Short-Term Exposure to Nitrogen Dioxide Provides Basal Pathogen Resistance

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.

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.

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.

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

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.

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.

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.

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.

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.