Detection of Nitric Oxide from Chickpea Using DAF Fluorescence and Chemiluminescence Methods

Abstract

The free radical nitric oxide (NO) has emerged as an important signal molecule in plants, due to its involvement in various plant growth, development, and stress responses. For elucidating the role of NO, it is very important to precisely determine, localize, and quantify NO levels. Due to a relatively short half‐life and its rapid, complex reactivity with other radicals, together with its capacity to diffuse from the source of production, the quantification of NO in whole plants, tissues, organelles, and extracts is notoriously difficult. Hence, it is essential to employ sensitive procedures for precise detection of NO. Currently available methods can fulfill many requirements to precisely determine NO, but each method has several advantages and pitfalls. In this article, we describe a detailed procedure for the measurement of NO by diaminofluorescein (DAF) in cell‐permeable forms (DAF‐FM‐DA). In this method, the tissues are immersed in DAF‐FM DA, leading to their diffusion from the plasma membrane to the inside of the cell, where intracellular esterases cleave the ester bonds, leading to DAF‐FM release. The resulting DAF‐FM reacts with intracellularly generated NO and forms highly fluorescent triazolofluorescein (DAF‐FMT), which can be localized and monitored by fluorescence or confocal microscopy, and can also be detected via fluorimetry and flow cytometry. DAF dyes are very popular as they are non‐invasive, relatively easy to handle, and commercially available. Another precise and very sensitive method is chemiluminescence detection of NO, where NO reacts with ozone (O3), leading to emission of a quantum of light from which NO can be calculated. Using chickpea seedlings, we describe in detail the measurement of NO using DAF‐FM‐DA and chemiluminescence methods. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Measurement of nitric oxide from chickpea seedlings using DAF‐FM DA fluorescence with fluorescence and confocal microscopy Basic Protocol 2: Chemiluminescence detection of nitric oxide from chickpea seedlings

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“…To gain mechanistic insights into the possible role(s) of NO in PTI, we first explored NO production in Arabidopsis upon flagellin peptide flg22 treatment. Following application of this well-established P/MAMP, a rapid nitrosative burst was detected using the NO-sensitive reporter, diaminofluorescein-FM diacetate (DAF-FM DA) ( 23 ), within 10 min post-flg22 treatment (Fig. 1A and fig.…”

“…S2, A and B). S -nitrosylation can be promoted by applying well-established NO donors, including GSNO or sodium nitroprusside (SNP) ( 23 ). We therefore tested whether NO is required for flg22-mediated ROS production by using these donors.…”

NIR-Triggered Release of Nitric Oxide by Upconversion-Based Nanoplatforms to Enhance Osteogenic Differentiation of Mesenchymal Stem Cells for Osteoporosis Therapy

“…A particular NO probe, DAF-FM DA, was used to identify intracellular NO release. DAF-FM DA can easily permeate live cells where it is rapidly catalyzed to transform into DAF-FM, which cannot infiltrate the cell membrane and then reacts with NO to produce intense green fluorescence [ 41 ]. As illustrated in Fig.…”

Determination of Nitric Oxide and Its Metabolites in Biological Tissues Using Ozone-Based Chemiluminescence Detection: A State-of-the-Art Review

“…Regarding nitrate and nitrite levels in the circulation, it is important to note that a main factor in modulating those levels is the dietary intake of nitrate. There are multiple studies using CLD for analyzing nitrate and nitrite levels in food and drinking water [136], which is also important due to the fact that a high intake of nitrate has been considered harmful and cancerogenic due to the conversion of nitrate into nitrite by bacteria and further metabolism into nitrosamine [137,138]. Furthermore, sodium nitrate and potassium nitrate as well as sodium nitrite and potassium nitrite are used in the food industry, especially for the preservation of meat as additives [139].…”