The Role of a Glucosinolate-Derived Nitrile in Plant Immune Responses

Abstract

Glucosinolates are defense-related secondary metabolites found in Brassicaceae. When Brassicaceae come under attack, glucosinolates are hydrolyzed into different forms of glucosinolate hydrolysis products (GHPs). Among the GHPs, isothiocyanates are the most comprehensively characterized defensive compounds, whereas the functional study of nitriles, another group of GHP, is still limited. Therefore, this study investigates whether 3-butenenitrile (3BN), a nitrile, can trigger the signaling pathways involved in the regulation of defense responses in Arabidopsis thaliana against biotic stresses. Briefly, the methodology is divided into three stages, (i) evaluate the physiological and biochemical effects of exogenous 3BN treatment on Arabidopsis, (ii) determine the metabolites involved in 3BN-mediated defense responses in Arabidopsis, and (iii) assess whether a 3BN treatment can enhance the disease tolerance of Arabidopsis against necrotrophic pathogens. As a result, a 2.5 mM 3BN treatment caused lesion formation in Arabidopsis Columbia (Col-0) plants, a process found to be modulated by nitric oxide (NO). Metabolite profiling revealed an increased production of soluble sugars, Krebs cycle associated carboxylic acids and amino acids in Arabidopsis upon a 2.5 mM 3BN treatment, presumably via NO action. Primary metabolites such as sugars and amino acids are known to be crucial components in modulating plant defense responses. Furthermore, exposure to 2.0 mM 3BN treatment began to increase the production of salicylic acid (SA) and jasmonic acid (JA) phytohormones in Arabidopsis Col-0 plants in the absence of lesion formation. The production of SA and JA in nitrate reductase loss-of function mutant (nia1nia2) plants was also induced by the 3BN treatments, with a greater induction for JA. The SA concentration in nia1nia2 plants was lower than in Col-0 plants, confirming the previously reported role of NO in controlling SA production in Arabidopsis. A 2.0 mM 3BN treatment prior to pathogen assays effectively alleviated the leaf lesion symptom of Arabidopsis Col-0 plants caused by Pectobacterium carotovorum ssp. carotovorum and Botrytis cinerea and reduced the pathogen growth on leaves. The findings of this study demonstrate that 3BN can elicit defense response pathways in Arabidopsis, which potentially involves a coordinated crosstalk between NO and phytohormone signaling.

Keywords: glucosinolates; metabolomics; nitriles; plant innate immunity; secondary metabolites; transcriptomics.

FIGURE 1

3BN treatment setup and response in Arabidopsis col-0 (wild-type). (A) Three weeks old Arabidopsis (wild-type) plants were separately treated with different concentrations of 3BN in closed containers for 24 h. (B) The lesion phenotype was observed for 2.5 mM 3BN treatment (red arrows) and severe necrosis was observed for 3BN concentrations ≥ 5.0 mM. Photographs were taken 2 days post-3BN treatment.

FIGURE 2

Comparison of physiological and biochemical effects of 2.5 mM 3BN treatment between Arabidopsis Col-0 wild-type and nia1nia2 mutant. (A) Signs of lesion formation (red arrows) were spotted on the wild-type leaves upon 3BN treatment. Photographs were taken 2 days post-3BN treatment. (B) Trypan blue staining revealed the presence of dark-blue stained spots, indicating lesion formation in Col-0 wild-type but not nia1nia2 leaves upon 3BN treatment. Lesion was visualized on rosette leaves 2 days post-3BN vapors treatment.

FIGURE 3

Electrolyte leakage analysis of Arabidopsis Col-0 wild-type and nia1nia2 mutant when subjected to different concentrations of 3BN treatments. This analysis was carried out 2 days post-3BN treatment. 2.5 mM 3BN treatment caused significant electrolyte leakage in wild-type but not in nia1nia2 mutant. Values for electrolyte leakage are the average (error bars indicate SD) of six plants. Stars indicate a statistically significant difference (One-way ANOVA, **P < 0.01, ***P < 0.001) to the control treatment.

FIGURE 4

An overview of microarray analysis results for Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.5 mM 3BN treatment for 24 h. (A) The comparison of the number of differentially expressed genes (DEGs) under the 3BN treatment between Col-0 and nia1nia2. (B) GO enrichment analysis of DEGs in Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.5 mM 3BN treatment. Enrichment of monosaccharide metabolic process and fatty acid beta-oxidation was uniquely found in the upregulated genes of the wild-type plants, while enrichment of carbohydrate metabolic process and sulfur compound metabolic process was uniquely found in the upregulated genes of the mutant plants.

FIGURE 5

An overview of metabolite profiling data for Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.5 mM 3BN treatment for 24 h. PCA plot depicting the metabolites with variable contents between any combination of paired samples.

FIGURE 6

Metabolite profiling of soluble sugars (including sorbitol, a sugar alcohol) for Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.5 mM 3BN treatment for 24 h. For sucrose, One-way ANOVA and Tukey post hoc test were used with different letters denoting a significant difference (P < 0.05). For glucose, fructose and sorbitol, where the homogeneity of variance assumption could not be fulfilled, Welch one-way test and pairwise t-test were used with different letters denoting a significant difference (P < 0.05).

FIGURE 7

Metabolite profiling of Krebs cycle-associated carboxylic acids for Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.5 mM 3BN treatment for 24 h. One-way ANOVA and Tukey post hoc test with different letters denoting a significant difference (P < 0.05).

FIGURE 8

Metabolite profiling of amino acids for Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.5 mM 3BN treatment for 24 h. One-way ANOVA and Tukey post hoc test with different letters denoting a significant difference (P < 0.05).

FIGURE 9

Phytohormone profiling of Arabidopsis Col-0 wild-type and nia1nia2 mutant upon 2.0 and 2.5 mM 3BN treatments for 24 h. (A) SA, (B) JA, and (C) ABA (t-test, *P < 0.05, n = 3 per sample).

FIGURE 10

Pathogen assays of Arabidopsis Col-0 with the necrotrophic bacterium Pectobacterium carotovorum ssp. Carotovorum (Pcc). (A) Arabidopsis Col-0 was challenged with the bacterium after having been exposed to a mock or 2.0 mM 3BN treatment for 24 h. (B) Representative pictures of plants exposed to a mock or 2.0 mM 3BN treatment for 24 h before pathogen inoculation. (C) Representative pictures of rosette leaves showing lesions post 16 h of inoculation with Pcc. (D) The lesion area at 16 h post-infection (t-test, ***P < 0.001, n = 32 leaves per treatment, with four batch data). (E) The bacterial population estimated by CFU at 16 h post-infection (t-test, ***P < 0.001, n = 32 leaves per treatment, with three batch data).

FIGURE 11

Pathogen assays of Arabidopsis Col-0 with the necrotrophic fungus Botrytis cinerea (Bc). (A) Arabidopsis Col-0 was challenged with the fungus after having been exposed to a mock or 2.0 mM 3BN treatment for 24 h. (B) Representative pictures of plants exposed to a mock or 2.0 mM 3BN treatment for 24 h before pathogen inoculation. (C) Representative pictures of rosette leaves showing lesions post 48 h of inoculation with Bc. (D) The lesion area at 48 h post-infection (t-test, **P < 0.01, n = 48 leaves per treatment). (E) Quantification of Bc growth at 48 h post-infection estimated by relative transcript levels of BcActin and AtActin using qRT-PCR (t-test, **P < 0.01, n = 5 plants per treatment).