Detection of Nitric Oxide and Determination of Nitrite Concentrations in Arabidopsis thaliana and Azospirilum brasilense

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 different opinions on the importance and functions of NO in plants. Partly, this may be due to the difficulties in detecting and even more in quantifying NO. Here, we summarize protocols for detecting NO and quantifying nitrite concentration in Arabidopisis seedlings and for the NO real time measurement in biofilms formed by the plant growth promoting rhizobacteria Azospirillum brasilense (A. brasilense). NO in oxygen-containing aqueous solution 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 different tissues of plants, and for microorganisms.