Salicylic Acid Modulates Volatile Organic Compound Profiles During CEVd Infection in Tomato Plants

Abstract

Background:Citrus Exocortis Viroid (CEVd) is a non-coding RNA pathogen capable of infecting a wide range of plant species, despite its lack of protein-coding ability. Viroid infections induce significant alterations in various physiological and biochemical processes, particularly impacting plant metabolism. This study shows the metabolic changes upon viroid infection in tomato plants (Solanum lycopersicum var. 'MoneyMaker') exhibiting altered levels of salicylic acid (SA), a key signal molecule involved in the plant defence against this pathogen. Methods: Transgenic RNAi_S5H lines, which have the salicylic acid 5-hydroxylase gene silenced to promote SA accumulation, and NahG lines, which overexpress a salicylate hydroxylase to degrade SA into catechol and prevent its accumulation, were used to establish different SA levels in plants, resulting in varying degrees of resistance to viroid infection. The analysis was performed by using gas chromatography-mass spectrometry (GC-MS) to explore the role of volatile organic compounds (VOCs) in plant immunity against this pathogen. Results: Our results revealed distinct volatile profiles associated with plant immunity, where RNAi_S5H-resistant plants showed significantly enhanced production of monoterpenoids upon viroid infection. Moreover, viroid-susceptible NahG plants emitted a broad range of VOCs, whilst viroid-tolerant RNAi_S5H plants exhibited less variation in VOC emission. Conclusions: This study demonstrates that SA levels significantly influence metabolic responses and immunity in tomato plants infected by CEVd. The identification of differential emitted VOCs upon CEVd infection could allow the development of biomarkers for disease or strategies for disease control.

Keywords: citrus exocortis viroid (CEVd); gas chromatography–mass spectrometry (GC-MS); metabolomics; salicylic acid; tomato plant defence; volatile organic compounds (VOCs).

Figure 1

Disease development of representative MoneyMaker (WT), NahG, and RNAi_S5H tomato plants 21 days after CEVd infection. (A) Phenotype of CEVd-infected tomato plants. (B) Height of mock-treated and CEVd-infected tomato plants. Error bars represent the standard error of the mean. The letters show the grouping information using Tukey’s range test (one-way ANOVA method) with a significance level of 5% (p-value < 0.05).

Figure 2

Principal Component Analysis (PCA) score plot based on the whole array of the mass spectra within an m/z range from 35 to 250 based on Pareto scaling. Samples included NahG, RNAi_S5H, and the genetic background ‘MoneyMaker’ (WT) plants, either infected with CEVd, including CEVd_NahG (full red circles), CEVd_S5H (full orange triangles), or CEVd_WT (full dark-green diamonds), or mock non-inoculated with Mock_NahG (open pink circles), Mock_S5H (open blue triangles), or Mock_WT (open green diamonds).

Figure 3

Hierarchical cluster heatmap of the VOCs in CEVd-infected tomato plants across genotypes. The log2-transformed ratios are represented as a heatmap according to the scale below. Red corresponds to higher values; green denotes lower values. Column 1 represents the ratios of the VOCs accumulated by CEVd-infected NahG tomato plants versus the mock-inoculated plants. Column 2 represents the ratios of the VOCs accumulated by CEVd-infected WT tomato plants versus the mock-inoculated plants. Column 3 represents the ratios of the VOCs accumulated by CEVd-infected RNAi_S5H tomato plants versus the mock-inoculated plants. * Tentative identification based on mass spectrum.

Figure 4

Relative accumulation levels of selected VOCs in tomato plants of different genotypes (WT, RNAi_S5H, and NahG) after 21 days of CEVd infection. (A) Relative quantification of nonanal, α-pinene, 3-carene, and α-terpinene, characteristic of both the aroma of death and resistance; (B) relative quantification of linalool oxide, octanal, guaiacol, and D-limonene, specific to the scent of death; and (C) relative quantification of β-myrcene and methyl salicylate, specific to the aroma of resistance. The accumulation levels were quantified based on the peak ion area corresponding to the VOCs from plants inoculated without the pathogen (mock) and inoculated with the CEVd pathogen. The letters show the grouping information using Tukey’s range test (one-way ANOVA method) with a significance level of 5% (p-value < 0.05).

Figure 4

Relative accumulation levels of selected VOCs in tomato plants of different genotypes (WT, RNAi_S5H, and NahG) after 21 days of CEVd infection. (A) Relative quantification of nonanal, α-pinene, 3-carene, and α-terpinene, characteristic of both the aroma of death and resistance; (B) relative quantification of linalool oxide, octanal, guaiacol, and D-limonene, specific to the scent of death; and (C) relative quantification of β-myrcene and methyl salicylate, specific to the aroma of resistance. The accumulation levels were quantified based on the peak ion area corresponding to the VOCs from plants inoculated without the pathogen (mock) and inoculated with the CEVd pathogen. The letters show the grouping information using Tukey’s range test (one-way ANOVA method) with a significance level of 5% (p-value < 0.05).

Figure 5

(A) Principal Component Analysis (PCA) score plot based on the normalized array of the mass spectra within an m/z range from 35 to 250 based on Pareto scaling. CEVd-infected NahG (red) and RNAi_S5H (green). (B) Loading plot derived from Principal Component Analysis with ion–volatile association.