Effect of Benzothiadiazole on the Metabolome of Tomato Plants Infected by Citrus Exocortis Viroid

Abstract

Benzothiadiazole (BTH) is a functional analogue of the phytohormone salycilic acid (SA) involved in the plant immune response. NahG tomato plants are unable to accumulate SA, which makes them hypersusceptible to several pathogens. Treatments with BTH increase the resistance to bacterial, fungal, viroid, or viral infections. In this study, metabolic alterations in BTH-treated Money Maker and NahG tomato plants infected by citrus exocortis viroid (CEVd) were investigated by nuclear magnetic resonance spectroscopy. Using multivariate data analysis, we have identified defence metabolites induced after viroid infection and BTH-treatment. Glycosylated phenolic compounds include gentisic and ferulic acid accumulated in CEVd-infected tomato plants, as well as phenylalanine, tyrosine, aspartate, glutamate, and asparagine. Besides, an increase of γ-aminobutyric acid (GABA), glutamine, adenosine, and trigonelline, contributed to a clear discrimination between the metabolome of BTH-treated tomato leaves and their corresponding controls. Among them, GABA was the only metabolite significantly accumulated in both genotypes after the chemical treatment. In view of these results, the addition of GABA was performed on tomato plants infected by CEVd, and a reversion of the NahG hypersusceptibility to CEVd was observed, indicating that GABA could regulate the resistance to CEVd induced by BTH.

Keywords: BTH; GABA; NMR; NahG plants; defence; metabolomics; tomato; viroid.

Figure 1

Chemical structures of BTH (A) and hydrolyzed BTH (B).

Figure 2

Multivariable data analysis based on the ¹H-NMR signals, in the range of 0.3–10.0 ppm, of the Money Maker (MM) and NahG tomato plants after viroid inoculation and BTH treatments at 3 weeks post infection (green; mock tomato leaves, yellow; tomato leaves infected by CEVd, red; leaves after BTH treatments blue; leaves after H₂O treatments). (A) Score plot of partial least square (PLS) (B) Score plot of orthogonal projection to latent structures-discriminant analysis (OPLS-DA) of the infection (R² = 0.95, Q² = 0.82). (C) Score plot of OPLS-DA of chemical treatment (R² = 0.99, Q² = 0.93). (D) Shared-and-unique-structures (SUS) plot correlating the two OPLS-DA models with the Y axis as CEVd infection and X axis as BTH treatment (¹H-NMR signals of GABA in red full circles and ¹H-NMR signals of hydrolyzed BTH in red open circles).

Figure 3

The metabolic inter-relationship between plant metabolism of malate, aspartate, GABA and glucose. 1: Citrate synthase (mCS), 2: Glutamate dehydrogenase (GDH), 3: Glutamate decarboxylase (GAD), 4: Glutamine synthetase (GS), 5: Aspartate aminotransferase (AST), 6: Fumarase (FH).

Figure 4

Expression levels of the tomato citrate synthase (mCS), glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), glutamine synthetase (GS), aspartate aminotransferase (AST), and fumarase (FH) genes in non-treated and BTH-treated (BTH) Money Maker and NahG mock tomato plants at 2.5 wpi, determined by real-time qRT-PCR analysis. Values were first normalized to the actine expression level. Expression levels are represented as mean ± standard error of three biological repetitions. An ANOVA analysis was performed for each gene, among the four samples (MM-non-treated, MM-BTH, NahG-non-treated and NahG-BTH). Different letters indicate the statistical significance differences among the four samples with p-value < 0.05.

Figure 5

Score plot of principal component analysis (PCA) based on the characteristic ion of the mass spectra from the primary metabolites measured in the m/z range 35–900, of the Money Maker (MM) and NahG tomato plants after at 48 h after BTH treatments (green; leaves after BTH treatments; blue; leaves after H₂O treatments).

Figure 6

Disease development in Money Maker and NahG plants infected with CEVd and treated with BTH (red), GABA (green) or water (blue). Evolution of tomato plants showing symptoms at the indicated weeks post inoculation. Data correspond to one representative experiment.

Figure 7

Growth of (A) Money Maker and (B) NahG plants following treatment with GABA or BTH and CEVd inoculation. Representative phenotype observed un infected plants 3 weeks after CEVd inoculation, and in equivalent plants pre-treated with 1 mM GABA or 1 mM BTH.

Figure 8

Expression levels of the tomato PAL, PR1 and P23 genes in Money Maker and NahG tomato plants, non-treated (CONTROL) and 48 h after BTH or GABA treatments. Values were first normalized to the actin expression level. Expression levels are represented as mean ± standard error of three biological repetitions. Asterisk (*), double asterisks (**) and triple asterisks (***) indicate significant differences between chemically-treated and non treated plants with p < 0.05, p < 0.01 and p < 0.001, respectively.