Guaiacol peroxidase assay
Guaiacol peroxidase activity was quantified as in MacAdam et al. (1992). Roots and shoots (from cotyledons upwards) of tomato seedlings were flash-frozen in liquid nitrogen 14 days after transplantation (24 h after second PA treatment) and ground in a liquid nitrogen-chilled mortar. ±75 mg powder was extracted with 8 µl mg-1cold extraction buffer (0.8 M KCl and 8 g l-1polyvinylpyrrolidone in 50 mM potassium phosphate buffer, pH 6.0). After centrifugation (10 minutes, 19 330g, 4°C), 10 µl extract was mixed with 990 µl assay buffer (3.3 mM guaiacol and 0.4 mM H2O2 in 100 mM potassium phosphate buffer, pH 6.0). Absorbance increase at 436 nm was recorded every 15 seconds for 3 minutes. Six biological replicates were used per treatment (each consisting of 2-3 pooled seedlings) and the experiment was repeated twice. The slope of the resulting curve was used as a measure of guaiacol peroxidase activity.