The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants.
MetadataShow full item record
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.
Place of Publication
The following license files are associated with this item:
Showing items related by title, author, creator and subject.
Radakovits, R; Barros, CS; Belvindrah, R; Patton, B; Müller, U (United StatesUnited StatesUnited StatesUnited StatesUnited StatesUnited States, 2009-06-17)Radial glial cells (RGCs) in the developing cerebral cortex are progenitors for neurons and glia, and their processes serve as guideposts for migrating neurons. So far, it has remained unclear whether RGC processes also ...
Parkinson, DB; Bhaskaran, A; Arthur-Farraj, P; Noon, LA; Woodhoo, A; Lloyd, AC; Feltri, ML; Wrabetz, L; Behrens, A; Mirsky, R; Jessen, KR (United StatesUnited StatesUnited StatesUnited StatesUnited StatesUnited StatesUnited States, 2008-05-19)Schwann cell myelination depends on Krox-20/Egr2 and other promyelin transcription factors that are activated by axonal signals and control the generation of myelin-forming cells. Myelin-forming cells remain remarkably ...
Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport. Barros, CS; Phelps, CB; Brand, AH (United StatesUnited StatesUnited StatesUnited StatesUnited StatesUnited States, 2003-12)Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb ...