Fig. 5
Glycolate oxidase activity in plants inoculated with avirulent and virulent Pseudomonas. The leaves of four week old Col-0 plants were pressure inoculated with either 106 cfu ml−1 avirulent Pst DC3000 AvrRpm1 or virulent Pst DC3000 or 10 mM MgCl2 (control). The effect of NO and cGMP during infection was examined by including 50 μM 8-Br cGMP or 50 μM diethylamine NONOate either separately or in combination, in the bacterial suspension at the time of infection. The infected leaves were harvested at 24 h post infection and GOX activity measured. Leaf proteins were extracted and incubated with sodium glycolate, O-dianisidine and horseradish peroxidase. GOX activity catalyses the conversion of the sodium glycolate substrate to glyoxylate, releasing H2O2. The H2O2 reacts with O-dianisidine in the presence of horseradish peroxidase to produce the coloured O-dianisidine radical which can be quantified spectrophotometrically at 440 nm. GOX activity was found to be significantly induced (p = 0.0276) by ArvRpm1 relative to the MgCl2 control and this activity was significantly suppressed (p = 0.0400) by the combined treatment with NO and cGMP. Error bars represent standard error of the mean (n = 6) and statistical significance was determined using a student’s t-test with the asterisks denoting significant p values < 0.05