Estimation of Proline Content

Proline content was estimated by using the method described by Ábrahám et al. (2010). Acidic ninhydrin reagent was used to estimate the total proline content of the plants. The reagent was prepared by dissolving 2.5 g of ninhydrin in 100 ml of the solution containing glacial acetic acid: distilled water: 85% orthophosphoric acid at a ratio of 6:3:1. One hundred milligrams of leaf samples were homogenized in 1 ml of 3% (w/v) aqueous sulfosalicylic acid solution and centrifuged at 12,000 × g for 15 min at 4°C. Following incubation, 1 ml of supernatant was collected and mixed with equal volume of glacial acetic acid and ninhydrin reagent and kept in a boiling water bath for 1 h. The reaction was stopped by incubating the reaction in a water bath at room temperature (21°C) for 5 min. The absorbance was measured immediately at 546 nm using Jasco V730 IRM spectrophotometer. Total proline content was estimated by using a proline standard curve as a reference and expressed in mg/g fresh tissue.