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.