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A fluorometer-based method for monitoring oxidation of redox-sensitive GFP (roGFP) during development and extended dark stress
Year:
2010
Source of publication :
Physiologia Plantarum
Authors :
Friedman, Haya
;
.
Rot, Ilona
;
.
Volume :
138
Co-Authors:
Rosenwasser, S., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel, Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Kennedy-Leigh Centre for Horticultural Research, Faculty of Agriculture, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel
Rot, I., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel
Meyer, A.J., Heidelberg Institute for Plant Science (HIP), Heidelberg University, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany
Feldman, L., Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720-3102, United States
Jiang, K., Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720-3102, United States
Friedman, H., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel
Facilitators :
From page:
493
To page:
502
(
Total pages:
10
)
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 H2O2 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 H2O2 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. Copyright © Physiologia Plantarum 2009.
Note:
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More details
DOI :
10.1111/j.1399-3054.2009.01334.x
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
19910
Last updated date:
02/03/2022 17:27
Creation date:
16/04/2018 23:32
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Scientific Publication
A fluorometer-based method for monitoring oxidation of redox-sensitive GFP (roGFP) during development and extended dark stress
138
Rosenwasser, S., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel, Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Kennedy-Leigh Centre for Horticultural Research, Faculty of Agriculture, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel
Rot, I., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel
Meyer, A.J., Heidelberg Institute for Plant Science (HIP), Heidelberg University, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany
Feldman, L., Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720-3102, United States
Jiang, K., Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720-3102, United States
Friedman, H., Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel
A fluorometer-based method for monitoring oxidation of redox-sensitive GFP (roGFP) during development and extended dark stress
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 H2O2 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 H2O2 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. Copyright © Physiologia Plantarum 2009.
Scientific Publication
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