DAF-fluorescence without NO: elicitor treated tobacco cells produce fluorescing DAF-derivatives not related to DAF-2 triazol.
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.
University of Wuerzburg, Julius-von-Sachs Institute of Biosciences, Julius-von-Sachs-Platz 2, D-97082 Wuerzburg, Germany.
Stefan
S
Rümer
Markus
M
Krischke
Agnes
A
Fekete
Martin J
MJ
Mueller
Werner M
WM
Kaiser
D002457:Cell Extracts / Q000737:chemistry; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism*; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014026:Nicotiana / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
C111205:4,5-diaminofluorescein; D002457:Cell Extracts; D005656:Fungal Proteins; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D012235:Rhodamines; C061032:cryptogein protein, Phytophthora cryptogea; C508638:diaminorhodamine-4M; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D009195:Peroxidase; D019793:Fluorescein