Overview of Nitric Oxide Detection Methods in Plants

The reactivity, short half-life, and low in vivo concentrations of NO make accurate detection in biological systems technically challenging. A wide range of methods—each with specific strengths and limitations—has been developed to study NO in plant tissues (Wishwakarma et al., 2019; Gupta et al., 2020).

1. Chemical and Colorimetric Methods

Griess Assay

•A classical, indirect colorimetric method that measures nitrite (NO₂⁻)—a stable oxidation product of NO.
•Simple and inexpensive, it is widely used but lacks specificity for NO because nitrite can originate from other metabolic pathways.

Oxyhemoglobin/Methemoglobin Assay

•Based on spectroscopic changes when NO reacts with oxyhemoglobin to form methemoglobin.
•Can provide more direct inference of NO but is sensitive to interference from other redox changes in plant extracts.

2. Fluorescent Probes

DAF-Based Probes

•DAF-2 DA (4,5-diaminofluorescein diacetate) and variants (e.g., DAF-FM DA) are cell-permeable fluorescent dyes that form highly fluorescent products in the presence of NO (via N₂O₃ intermediates).
•These allow real-time imaging and spatial localization of NO in living cells.

Limitations

•DAF dyes can respond to other reactive species and cellular conditions, leading to artefacts (Ruemer et al., 2016).
•Researchers have developed alternative fluorescent probes (e.g., the MNIP-Cu probe) with improved specificity for NO (Jain et al., 2016).

3. Electrochemical Approaches

NO-Selective Electrodes

•Electrodes detect NO based on its electrochemical oxidation or reduction.
•Provide real-time quantitative measures but can be invasive and technically demanding, especially for small tissues (Wishwakarma et al., 2019).

4. Spectroscopic Techniques

Electron Paramagnetic Resonance (EPR)

•EPR detects unpaired electrons using spin-trapping agents to stabilize the otherwise transient NO radical.
•Offers high specificity, but requires specialized equipment and expertise (Wishwakarma et al., 2019).

Laser Photoacoustics and Mass Spectrometry

Methods such as membrane inlet mass spectrometry (MIMS) and quantum cascade lasers permit highly sensitive NO detection in the gas phase (Wishwakarma et al., 2019).

5. Gas-Phase Detection

Chemiluminescence

•A sensitive technique where NO reacts with ozone, emitting light proportional to NO concentration.
•Considered a gold standard for quantification, capable of detecting NO at very low levels (Wishwakarma et al., 2019).

6. Reporter and Genetic Approaches

•Plants lack broadly adopted genetically encoded NO reporters comparable to calcium or ROS sensors, though NO sensor proteins (e.g., Lb²⁺NO) show promise for sensitive, spatiotemporal detection under appropriate promoters (Safavi-Rizi 2021).
•Genetic manipulation or use of NO-responsive gene expression markers can support physiological interpretation but do not provide direct NO quantification (Lindermayr and Durner, 2018; Safavi-Rizi 2021).

7. NO-Sensitive Nanosensors

NO-specific nanosensors—particularly SWCNT-based sensors such as SWCNTDAP-dex (Kim et al., 2009)—offer a promising solution because they can detect NO in real time, non-destructively, and within specific cellular compartments. Although this technology has so far been demonstrated in only a few studies, it shows strong potential to quantitatively monitor plant NO signaling and translate chemical signals into digital data (Kolbert et al., 2021).

Summary of Strengths & Limitations

Given the limitations of individual methods, multimethod approaches are increasingly recommended. For example, pairing fluorescent imaging with chemiluminescence or EPR helps confirm that signals truly represent NO.

Method	Strengths	Limitations
Griess assay	Simple, low cost	Indirect; vulnerable to artifacts
DAF fluorescence	Spatial imaging	Probe specificity issues
Electrochemical sensors	Real-time quantification	Invasive; technical
EPR	Highly specific	Specialized, not routine
Chemiluminescence	Highly sensitive, quantitative	Requires specialized setup
Genetic/Reporter methods	Physiologically informative	Indirect for quantification
Nanosensors	Allows subcellular localization	Lack of standardized protocols

