Methods to Detect Nitric Oxide in Plants: Are DAFs Really Measuring NO?
Nitric oxide, a gaseous radical molecule, appears involved in many reactions in all living organisms. Fluorescent dyes like DAF-2 and related compounds are still widely used to monitor NO production inside or outside cells, although doubts about their specificity have recently been raised. We present evidence that DAF dyes do not only react with nitric oxide but also with peroxidase enzyme and hydrogen peroxide. Both are secreted in the case of elicitation of tobacco suspension cells with cryptogein, with a fluorescence increase mimicking NO release from cells. However, HPLC separation shows that fluorescence outside cells does not at all originate from DAF-2T, the product of DAF-2 and NO, but from other yet unidentified compounds. Inside cells, other DAF molecules are formed but only a minor part is DAF-2T. The chemical nature of the novel DAF derivatives still needs to be determined.
Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany.
Stefan
S
Ruemer
Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany.
Markus
M
Krischke
Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany.
Agnes
A
Fekete
Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany.
Maria
M
Lesch
Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany.
Marin J
MJ
Mueller
Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany. kaiser@botanik.uni-wuerzburg.de.
Werner M
WM
Kaiser
D019793:Fluorescein / Q000378:metabolism*; D005453:Fluorescence; D009569:Nitric Oxide / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*
C111205:4,5-diaminofluorescein; D009569:Nitric Oxide; D019793:Fluorescein
Cryptogein; Diaminofluorescein; Nitrate reductase; Nitric oxide; Nitric oxide synthase; Nitrite