U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska


Date of this Version



Published in Physiological and Molecular Plant Pathology 64 (2004) 255–261.


Detection of H2O2 in the supernatant of plant suspension cells is often used to indicate the time and extent of the oxidative burst during interactions with either bacteria or pathogen-related elicitors. We have found that suspensions of plant cells, depending on conditions, may produce considerable levels of extracellular phenolics that can function as antioxidants and prevent or suppress the detection of H2O2. These compounds can be used as substrates by extracellular peroxidases to scavenge stoichiometric amounts of H2O2. When this occurs during plant/pathogen interactions it can mask both the timing and extent of the oxidative burst if detection of free H2O2 is the only technique used. We have developed a chemiluminescent technique that will account for the H2O2 scavenged by these extracellular metabolites. A known quantity of H2O2 is added to samples and allowed to react with the extracellular antioxidants. The amount of H2O2 that remains is then determined by adding luminol to the sample and measuring luminol-dependent-chemiluminescence. The difference between treated and control samples represents the amount of H2O2 that has been produced by the cells in response to the treatment. We have found that this technique provides a better estimate of both the magnitude and timing of the oxidative burst in bacterial/suspension cell systems.