Functional Characterization of the Plant NADPH oxidase by Heterologous Expression

Abstract

Rapid generation of reactive oxygen species (ROS) is an important component of plant defense responses against pathogens. ROS production is inhibited by diphenylene iodonium (DPI), suggesting that NADPH oxidase-like enzyme mediates generation of ^.O₂^-. Plant NADPH oxidase homologs (rboh; respiratory burst oxidase homolog) possess hydrophilic N-terminal domain containing two Ca²⁺ binding EF-hand motifs. However, regulatory mechanisms and physiological functions of rboh are still largely unknown. For functional analyses, heterologous expression system was applied. AtrbohD was transiently transfected to human embryonic kidney (HEK) 293T cells, and ROS generation was detected by chemiluminescence. Treatment with a Ca²⁺ ionophore induced rapid and transient production of ^.O₂^-. This activity was completely inhibited by DPI, suggesting that rboh is directly activated by binding of Ca²⁺ to its EF-hands to show NADPH oxidase activity.