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DAF-fluorescence without NO: Elicitor treated tobacco cells produce fluorescing DAF-derivatives not related to DAF-2 triazol.
MedLine Citation:
PMID:  22683597     Owner:  NLM     Status:  Publisher    
Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO(•) production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H(2)O(2) plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H(2)O(2) also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H(2)O(2). DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers. In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H(2)O(2), and another, minor peak eluted at or close to DAF-2T. Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO(•) dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO(•) production.
Stefan Rümer; Markus Krischke; Agnes Fekete; Martin J Mueller; Werner M Kaiser
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-6-5
Journal Detail:
Title:  Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society     Volume:  -     ISSN:  1089-8611     ISO Abbreviation:  -     Publication Date:  2012 Jun 
Date Detail:
Created Date:  2012-6-11     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9709307     Medline TA:  Nitric Oxide     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012. Published by Elsevier Inc.
University of Wuerzburg, Julius-von-Sachs Institute of Biosciences, Julius-von-Sachs-Platz 2, D-97082 Wuerzburg, Germany.
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