Using Hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: An updated method

Reactive oxygen species (ROS) are highly reactive reduced oxygen molecules that play a myriad of roles in animal and plant cells. In plant cells the production of ROS results from aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, ROS can also be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses or during cell wall organization and plant morphogenesis. The study of ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes prevents the visualization of dynamic changes. We have previously reported that Hyper 1 is a biosensor for H₂O₂ and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H₂O₂) sensor protein OxyR rendering it an H₂O₂-specific quantitative probe (Bilan & Belousov, 2018; Hernandez-Barrera et al., 2015). Herein we describe an updated protocol for using the improved new version of Hyper 2 and Hyper 3 as a dynamic biosensor for H₂O₂ in Arabidopsis with virtually unlimited potential to detect H₂O₂ throughout the plant and under a broad range of developmental and environmental conditions (Bilan et al., 2013).