Determination of Leaf Proline Content

Proline concentration was determined according the methods of Ortiz et al. [26] with little modifications. Briefly, 1.25 g of ninhydrin was dissolved in 20 ml of 6 M phosphoric acid and 30 ml of glacial acetic acid by heating on a hot-plate with agitation. The solution was allowed to cool and kept at 4 °C and the solution became stable after 24 h. Approximately 500 mg of fresh soybean leaf sample was ground in 10 ml of 3% aqueous sulfo-salicyclic acid and centrifuged at 10000×g for 10 min. Two ml (2 ml) of the supernatant was reacted with 2 ml of glacial acetic acid and 2 ml of acid-ninhydrin solution in 45 ml falcon tubes at 100 °C in a water bath for 60 min and the reaction was stopped in an ice box. Four ml (4 ml) of toluene was added to extract the mixture and agitated vigorously for 15–20 s in a shaker incubator at 250 rpm. The mixture was kept in the dark for 30 min and the ‘chromophore containing toluene was aspirated from the aqueous phase and the absorbance was read at 520 nm using toluene for a blank’. The concentration of proline was estimated from a standard curve ‘established with a reference proline solution’. Briefly, 1 mg/ml stock solution of proline was prepared by weighing 10 mg of proline (DL-Proline, China) in 10 ml of sterile water. 0, 50, 100, 150, 200, 250, 300 µl of the stock solution was pipetted into seven tubes containing 300, 250, 200, 150, 100, 50 and 0 µl of sterile water, respectively. The mixtures were then reacted with 2 ml of glacial acetic acid and 2 ml of acid-ninhydrin solution in 45 ml falcon tubes at 100 °C in a water bath for 60 min and the reaction was stopped in an ice box. The mixture was vigorously agitated using a vortex (Vortex Genie, U.S.A) after adding 4 ml of toluene. The mixture was kept in the dark for 30 min and the absorbance of the proline-containing upper layer ‘was read at 520 nm using toluene for a blank’ and proline standard curve was plotted from the absorbance values.