References

Kolbert Zs, Szőllősi R, Feigl G, Kónya Z, Rónavári A Nitric oxide signalling in plant nanobiology: current status and perspectives.. Journal of Experimental Botany 72(3), 928–940 (2021). https://doi.org/10.1093/jxb/eraa470
Safavi-Rizi V Towards genetically encoded sensors for nitric oxide bioimaging in planta.. Plant Physiology 187(2), 477–479 (2021). https://doi.org/10.1093/plphys/kiab232
Vishwakarma A, Wany A, Pandey S et al. Current approaches to measure nitric oxide in plants.. J Exp Bot 70(17), 4333–4343 (2019). https://doi.org/10.1093/jxb/erz242
Jain P, David A, Bhatla SC A Novel Protocol for Detection of Nitric Oxide in Plants.. Methods Mol Biol 1424, 69-79 (2016). https://doi.org/10.1007/978-1-4939-3600-7_7
Ruemer S, Krischke M, Fekete A, Lesch M, Mueller MJ, Kaiser WM Methods to Detect Nitric Oxide in Plants: Are DAFs Really Measuring NO?. Methods Mol Biol 1424, 57-68 (2016). https://doi.org/10.1007/978-1-4939-3600-7_6
Mur LAJ, Mandon J, Cristescu SM, Harren FJM, Prats E Methods of nitric oxide detection in plants: A commentary.. Plant Science 181(5), 509-519 (2011). https://doi.org/10.1016/j.plantsci.2011.04.003
Lindermayr C, Durner J Nitric oxide sensor proteins with revolutionary potential.. Journal of Experimental Botany 69(15), 3507–3510 (2018). https://doi.org/10.1093/jxb/ery193
Kim JH, Heller DA, Jin H, Barone PW, Song C, Zhang J, Trudel LJ, Wogan GN, Tannenbaum SR, Strano MS The rational design of nitric oxide selectivity in single-walled carbon nanotube near-infrared fluorescence sensors for biological detection.. Nature Chemistry 1, 473–481 (2009).
Gupta KJ, Hancock JT, Petrivalsky M et al. Recommendations on terminology and experimental best practice associated with plant nitric oxide research.. New Phytol 225(5), 1828-1834 (2020). https://doi.org/10.1111/nph.16157

Overview of Nitric Oxide Detection Methods in Plants

Method

Strengths

Limitations

Griess assay

Simple, low cost

Indirect; vulnerable to artifacts

DAF fluorescence

Spatial imaging

Probe specificity issues

Electrochemical sensors

Real-time quantification

Invasive; technical

EPR

Highly specific

Specialized, not routine

Chemiluminescence

Highly sensitive, quantitative

Requires specialized setup

Genetic/Reporter methods

Physiologically informative

Indirect for quantification

Nanosensors

Allows subcellular localization

Lack of standardized protocols

References

Kolbert Zs, Szőllősi R, Feigl G, Kónya Z, Rónavári A Nitric oxide signalling in plant nanobiology: current status and perspectives.. Journal of Experimental Botany 72(3), 928–940 (2021). https://doi.org/10.1093/jxb/eraa470

Safavi-Rizi V Towards genetically encoded sensors for nitric oxide bioimaging in planta.. Plant Physiology 187(2), 477–479 (2021). https://doi.org/10.1093/plphys/kiab232

Vishwakarma A, Wany A, Pandey S et al. Current approaches to measure nitric oxide in plants.. J Exp Bot 70(17), 4333–4343 (2019). https://doi.org/10.1093/jxb/erz242

Jain P, David A, Bhatla SC A Novel Protocol for Detection of Nitric Oxide in Plants.. Methods Mol Biol 1424, 69-79 (2016). https://doi.org/10.1007/978-1-4939-3600-7_7

Ruemer S, Krischke M, Fekete A, Lesch M, Mueller MJ, Kaiser WM Methods to Detect Nitric Oxide in Plants: Are DAFs Really Measuring NO?. Methods Mol Biol 1424, 57-68 (2016). https://doi.org/10.1007/978-1-4939-3600-7_6

Mur LAJ, Mandon J, Cristescu SM, Harren FJM, Prats E Methods of nitric oxide detection in plants: A commentary.. Plant Science 181(5), 509-519 (2011). https://doi.org/10.1016/j.plantsci.2011.04.003

Lindermayr C, Durner J Nitric oxide sensor proteins with revolutionary potential.. Journal of Experimental Botany 69(15), 3507–3510 (2018). https://doi.org/10.1093/jxb/ery193

Kim JH, Heller DA, Jin H, Barone PW, Song C, Zhang J, Trudel LJ, Wogan GN, Tannenbaum SR, Strano MS The rational design of nitric oxide selectivity in single-walled carbon nanotube near-infrared fluorescence sensors for biological detection.. Nature Chemistry 1, 473–481 (2009).

Gupta KJ, Hancock JT, Petrivalsky M et al. Recommendations on terminology and experimental best practice associated with plant nitric oxide research.. New Phytol 225(5), 1828-1834 (2020). https://doi.org/10.1111/nph.16157