Measuring NO Production by Plant Tissues and Suspension Cultured Cells

Abstract

We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants. The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap. The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors. We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources. NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected, as was the production of NO from nitrite by the incubation medium of barley aleurone. Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up. We could also measure NO production from elicitor-treated suspension cultured cells using this set-up. Further, we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues. In addition to the dramatic effects that pH can have on fluorescence from DAF-FM, the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results. Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

Nitric oxide production and sequestration in the sinus gland of the green shore crab, Carcinus maneas

“…5), indicating that CuFL outcompeted cPTIO for binding to free NO. Similar results were found when DAF-2 was used to quantify NO in plant tissues; at high NO levels cPTIO increased DAF-2 NO fluorescence (Vitecek et al, 2008). This effect was attributed to DAF-2 binding to N 2 O 3 , a by-…”

Heat Reduces Nitric Oxide Production Required for Auxin-Mediated Gene Expression and Fate Determination in Tree Tobacco Guard Cell Protoplasts

“…DAF-2T fluorescence can be quenched drastically at lower pH levels (Hilderbrand et al, 2005), and the reactive oxygen chemistry of some cellular and/or subcellular compartments (Planchet and Kaiser, 2006a), ascorbic acid and dehydroascorbic acid (Planchet and Kaiser, 2006a), PTIO (Vitecek et al, 2008), and unidentified compounds (Planchet and Kaiser, 2006b) can yield false-positive readings with at least some diaminofluorescein dyes. Thus, full controls are required when diaminofluorescein dyes are used to evaluate cellular NO levels.…”

A Ratiometric Sensor Using Single Chirality Near‐Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring

“…[ 42 ] The cell permeable 2′,7′-dichlorodihydrofl uorescein diacetate (H 2 DCF-DA) dye has been demonstrated to detect H 2 O 2 in living organisms despite its relatively nonselectivity to reactive oxygen species (ROS) [ 23 ] and susceptibility to photo-oxidation and photobleaching. [ 40 ] Imaging of NO in living systems has been performed with diamionofl uoresceins with the disadvantage that their fl uorescein chromophore is responsive to changes in pH [ 43 ] and reacts with dehydroascorbic and ascorbic acid. [ 41 ] Plants are optically dense living organisms due to thick tissues and photosynthetic pigments, making it diffi cult to detect analytes in vivo.…”