3.4. Determination of L-Phenylalanine Ammonia-Lyase (PAL) Activity

The enzymatic activity was analyzed on the same plant material used for RNA extractions for each treatment. Tissue was homogenized in 3 mL of 0.1 M trisodium borate buffer (pH 8.5) containing 1.4 mM 2-mercaptoethanol and 0.1 g of insoluble polyvinylpyrrolidone. The extract was filtered through cheesecloth, and centrifugation was carried out at full speed for 15 min. The determination of PAL activity was analyzed as the rate of conversion of _L-phenylalanine into trans-cinnamic acid at 270 nm in a spectrophotometer. A 200 μL aliquot of the sample was added to 400 μL of borate buffer and 200 μL of 40 mM _L-phenylalanine, and the reaction mixture was incubated at 37 °C for 15 min. The reaction was stopped with an equal volume of 10% (w/v) of trichloroacetic acid (TCA), and samples were centrifuged at full speed for 15 min. The supernatants were used to measure the absorbance at 270 nm, and trans-cinnamic acid was calculated using a standard curve according to Lee et al. [69]. The results are presented as μmol/min (U) per g of fresh tissue.