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Methods to Detect Nitric Oxide in Plants: Are DAFs Really Measuring NO?
Stefan Ruemer1, Markus Krischke1, Agnes Fekete1
1Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|April 21, 2016
Summary
Fluorescent dyes like DAF-2, commonly used to detect nitric oxide (NO), also react with peroxidase and hydrogen peroxide. This challenges their accuracy in measuring NO production in biological systems.
Area of Science:
- Biochemistry
- Plant Science
- Cell Biology
Background:
- Nitric oxide (NO) is a crucial signaling molecule in biological systems.
- Fluorescent dyes, such as DAF-2, are widely employed for NO detection.
- Concerns regarding the specificity of DAF dyes have recently emerged.
Purpose of the Study:
- To investigate the specificity of DAF dyes in detecting nitric oxide.
- To identify potential interfering substances that affect DAF dye fluorescence.
- To clarify the origin of fluorescence signals in plant cells stimulated with cryptogein.
Main Methods:
- Utilized DAF-2 and related fluorescent dyes for detection.
- Employed high-performance liquid chromatography (HPLC) for compound separation.
- Analyzed fluorescence signals in elicited tobacco suspension cells.
Main Results:
- DAF dyes exhibit cross-reactivity with peroxidase and hydrogen peroxide.
- Fluorescence observed outside elicited cells does not originate from the expected DAF-2-NO adduct.
- Inside cells, unidentified DAF derivatives are formed, with only a minor fraction being the NO adduct.
Conclusions:
- The specificity of DAF dyes for nitric oxide detection is questionable due to cross-reactivity.
- Fluorescence signals in elicited plant cells may be artifacts of non-specific reactions.
- Further research is required to determine the chemical identity of novel DAF derivatives and validate NO detection methods.
