Phenylalanine Ammonia-Lyase Activity (PAL)

The PAL activity was measure according to Beaudoin-Eagan and Thorpe (1985). The enzyme activity was expressed in nmol of trans-cinnamic acid.mg protein^-1.min^-1, where 1 U is defined as 1 nmol trans-cinnamic acid.mg protein^-1.min^-1. 300 mg of leaf samples were homogenized in 3 mL of Tris-HCl buffer 50 mM pH 8.5 with 2-mercaptoethanol 14.4 mM and 100 mg PVPP, and centrifuged at 6,000 × g for 10 min at 4°C. The supernatant were passed through a column of celite and centrifuged at 10,000 × g for 15 min at 4°C. Total protein concentration was determined using the Bradford (1976) assay.