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Open Access

Short-Term Exposure to Nitrogen Dioxide Provides Basal Pathogen Resistance

Dörte Mayer, Axel Mithöfer, Erich Glawischnig, Elisabeth Georgii, Andrea Ghirardo, Basem Kanawati, Philippe Schmitt-Kopplin, Jörg-Peter Schnitzler, Jörg Durner, Frank Gaupels
Dörte Mayer
Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Axel Mithöfer
Max Planck Institute for Chemical Ecology, Department Bioorganic Chemistry, D-07745 Jena, Germany
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Erich Glawischnig
Department of Plant Sciences, Technical University of Munich, D-85354 Freising, Germany
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Elisabeth Georgii
Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Andrea Ghirardo
Research Unit Environmental Simulation, Institute of Biochemical Plant Pathology, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Basem Kanawati
Analytical BioGeoChemistry, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Philippe Schmitt-Kopplin
Analytical BioGeoChemistry, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Jörg-Peter Schnitzler
Research Unit Environmental Simulation, Institute of Biochemical Plant Pathology, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Jörg Durner
Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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Frank Gaupels
Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
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  • For correspondence: frank.gaupels@helmholtz-muenchen.de

Published September 2018. DOI: https://doi.org/10.1104/pp.18.00704

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  • Figure 1.
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    Figure 1.

    NO2 triggers a rapid and transient defense response. Arabidopsis Col-0 plants were fumigated with 10 µL L−1 NO2 or air for 1 h. A, NO2 caused no visible leaf damage (Supplemental Fig. S1) but a transient increase in red chlorophyll autofluorescence under UV light (white arrows) indicative of stress-induced photoprotective energy dissipation. B, Leaf material was harvested in quadruplicates for microarray analysis immediately or 6 h after fumigation. Volcano plots visualize the changes in gene expression at 0 and 6 h after fumigation by plotting the adjusted P value over the fold change. Horizontal dashed lines mark P = 0.05; vertical dashed lines indicate log2 fold change [log2(FC)] ± 1. Data points represent the expression of individual genes. The expression of genes appearing in the colored left sections was significantly down-regulated [P < 0.05, log2(FC) < −1], whereas the expression of genes within the colored right sections showed significant up-regulation [P < 0.05, log2(FC) > 1]. C, Venn diagrams illustrate the number of genes that were significantly up-regulated (top) or down-regulated (bottom) after NO2 exposure with P < 0.05 and log2(FC) ± 1. The color code is consistent in B and C, indicating genes down-regulated immediately (gray) and 6 h (green) after fumigation or up-regulated immediately (blue) and 6 h (yellow) after fumigation.

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    Figure 2.

    NO2-induced genes are related to pathogen defense. A, GO term enrichment of genes up-regulated directly (0 h) after fumigation. Enriched GO terms (P < 0.05) were identified using the PANTHER 11.0 overrepresentation test and visualized in scatterplots using the REVIGO tool. Each circle represents a GO term, and circle size represents the number of genes encompassed. The color code depicts the fold enrichment of the respective GO term within the data set compared with the PANTHER Arabidopsis reference list. Circles are clustered according to the distance of the respective GO terms within the GO hierarchical tree. Highly enriched or interesting GO terms were labeled. B, Principal component analysis of Arabidopsis gene expression responses to NO2 fumigation, biotic stress, and abiotic stress. Data from microarray analysis after NO2 fumigation were combined with previously published data sets representing responses to different stresses and elicitors (115 samples in total). The overall expression response similarities between samples of the combined data set are visualized using the top two principal components (PC1 and PC2), capturing 22% and 14% of the total variation, respectively. NO2, NO2 fumigation; Bc, B. cinerea infection, ArrayExpress accession number E-GEOD-5684; Ps, P. syringae infection, E-GEOD-6176; Chitin, chitin treatment, E-GEOD-2538; Flg22, flagellin epitope 22 treatment, E-GEOD-17382; AS, abiotic stress treatment study, E-MTAB-4867. For each study, treated samples are marked by triangles and controls by circles.

