Ascorbate peroxidase 1 allows monitoring of cytosolic accumulation of effector-triggered reactive oxygen species using a luminol-based assay

Abstract

Biphasic production of reactive oxygen species (ROS) has been observed in plants treated with avirulent bacterial strains. The first transient peak corresponds to pattern-triggered immunity (PTI)-ROS, whereas the second long-lasting peak corresponds to effector-triggered immunity (ETI)-ROS. PTI-ROS are produced in the apoplast by plasma membrane-localized NADPH oxidases, and the recognition of an avirulent effector increases the PTI-ROS regulatory module, leading to ETI-ROS accumulation in the apoplast. However, how apoplastic ETI-ROS signaling is relayed to the cytosol is still unknown. Here, we found that in the absence of cytosolic ascorbate peroxidase 1 (APX1), the second phase of ETI-ROS accumulation was undetectable in Arabidopsis (Arabidopsis thaliana) using luminol-based assays. In addition to being a scavenger of cytosolic H2O2, we discovered that APX1 served as a catalyst in this chemiluminescence ROS assay by employing luminol as an electron donor. A horseradish peroxidase (HRP)-mimicking APX1 mutation (APX1W41F) further enhanced its catalytic activity toward luminol, whereas an HRP-dead APX1 mutation (APX1R38H) reduced its luminol oxidation activity. The cytosolic localization of APX1 implies that ETI-ROS might accumulate in the cytosol. When ROS were detected using a fluorescent dye, green fluorescence was observed in the cytosol 6 h after infiltration with an avirulent bacterial strain. Collectively, these results indicate that ETI-ROS eventually accumulate in the cytosol, and cytosolic APX1 catalyzes luminol oxidation and allows monitoring of the kinetics of ETI-ROS in the cytosol. Our study provides important insights into the spatial dynamics of ROS accumulation in plant immunity.