Abstract
There is now general agreement that nitric oxide (NO) is an important and almost ubiquitous signal in plants. Nevertheless, there are still many controversial observations and differing opinions on the importance and functions of NO in plants. Partly, this may be due to the difficulties in detecting and quantifying NO. Here, we summarize protocols for detecting NO and quantifying nitrite concentration in Arabidopsis seedlings. We also present a method to measure NO in biofilms formed by the plant growth promoting rhizobacteria Azospirillum brasilense (A. brasilense). NO in oxygen-containing aqueous solutions has a short half-life that is often attributed to a rapid oxidation to nitrite. Here we detail the use of the fluorescent probe DAF-FM DA and the electrochemical method for directly detecting and quantifying NO, respectively, and the Griess reagent to indirectly detect NO through its oxidized nitrite form. These protocols could be useful in a variety of cell types and plant tissues, as well as for microorganisms.
Keywords: Nitric oxide, Nitrite, Plants, Rhizosphere microorganisms
Part I. In vitro determination of nitrite concentration
Materials and Reagents
Equipment
Procedure
Recipes
Part II. In vivo detection of NO Method 1. Electrochemical detection of NO
Software
Method 2. NO Fluorometric assay
Acknowledgments
This research was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT: PICTs 2383/2011 and 2621/2011 to L.L. and N.C.-A., respectively), the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0903/2011 to L. L.), and institutional grants from the Universidad Nacional de Mar del Plata, Argentina. This protocol was adapted from the published work (Palma et al., 2013; Foresi et al., 2015).
References
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