title: FNOCT as a fluorescent probe for in vivo localization of nitric oxide distribution in tobacco roots.
abstract: The nitric oxide-specific fluorescent probe Fluorescent Nitric Oxide Cheletropic Trap (FNOCT) 8a was applied to intact tobacco (Nicotiana tabacum cv. Samsun) roots to detect sites of nitric oxide formation and NO distribution. Three week old tobacco seedlings were gently removed from the sand culture pots with intact roots and transferred to small Petri dishes, whose base was replaced by a thin coverslip. Intact roots were subjected to FNOCT 8a to localize NO-dependent fluorescence in these roots; controls with an exogenous NO donor confirmed the presence and distribution of the probe in the roots. To confirm the NO-dependent fluorescence, roots were incubated with the three different NO scavengers cPTIO {2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-L-oxyl-3-oxide}, methylene blue and sodium diethyl dithiocarbamate (DCC) followed by incubation with FNOCT 8a. Methylene blue and DCC were able to completely quench NO-dependent fluorescence, cPTIO quenched partially. The roots were incubated in the presence of NaNO₂ and NaNO₃, which are substrates for nitrite:nitric oxide reductase (NI-NOR) and plasma membrane-bound nitrate reductase (PM-NR), respectively. The NO-dependent fluorescence was more or less same at the root tips upon treatment with NaNO₂, while the overall fluorescence was reduced in the presence of NaNO. Fluorescence from the living roots was visualized by inverted confocal laser scanning microscope (CLSM) using UV laser (excitation 360 nm and emission 408 nm). A specialized apparatus has been devised by the authors for analysis of intact roots as described in the methods section of this paper. Intact roots were chosen for microscopic observation rather than incised roots to avoid production of NO due to stress or physical injury.
authors.affiliation: Institute of Botany & Landscape Ecology, Ernst-Moritz-Arndt-University, Grimmer Strasse 88, D17487 Greifswald, Germany. shwetavandana@gmail.com
authors.forename: Shweta
authors.initials: S
authors.lastname: Vandana
authors.forename: Reiner
authors.initials: R
authors.lastname: Sustmann
authors.forename: Ursula
authors.initials: U
authors.lastname: Rauen
authors.forename: Christine
authors.initials: C
authors.lastname: Stöhr
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D050901:Nitrate Reductase / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D036226:Seedlings / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D012977:Sodium Nitrite / Q000494:pharmacology*; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009566:Nitrates; D010940:Plant Proteins; D009569:Nitric Oxide; C031618:sodium nitrate; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D050901:Nitrate Reductase; D012977:Sodium Nitrite