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JXB Advance Access originally published online on August 7, 2006
Journal of Experimental Botany 2006 57(12):3043-3055; doi:10.1093/jxb/erl070
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources

Elisabeth Planchet and Werner M. Kaiser*

Julius-von-Sachs Institute for Biosciences, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany

*To whom correspondence should be addressed. E-mail: kaiser{at}botanik.uni-wuerzburg.de

Because of controversies in the literature on nitric oxide (NO) production by plants, NO detection by the frequently used diaminofluorescein (DAF-2 and DAF-2DA) and by chemiluminescence were compared using the following systems of increasing complexity: (i) dissolved NO gas; (ii) the NO donor sodium nitroprusside (SNP); (iii) purified nitrate reductase (NR); and (iv) tobacco cell suspensions. Low (physiological) concentrations (≤1 nM) of dissolved NO could be precisely quantified by chemiluminescence, but caused no DAF-2 fluorescence. In contrast to NO gas, SNP, NR, or cell suspensions produced both good DAF fluorescence and chemiluminescence signals which were completely (chemiluminescence) or partly (DAF fluorescence) prevented by NO scavengers. Signal strength ratios between the two methods were variable depending on the NO source, and eventually reflect variable NO oxidation. DAF fluorescence in cell suspension cultures was also increased by an as yet unidentified compound(s) released from cells into the medium. These compounds gave no chemiluminescence signal and were not produced by NR-free mutants. Their production was stimulated by anoxia, by inhibitors of mitochondrial electron transport, and by the fungal elicitor cryptogein. Thus, changes in DAF fluorescence are not necessarily indicative for NO production, but may also reflect NO oxidation and/or production of other DAF-reactive compounds.

Key words: Anoxia, chemiluminescence, cryptogein, DAF fluorescence, mitochondria, nitrate reductase, nitric oxide, NO oxidation


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