A fluorometer‐based method for monitoring oxidation of redox‐sensitive GFP (roGFP) during development and extended dark stress

Abstract

Redox-sensitive GFP (roGFP) localized to different compartments has been shown to be suitable for determination of redox potentials in plants via imaging. Long-term measurements bring out the need for analyzing a large number of samples which are averaged over a large population of cells. Because this goal is too tedious to be achieved by confocal imaging, we have examined the possibility of using a fluorometer to monitor changes in roGFP localized to different subcellular compartments during development and dark-induced senescence. The degree of oxidations determined by a fluorometer for different probes was similar to values obtained by confocal image analysis. Comparison of young and old leaves indicated that in younger cells higher levels of H(2)O(2) were required to achieve full roGFP oxidation, a parameter which is necessary for calculation of the degree of oxidation of the probe and the actual redox potential. Therefore, it is necessary to carefully determine the H(2)O(2) concentration required to achieve full oxidation of the probe. In addition, there is an increase in autofluorescence during development and extended dark stress, which might interfere with the ability to detect changes in oxidation-reduction dependent fluorescence of roGFP. Nevertheless, it was possible to determine the full dynamic range between the oxidized and the reduced forms of the different probes in the various organelles until the third day of darkness and during plant development, thereby enabling further analysis of probe oxidation. Hence, fluorometer measurements of roGFP can be used for extended measurements enabling the processing of multiple samples. It is envisaged that this technology may be applicable to the analysis of redox changes in response to other stresses or to various mutants.

Nitric Oxide-Mediated Maintenance of Redox Homeostasis Contributes to NPR1-Dependent Plant Innate Immunity Triggered by Lipopolysaccharides

“…The roGFP1 degree of oxidation was determined as described (Rosenwasser et al, 2010). Leaf discs were excited by using 400-6 15-nm and 485-6 10-nm filters, and fluorescence values were measured using a 525-6 10-nm emission filter.…”

Organelles Contribute Differentially to Reactive Oxygen Species-Related Events during Extended Darkness

“…To determine the redox changes in specific cellular compartments during darkness, we measured the degree of oxidation of the roGFP probe in Arabidopsis transgenic lines, which included probes localized in the mitochondria (mit-roGFP1, mit-roGFP2), plastids (pla-roGFP2), cytoplasm (cyt-GRX1-roGFP2), and peroxisomes (per-GRX1-roGFP2; for source description, see Supplemental Table S5). Both roGFP1 and roGFP2 were found to be suitable to monitor the glutathione redox potential and display different degrees of oxidation at the steady state due to their different midpoint redox potentials (Schwarzländer et al, 2008;Rosenwasser et al, 2010b). Quantitative assessment provided by direct fluorometric measurements was limited to 3 d of darkness treatment, as longer periods were found to induce high levels of autofluorescence of unknown origin at 405 nm that obviates measurements (Rosenwasser et al, 2010a(Rosenwasser et al, , 2010b.…”

“…Both roGFP1 and roGFP2 were found to be suitable to monitor the glutathione redox potential and display different degrees of oxidation at the steady state due to their different midpoint redox potentials (Schwarzländer et al, 2008;Rosenwasser et al, 2010b). Quantitative assessment provided by direct fluorometric measurements was limited to 3 d of darkness treatment, as longer periods were found to induce high levels of autofluorescence of unknown origin at 405 nm that obviates measurements (Rosenwasser et al, 2010a(Rosenwasser et al, , 2010b. The results showed a significant increase in the degree of probe oxidation in two mitochondria reporter lines (mit-roGFP1 and mit-roGFP2) as early as the first day under darkness (Fig.…”

“…Kinetic analysis of the purified protein as well as in vivo experiments have confirmed that roGFP is in equilibrium with the glutathione redox potential and therefore can serve as a sensor for measuring the potential of the glutathione redox buffer (Meyer et al, 2007). We have recently reported that roGFP-expressing plants can also be analyzed ratiometrically by a fluorometerbased assay (Rosenwasser et al, 2010b).…”

Mitochondrial Energy and Redox Signaling in Plants

“…These probes can be targeted to mitochondria, solving the problem of insufficient spatial specificity. Additionally, they are largely nontoxic and can be monitored in living plant cells or tissues, by microscopy or fluorimetry (185). An important feature of these sensors is the rapid and reversible equilibration with their direct environment, allowing live monitoring of physiological changes.…”