline (Sigma‐Aldrich).