Quantitative determination of superoxide in strawberry leaves using a modified NBT staining method

GRELLET, CARLOS; CASTAGNARO, ATILIO P.; DIAZ RICCI, JUAN C

Villa Carlos Paz, Córdoba.

Congreso; 44º Reunión Anual de la Sociedad de Bioquímica y Biología Molecular; 2008

Sociedad de Bioquímica y Biología Molecular

The interest of studying the reactive oxygen species (ROS, i.e. H2O2, O2.-) in plants has increased lately for they are involved in different physiological and stress mechanisms. Although a variety of methods have been developed to determine the production of ROS in plants, its evaluation in leaves of certain species is difficult and scarcely reliable due to a great number of interferents. During the study of the defense reponse in strawberry we were interested in the determination of the anion superoxide (O2.-). The determination of O2.- is usually carried out by direct histochemical staining, but these methods allow only to a visual observation of O2.- production unless special computer programs are used to quantify the histological preparations. However, the accuracy of this prodecure of evaluation is always debatable. In this work, we present a protocol for the quantitative determination of the anion O2.- based on a modification of a classic histological method reported earlier by Jabs et. al. (1996) that uses a tetrazolium salt (NBT). The modification consists on the selective solubilization of the reduced Formazan produced during the O2.- formation, allowing to make reliable spectrophotometrics readings (Muñoz et. al. 2000). The method was tested and validated on strawberry leaves subjected to biotic and abiotic stress produced by infection and an herbicide, respectively.