Using hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: a method with virtually unlimited potential in plant biology

Methods Enzymol. 2013;527:275-90. doi: 10.1016/B978-0-12-405882-8.00015-5.


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 occurs as a result of aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, they can be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses, or during cell wall organization and plant morphogenesis. Monitoring ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes make it impossible to visualize dynamic changes of ROS. Hyper is a recently developed live cell probe for H2O2 and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H2O2) sensor OxyR rendering it a H2O2 specific ratiometric, and therefore quantitative probe. Herein, we describe a protocol for using Hyper as a dynamic probe for H2O2 in Arabidopsis with virtually unlimited potential to detect H2O2 throughout the plant and under a broad range of developmental and environmental conditions.

Keywords: Hydrogen peroxide; Hyper; NADPH oxidase; Plant root; Pollen tube; Ratiometric; Reactive oxygen species; Root hair.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Arabidopsis / cytology
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / genetics
  • Biosensing Techniques
  • Escherichia coli Proteins / biosynthesis
  • Escherichia coli Proteins / genetics
  • Hydrogen Peroxide / metabolism*
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • NADPH Oxidases / metabolism
  • Plant Proteins / metabolism
  • Plant Roots / cytology
  • Plant Roots / genetics
  • Plant Roots / metabolism
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Pollen Tube / cytology
  • Pollen Tube / genetics
  • Pollen Tube / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Repressor Proteins / biosynthesis
  • Repressor Proteins / genetics
  • Tobacco / cytology
  • Tobacco / genetics
  • Tobacco / metabolism
  • Transformation, Genetic


  • Bacterial Proteins
  • Escherichia coli Proteins
  • Luminescent Proteins
  • Plant Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • oxyR protein, E coli
  • yellow fluorescent protein, Bacteria
  • Hydrogen Peroxide
  • NADPH Oxidases