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    Figure 3.

    NO2 induces resistance against B. cinerea and P. syringae. A, Col-0 plants were fumigated or not (control) with 10 µL L−1 NO2 for 1 h, followed by droplet infection of detached leaves with approximately 1,000 spores of B. cinerea 6 h after fumigation. Necrotic lesion area was measured 3 d later using ImageJ. Columns represent means of 18 independent experiments ± se (n = 624–640). Asterisks indicate significant differences from the control according to the Mann-Whitney rank-sum test (***, P < 0.001). Representative photographs of necrotic lesions 3 d after droplet infection with B. cinerea are shown. Bars = 5 mm. B, Col-0 plants were fumigated with 10 µL L−1 NO2 for 1 h and syringe infiltrated with 1 × 105 cfu mL−1 P. syringae pv tomato DC3000 4 h after fumigation. Leaf discs from infected leaves were obtained 2 h or 1 and 2 d after infection to determine the bacterial titer (cfu cm−2 leaf material). Columns represent means ± se from seven independent experiments (n [2 h post infection] = 26–27, n [1 dpi] = 72, and n [2 dpi] = 66). Asterisks indicate significant differences of all pairwise comparisons via two-way ANOVA plus the Holm-Sidak posthoc test (*, P < 0.05 and ***, P < 0.001); n.s., not significant. White columns, unfumigated; black columns, 10 µL L−1 NO2.

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    Figure 4.

    NO2 induces signaling by SA. SA levels at different time points after fumigation with air or 10 µL L−1 NO2 were measured via LC-MS/MS and normalized to the samples’ fresh weight (FW). Columns represent means ± sd (n = 5). Asterisks indicate significant differences within the time points as determined by two-way ANOVA plus the Holm-Sidak posthoc test (**, P < 0.01 and ***, P < 0.001). White columns, air; black columns, 10 µL L−1 NO2.

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    Figure 5.

    JA biosynthesis and degradation pathways are up-regulated simultaneously in response to NO2. The schematic pathway of jasmonate metabolism illustrates the change in expression levels [log2(FC)] of the respective genes obtained from the microarray analysis immediately (0 h, left part of the colored sections) or 6 h (right part of the colored sections) after fumigation with 10 µL L−1 NO2. Expression levels of all depicted genes can be found in Supplemental Table S1. JAR1, Jasmonate-amido synthetase; JMT, JA-carboxyl methyltransferase; MeJA, methyl jasmonate

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    Figure 6.

    JA degradation products accumulate in response to NO2. Various jasmonates were measured by LC-MS/MS at different time points after fumigation with air or 10 µL L−1 NO2. Concentrations were normalized to the leaf sample fresh weight (FW). A, OPDA. B, JA. C, JA-Ile. D, 12-OH-JA. E, 12-OH-JA-Ile. F, 12-COOH-JA-Ile. A to C show products of the JA biosynthesis pathway, and D to F show JA catabolism products. Columns represent means ± sd (n = 5). Asterisks indicate significant differences within the time points according to two-way ANOVA plus the Holm-Sidak posthoc test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). White columns, air; black columns, 10 µL L−1 NO2.

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    Figure 7.

    SA and JA function in NO2-induced resistance against B. cinerea. Mutants were subjected to B. cinerea droplet infection 6 h after fumigation with 10 µL L−1 NO2 for 1 h. Necrotic areas were measured 3 d later and were normalized to the mean necrotic area of the respective unfumigated wild type. A, SA-deficient (NahG and sid2) or SA-signaling (npr1) mutants and the corresponding Col-0 wild type. Columns represent means of at least three independent experiments ± se (n = 95–331). B, JA-deficient (aos and opr3) or JA-signaling (coi-1) mutants and corresponding wild-types (Col-gl for aos, Wassilewskija [WS] for opr3, and Col-0 for coi-1). Columns represent means of three independent experiments ± se (n = 66–126). Letters indicate significant differences of all pairwise comparisons via the Kruskal-Wallis test plus Dunn’s posthoc test (P < 0.05). White columns, unfumigated; black columns, 10 µL L−1 NO2.

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    Figure 8.

    NO2 exposure induces volatile emissions. A, Emission of the monoterpene α-pinene. B, Emission of the sesquiterpene longifolene. After 1 h of fumigation with 10 µL L−1 NO2, Arabidopsis Col-0 plants were enclosed in a flow-through cuvette system and volatile emissions were collected and analyzed successively by thermal desorption-gas chromatography-mass spectrometry and multivariate data analysis (Supplemental Figs. S5 and S6). Columns represent means ± se (n = 10–12). Significant main effects (NO2 and Time) and interactions (NO2 × Time) are shown (two-way ANOVA, all pairwise multiple comparison Holm-Sidak posthoc test). *, P < 0.05, **, P < 0.01; and n.d., not detected. White columns, control (air); black columns, 10 µL L−1 NO2.

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    Figure 9.

    NO2-induced resistance against B. cinerea is dependent on CYP79B2, CYP79B3, and PAD3 but independent of camalexin. A, The expression of genes related to the biosynthesis of Trp-derived indole glucosinolates and camalexin was strongly up-regulated after fumigation with 10 µL L−1 NO2 for 1 h. Colored areas indicate gene expression [log2(FC)] immediately (left) or 6 h (right) after the NO2 treatment. Genes that were investigated further are highlighted in boldface letters. Gene regulation by transcription factors is indicated by dashed-line arrows. B, The cyp79b2/b3 double mutant and the myb51, cyp81f2, and pad3 mutants were subjected to B. cinerea droplet infection 6 h after fumigation with 10 µL L−1 NO2 for 1 h. Necrotic areas were measured 3 d later and were normalized to the mean necrotic area of the unfumigated Col-0 wild type. Columns represent means of three independent experiments ± se (n = 81–418). Letters indicate significant differences of all pairwise comparisons via the Kruskal-Wallis test plus Dunn’s posthoc test (P < 0.01). C, NO2-exposed or control (unfumigated) Col-0 plants were spray infected with 2 × 105 B. cinerea spores 6 h after fumigation. PAD3 transcript levels were quantified 16, 24, or 48 h after infection relative to housekeeping gene expression via RT-qPCR. Columns represent means of two independent experiments ± sd (n = 5). Letters indicate significant differences of all pairwise comparisons within the time points via two-way ANOVA plus the Holm-Sidak posthoc test (P < 0.01). D, Plants were spray infected with B. cinerea at 6 h after NO2 or air fumigation. Camalexin levels were measured by HPLC-MS 24 and 48 h after infection. Columns represent means ± se (n = 12). Letters indicate significant differences of all pairwise comparisons within the time points via two-way ANOVA plus the Holm-Sidak posthoc test (P < 0.01). FW, Fresh weight.

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    Figure 10.

    Plants impaired in callose formation display a loss in NO2-induced resistance against B. cinerea. A, Col-0 and callose-deficient pmr4 plants were subjected to B. cinerea droplet infection 6 h after fumigation with 10 µL L−1 NO2 for 1 h. Necrotic areas formed on fumigated leaves after 3 d were normalized to the mean necrotic areas of the respective unfumigated leaves. Columns represent means of four independent experiments ± se (n = 135–145). B, Relative necrotic areas determined on Col-0 plants that were infiltrated with 1.2 mm of the callose synthesis inhibitor 2-DDG (water as a control) 24 h before fumigation followed by B. cinerea infection. Columns represent means ± se (n = 70–130). Letters indicate significant differences of all pairwise comparisons via the Kruskal-Wallis test plus Dunn’s posthoc test (P < 0.05). White columns, unfumigated; black columns, 10 µL L−1 NO2.

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    Figure 11.

    NO2 pretreatment enhances early callose deposition upon treatment with the fungal elicitor chitosan. Plants were fumigated with 10 µL L−1 NO2 for 1 h and infiltrated with 500 µg mL−1 chitosan (0.04% acetic acid as a control) 4 h after fumigation. Leaf discs were obtained for callose quantification with Aniline Blue 4 or 16 h after chitosan treatment. A, Callose quantification in Col-0. Columns represent means ± se (n = 34–44 from 10 plants per time point and treatment). A.U., Arbitrary units; hpi, hours after infection. B, Detection of Aniline Blue-stained callose by confocal laser scanning microscopy. Fluorescence and bright-field channels were merged using ImageJ software. Representative photographs were taken of NO2-fumigated or unfumigated Col-0 or pmr4 (right section) 4 h after treatment with chitosan. Bars = 100 μm. C, Callose quantification in mutants impaired in SA synthesis (sid2), SA signaling (npr1), JA signaling (coi1), camalexin synthesis (pad3), and callose deposition (pmr4). Columns represent means ± se (n = 103–159 for Col-0 and pmr4; n = 57–65 for other mutants). White columns, unfumigated; black columns, 10 µL L−1 NO2. C, Infiltration control; E, elicitor chitosan. Letters indicate significant differences of all pairwise comparisons within time points via the Kruskal-Wallis test plus Dunn’s posthoc test (P < 0.05). White columns, unfumigated; black columns, 10 µL L−1 NO2. D, Detection of Aniline Blue-stained callose in NO2-fumigated or unfumigated cyp81f2 and cyp79b2/b3 mutant plants 4 h after treatment with chitosan. Col-0 stained in the same experiment is shown for comparison. Bars = 100 μm.

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    Table 1. CYP79B2/B3-dependent accumulation of metabolites 6 h after fumigation with 10 µL L−1 NO2

    Metabolites were not detected in the cyp79b2/b3 double mutant. NO2-induced up-regulation in wild-type plants is given as fold change of median spectral count. See Supplemental Table S1 for the complete data set, including statistics. Formulae and tentative annotations were deduced from the exact masses as determined by FT-ICR-MS.

    Mass m/z [M-H]−Up-Regulation by 10 µL L−1 NO2Formula [M-H]Tentative Annotation
    MeasuredΔ µL L−1
    447.0537−0.051.8C16H20N2O9S2Glucobrassicin, indol-3-ylmethylglucosinolate
    367.0783±0.003.0C15H16N2O9Unknown CYP79B2/B3-dependent metabolite
    364.1038±0.002.7C17H19NO81,4-Dimethoxyindol-3-ylmethylascorbate
    304.0826−0.042.8C15H15NO6Ascorbigen, indol-3-ylmethylascorbate
    232.0463±0.002.2C8H11NO7Unknown CYP79B2/B3-dependent metabolite

Additional Files

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    Supplemental Figures, Tables, and Data Set

    Files in this Data Supplement:

    • Supplemental Data - Supplemental Figures 1-8
    • Supplemental Data - Supplemental Tables 1 and 2
    • Supplemental Data - Supplemental Data Set
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Short-Term Exposure to Nitrogen Dioxide Provides Basal Pathogen Resistance
Dörte Mayer, Axel Mithöfer, Erich Glawischnig, Elisabeth Georgii, Andrea Ghirardo, Basem Kanawati, Philippe Schmitt-Kopplin, Jörg-Peter Schnitzler, Jörg Durner, Frank Gaupels
Plant Physiology Sep 2018, 178 (1) 468-487; DOI: 10.1104/pp.18.00704

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Short-Term Exposure to Nitrogen Dioxide Provides Basal Pathogen Resistance
Dörte Mayer, Axel Mithöfer, Erich Glawischnig, Elisabeth Georgii, Andrea Ghirardo, Basem Kanawati, Philippe Schmitt-Kopplin, Jörg-Peter Schnitzler, Jörg Durner, Frank Gaupels
Plant Physiology Sep 2018, 178 (1) 468-487; DOI: 10.1104/pp.18.00704
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Plant Physiology: 178 (1)
Plant Physiology
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Sep 2018
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