Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca2+, phosphorylation, and protein-protein interactions on RBOH-dependent ROS production.

Department of Applied Biological Science Tokyo University of Science Noda Chiba Japan

Department of Food Production Science Ehime University Matsuyama Ehime Japan

Institute of Plant Molecular Biology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Organismal and Evolutionary Biology Research Programme Viikki Plant Science Centre University of Helsinki Helsinki Finland

Ritsumeikan Global Innovation Research Organization Ritsumeikan University Kusatsu Shiga Japan

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