Measuring NO Production by Plant Tissues and Suspension Cultured Cells Vitecek Jan , Reinohl Vilem , Jones Russell L.   Molecular Plant  Volume 1, Issue 2, Pages 270-284 (March 2008) DOI: 10.1093/mp/ssm020 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Diagram of the Oxidizer Column Set-Up for Detecting HNO2/NO2 and NO. The chemistry of the reactions that occur in the system is also shown. HNO2 or NO2 is captured in the first trap in the form of nitrite, whereas NO enters the oxidizer column, where it is converted to NO2 and captured in the second trap. Both traps contain Griess reagent to convert nitrite formed to an intensely colored azo dye. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Characteristics of the NO Detection System. (A) Influence of airflow rate at a constant NO concentration of 9.9 ppm on the ratio of nitrogen equivalents captured. (B) Influence of NO concentration at a constant flow rate of 40 ml min−1on the ratio of nitrogen equivalents captured. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Validation of the Detection System. (A) Production of HNO2and NO by tobacco (N. benthamiana) leaves infiltrated with 50 mM KNO3. (B) Production of HNO2 and NO after addition of nitrite to the incubation medium (pH ∼ 4.0) from barley aleurone layers. (C) Production of NO by barley aleurone tissue treated with 50 mM KNO3 under aerobic conditions. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Endogenous Production of NO by Intact Arabidopsis Leaves. Freshly detached leaves from WT, and mutants Atnox1 (NO overprod) and Atnos1 ( NOS knockout), were assayed for NO production in our oxidizer set-up for 1 h. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Production of NO by Tobacco (N. tabacum) Cell Suspension Cultures Treated with 50 nM Cryptogein Compared to Control Water-Treated Cells. (A) Using the oxidizer column approach. (B) Detection with DAF-FM: mock (▪), cryptogein (▴), representative data from three measurements are shown. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 In Vitro Detection of NO with DAF-FM. (A) Time course of the reaction of 1 μM DAF-FM with 190 nM NO (▪) and 380 nM NO (▴). (B) The effect of pH of the reaction mixture on fluorescence from 1 μM DAF-FM incubated with 300 nM NO for 1 h. Background fluorescence (♦), total fluorescence in the presence of NO (▪). (C) The effect of MES (pH 5.8, ♦), MOPS (pH 7.0,▪), and phosphate (pH 7.0, ▴) in the reaction mixture on net fluorescence from 1 μM DAF-FM incubated with 300 nM NO for 1 h. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 Sensitivity of DAF-FM Fluorescence to Nitrite. Fluorescence from 1 μM DAF-FM incubated with 0.5 μM nitrite in water (pH ∼ 5.6, ♦), 10 mM phosphate buffer (pH 2.5, ▪), 10 mM HCl (pH 2.0, ▴) and 100 mM HCl (pH 1.0,×), and 100 μM nitrite in water (pH ∼ 5.6, ○ dashed line). Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 8 Effect of PTIO on the Kinetics of the Reaction of NO with DAF-FM and the Fluorescence of DAF-FM-T In Vitro. (A) 1 μM DAF-FM and 300 nM NO in the presence of 0 (♦), 5 (▪), and 100 (▴) μM PTIO. (B) Fluorescence of DAF-FM-T in the presence of PTIO. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 9 In-Vivo Fluorescence from Barley and Tobacco Tissue Loaded with DAF-FM DA. Phase contrast (A) and fluorescence (B–D) images of barley aleurone cells loaded with DAF-FM DA (B–D), and incubated in CaCl2 (B), 500 μM DEANO for 5 min (C) and 10 μM FDA (D). Phase contrast (E), and fluorescence (F–H) images of tobacco epidermis incubated in H2O (F), 50 nM cryptogein for 8 min (G), and 10 μM fluorescein diacetate (H). al, aleurone cell; c, cytoplasm; g, guard cell; n, nucleus; p, plastid; sa, subaleurone cell. Bars indicate 50 μm. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Scheme 1 Scheme for Reactions Leading to the Detection of NO with DAF: Formation of Fluorescent DAF Triazole (DAF-T). Adapted from data in Kojima et al. (1998). Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Scheme 2 Summary of the Most Critical Reactions for DAF-FM Nitrosation NO undergoes oxidation to NO2which, in turn, reacts with another molecule of NO to form N2O3which is capable of nitrosating several substrates including DAF-FM (Koppenol, 1998). Dinitrogen trioxide N2O3 may also undergo hydrolysis accompanied by NO2– formation, which can compete in nitrosation reactions. The NO scavenger PTIO (cPTIO) may facilitate formation of N2O3 by increasing the rate of NO oxidation (Arita et al., 2006). PTIO (cPTIO) can also open an alternative pathway (bold arrows), as it may be oxidized with NO2 (Goldstein et al., 2003). The oxidized form of PTIO or cPTIO (PTIO+ or cPTIO+) could convert DAF-FM to its free radical form (DAF-FM+•), which can readily react with NO (Jourd'heuil, 2002). Our data indicate the critical importance of hydroxyl anions in hydrolysis of N2O3because increasing pH decreases the effectiveness of detection of NO by DAF-FM. Nitrite may be protonated to HNO2 at physiologically relevant pH. Nitrous acid may produce N2O3 (Grossi and Montevecchi, 2002; Lancaster Jr, 2003), or may undergo reduction to nitric oxide (Yamasaki, 2000), thereby recycling nitrite and increasing the sensitivity of NO detection. Molecular Plant 2008 1, 270-284DOI: (10.1093/mp/ssm020) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions