A107 1 1

Approximately 100 mg of fresh leaves and roots was homogenized for determination of proline content following the manufacturer’s instructions (A107-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the absorbance was measured at 520 nm. For the analysis of soluble sugars, approximately 0.1 g of fresh samples was homogenized in 1 ml ddH₂O with a glass homogenizer. The tubes were boiled at 95°C for 10 min and cooled with tap water. After cooling, the homogenate was centrifuged at 4,000 × g for 10 min. Thereafter, the supernatant was diluted with ddH₂O at 1:9. The diluted extracts were used for determination of soluble sugar content using a commercially available test kit (A145-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Finally, the absorbance was measured at 620 nm and the soluble sugar concentration expressed and the results expressed in the fresh weight (FW) basis (Dai et al., 2018 (link); Kumar et al., 2021 (link)).

The water loss rate was estimated according to the methods described by Wei et al. [46 (link)]. Leaves of the seedlings in the same position were detached and their fresh weight was measured. Then, the detached leaves were exposed in the air to induce dehydration. Desiccated weight was assessed at 1 h, 2 h and 3 h, respectively. Water loss rate (%) = (fresh weight – desiccated weight)/FW × 100. Proline content was determined following the proline assay kit (A107-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), with homogenized 0.1 mg of homogenized fresh leaves. Absorbance values were measured at 520 nm.

Half of the leaf samples were frozen in liquid nitrogen and stored at −80 °C. Fresh leaves were weighed; proline and superoxide dismutase (SOD) contents in the remaining samples were evaluated using respective assay kits (Cat. Nos. A107-1-1 and A101-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as described by the manufacturer.