No abstract

Biphasic production of reactive oxygen species (ROS) has been observed in plants treated with avirulent bacterial strains. The first transient peak corresponds to PTI (pattern-triggered immunity)-ROS, whereas the second long-lasting peak corresponds to ETI (effector-triggered immunity)-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.

Breeding crops with enhanced immunity is an effective strategy to reduce yield loss caused by pathogens. The constitutive expresser of pathogenesis-related genes (cpr5) mutant shows enhanced pathogen resistance but retarded growth; thus, it restricts the application of cpr5 in breeding crops with disease resistance. Reactive oxygen species (ROS) play important roles in plant growth and defense. In this study, we determined that the cpr5 mutant exhibited excessive ROS accumulation. However, the mutation of respiratory burst oxidase homolog D (RBOHD), a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for the production of ROS signaling in plant immunity, did not suppress excessive ROS levels in cpr5. Furthermore, the cpr5 mutant showed low levels of ascorbate peroxidase 1 (APX1), an important cytosolic ROS-scavenging enzyme. APX1 overexpression in the cpr5 background removed excessive ROS and restored the pleiotropic growth phenotype. Notably, APX1 overexpression did not reduce the resistance of cpr5 mutant to virulent strain Pseudomonas syringae pv. tomato (Pst) DC3000 and avirulent strain Pst DC3000 (avrRpt2). These results suggest that the removal of excessive ROS by APX1 overexpression restored the cpr5 growth phenotype while conserving pathogen resistance. Hence, our study provides a theoretical and empirical basis for utilizing CPR5 in the breeding of crops with disease resistance by effective oxidative stress management via APX1 expression.