Phenylalanine Ammonia Lyase (PAL) Assay

Leaf sample (0.5 g) from each treatment was taken and homogenized in 4 ml of borate buffer (pH 8.7; 4°C) and centrifuged at 13000 rpm for 15 min at 4°C. The supernatant was used as enzyme source. The reaction mixture contained 0.2 ml of enzyme extract, 1.3 ml distilled water and 1.0 ml of 0.1 M phenylalanine. The reaction mixture was incubated at 32°C for 30–60 min after the incubation and 0.5 ml of 1 M TCA (trichloro acetic acid) was added immediately to stop the reaction. OD optical density was measured at 290 nm by taking the control containing all the solutions except enzyme extract. Phenylalanine ammonia lyase (PAL) (EC 4.1.3.5) activity was also measured at 290 nm following formation of trans-cinnamic acid (Havir, 1987) and was expressed in terms of μmol l^-1 TCA per g fresh weight (FW).