Establishment of a reliable protocol for the quantification of an oxidative burst in suspension-cultured wheat cells upon elicitation

Abstract

The rapid and transient accumulation of reactive oxygen species, termed ‘oxidative burst’, is one of the earliest observable reactions of plant cells to microbial infection or elicitation. Although its importance for plant defence is well recognized and there are many reports about the detection of an oxidative burst in dicotyledonous plants, evidence for reactive oxygen species in monocotyledonous plants is still sparse. Here, we describe a reliable protocol for the quantitative determination of hydrogen peroxide in suspension-cultured wheat cells ( Triticum aestivum L., cultivar Prelude-Sr5) by luminol-dependent chemiluminescence. Mixtures of chitin oligomers and chitosan polymers were chosen as elicitors. The amounts of H 2O 2 detected in the assay were much higher when the measurement was performed in a dedicated assay medium instead of the normal growth medium of the cells. The maximum of the oxidative burst depended on the pre-incubation time of the wheat cells. When purified chitin oligomers were tested for their elicitor activity, the chitin pentamer turned out to be the smallest active oligomer.