# Salicylic Acid Modulates Volatile Organic Compound Profiles During CEVd Infection in Tomato Plants
## 4 Discussion  
Our study highlights the distinct symptomatic (Figure 1) and metabolic (Figure 2) responses to _Citrus Exocortis Viroid_ (CEVd) infection of different tomato genotypes with varying levels of SA content: wild-type, SA-deficient (_NalG_), and SA-accumulating (_RNAi_55H_). Our results emphasize the role of VOCs as potential markers of plant defence, providing critical insights into the influence of SA on plant-pathogen interactions and metabolic reprogramming. These findings align with previous research demonstrating the pivotal role of SA in systemic acquired resistance (SAR) against biotrophic and hemibiotrophic pathogens [5,36]. Moreover, they underscore the potential of SA-mediated pathways to enhance defence responses while mitigating extensive metabolic shifts, offering promising strategies to improve plant resilience against pathogens. Within the _NalG_ genotype, which is unable to accumulate SA due to its conversion to catechol [37], a broad over-accumulation of VOCs was observed upon CEVd infection. This included significant increases in terpene compounds, various sesquiterpenes, aldehydes derived from fatty acids, and benzenoids (Figure S1B, Figure 3, column 1). These results suggest that _NalG_ plants appear to be activating an alternative defence mechanism in response to the absence of SA since some of these VOCs have been described to have a direct or indirect role in plant defence [38,39]. Particularly, monotereponoids have been described to play a role in tomato defence against bacterial infection. Specifically, \(\alpha\)-terpineol induces stomatal closure in an SA-independent manner and enhances bacterial resistance in tomato plants [30]. Moreover, JA and ethylene pathways could also be involved in compensating for the absence of SA in _NalG_ plants, which is consistent with their roles in defence against heterotrophic pathogens [40,41]. In this regard, ethylene  
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 _NalG_ (red) and _RNAi_55H_ (green). **(B)** Loading plot derived from Principal Component Analysis with ion–volatile association. production has been described to be dramatically increased in CEVd-infected _NahG_ plants, pointing to the role of this phytohormone in viroid infections [15]. The distinct VOC profile of _NahG_ plants reflects stress-induced metabolic reprogramming due to impaired defence, forcing the plant to adopt less specific defence mechanisms [4; 43; 42]. Interestingly, elevated levels of benzenoids such as guaiacol and eugenol were observed. Catechol detoxification through methylation likely explains guaiacol accumulation [15; 44], while eugenol has been described as a priming agent enhancing virus resistance by stimulating SA and nitric oxide levels, as well as the expression of _SlPer1_ resistance genes [45; 46]. In any case, VOCs forming part of the so-called \"aroma of death\" could be used as biomarkers of the viroid disease, since they are associated with viroid symptomatology. In contrast, _RNAi_55H_ plants, which accumulate elevated SA levels due to the silencing of the SA hydroxylase enzyme (5 SH), displayed a restrained metabolic response to CEVd infection. This was supported by PC2 of the PCA (Figure 2), with fewer differential VOCs detected, particularly among key terpenoids known for their signalling roles (Figure S1C, Figure 3, column 3). The limited metabolic shift observed in _RNAi_55H_ plants reflects an SA-primed state that enables a rapid yet targeted defence response, minimizing resource expenditure and metabolic disruption [36; 47]. These findings align with previous work which demonstrated that enhanced SA accumulation increased CEVd resistance through glycosylated SA [9]. Wild-type plants exhibited an intermediate response to CEVd infection, characterized by moderate changes in VOCs, including \(p\)-cymene and methyl salicate (MeSA) (Table 1).