The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants

Methods Enzymol. 2013:527:185-201. doi: 10.1016/B978-0-12-405882-8.00010-6.


Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.

Keywords: Arabidopsis thaliana; Chlorophyll fluorescence; Confocal microscopy; Cotyledons; Gene silencing; High light; HyPer; Hydrogen peroxide; Signaling; Yellow fluorescent protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis / radiation effects
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / genetics
  • Biosensing Techniques
  • Chlorophyll / metabolism
  • Cotyledon / genetics
  • Cotyledon / metabolism
  • Cotyledon / radiation effects
  • Escherichia coli Proteins / biosynthesis
  • Escherichia coli Proteins / genetics
  • Gene Expression Regulation, Plant / radiation effects
  • Hydrogen Peroxide / metabolism*
  • Light
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Plants, Genetically Modified / radiation effects
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Repressor Proteins / biosynthesis
  • Repressor Proteins / genetics
  • Seedlings / genetics
  • Seedlings / metabolism*
  • Seedlings / radiation effects


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