Proline determination

Proline was measured using spectrophotometry as described in the literature (Carillo and Gibon 2011, Bergman and Loxley 1970). Briefly, proline standard solution (0.01-0.04 mM) was prepared in ethanol : water (70 : 30 v/v). The reaction mixture was prepared by mixing ninhydrin 1% (w/v) in acetic acid 60% (v/v) and ethanol 20% (v/v). The 400 μl of standard proline solution was mixed with 100 μl ninhydrin reaction mixture and heated to 95°C for 20 min. After cooling and spinning for 1 min at 2500 rpm, the absorbance was taken at 520 nm. A standard curve was obtained and the generated linear equation (R² = 0.9993) was used to calculate proline in plant extracts.

Plant extract was prepared from 0.5 g fresh frozen plant tissue, and thawed and homogenized with a polytron homogenizer (PT10/35 GT, Kinematica, Zaragoza, Spain) in 5 mL ethanol : water (70 : 30 v/v) and centrifuged (PT10/35 GT, Kinematica, Zaragoza, Spain) at 10,000g for 15 min. The supernatant was taken and the pellet was twice re-extracted with 5 mL of the same solvent mixture. The combined supernatant was diluted to 100 mL with the same solvent mixture. The ninhydrin reaction mixture, prepared as above, (100 μl) was mixed with the plant extract (400 μl) and absorbance (520 nm) was used in the linear equation to calculate proline concentration. This procedure was repeated in triplicate on three different samples and SEM was taken.