Characterization of apoplast phenolics: In vitro oxidation of acetosyringone results in a rapid and prolonged increase in the redox potential

• Find similarities between the redox response of acetosyringone and cell suspensions.
• Oxidation of acetosyringone with H2O2 and peroxidase produces prolonged redox response.
• Equal concentration of reactants favors longer response.

Previous work demonstrated that when tobacco cell suspensions were inoculated with certain bacterial strains, the redox potential of the suspensions would increase (oxidative), as much as 100 mV, and in some cases last more than an hour. To discover possible contributors to this prolonged redox potential burst, we examined the oxidation of various plant phenolics that were similar to those found in the cell suspensions. Acetosyringone, one of the major extracellular phenolics in tobacco cell suspensions, was unique in causing a prolonged increase in redox potential of more than 100 mV when incubated with H2O2 and peroxidase. The increase in potential appears to be due to a relatively stable radical intermediate. The length of time that the redox potential increased was dependent on the ratio of H2O2 and acetosyringone. The most favorable was a 1:1 ratio. In vitro characterization showed that production of this intermediate is favored by relatively low pH (around 6) and cooler temperatures possibly due to slower kinetics. Phosphate buffer was substituted for MES buffer, which had been used initially but was found to interfere with the oxidation. The results of this study demonstrate that apoplastic phenolics, in addition to often playing a role as antioxidants that reduce H2O2, can also cause rapid transient periods of extreme redox potential creating a local environment hostile to pathogen ingress.