OG perception modulates tomato plant metabolism in an organ-specific manner
In order to characterize the responses elicited by OGs in tomato, we decided to carry out a non-targeted metabolomic analysis at 6 hpt, the time point showing the major changes according to the hormone profiles (Figure 1).
The sparse partial least square discriminant analysis (sPLS-DA) in root and shoot samples revealed important differences among organs and treatments (suppl. Fig. 3). The component 1, which explains the highest source of variability, clearly differentiated between root and leaf samples, coherent with specific metabolomic profiles in the different organs. Within leaves, variability was mainly related to the leaf age, since older leaves (4th leaf, chosen for OG local treatment and younger/upper leaves (true sixth leaf, chosen for the analysis of the systemic response displayed very distinctive metabolic profiles, despite of being both adult, fully expanded leaves. Regardless of this difference, responses to OGs in leaves appeared much subtler than responses in roots (suppl. Fig. S3). In roots, local and systemic responses to OGs showed marked differences (suppl. Fig. S3).
A more detailed analysis revealed that, in leaves, OG treatments had a stronger systemic than local impact in the metabolic profile (Figure 2a). The systemic response was also different when OG treatment was applied to leaves or roots. A heatmap analysis was carried out to identify the main features responsible for such differential profiles (Figure 2b). Different clusters showing signals with higher intensity due to OG treatments were selected for signal identification. Cluster 1 was selected to study local responses in leaves (LT-TL) and cluster 2 and 3 were selected for systemic responses: Cluster 2 for systemic responses in leaves after root treatments (RT-SL) and cluster 3 for systemic responses in leaves after leaf treatment (LT-SL). Tentative identification, based on exact mass accuracy and on-line fragmentation spectra of the signals was performed, and the pathways of the identified metabolites that are differentially accumulated in the different treatments are shown in Table 2. Remarkably, while almost 6% and 3.5% of the total detected signals were significantly more accumulated in roots as a local or systemic response to OGs, respectively, only about 2% were significantly more accumulated in the local or systemic responses in leaves.
Regarding the metabolic pathways, local leaf responses to OGs included changes in flavonoid biosynthesis and in porphyrin-chlorophyll metabolism (Table 2). Considering systemic leaf responses to root and leaf treatment, the comparison between cluster 2 (systemic, RT) and cluster 3 (systemic LT) revealed that none of the signals tentatively identified were shared, explaining the difference observed in the sPLS-DA. Systemic leaf responses upon leaf treatment included flavonoid accumulation and changes in purine, amino acid and fatty acid biosynthesis. In contrast, systemic leaf responses to root treatment mostly involved tropane alkaloids, although it also impacted amino acids metabolism (Table 2). Remarkably, two of the three putative identified amino acids belong to the arginine and proline metabolism, known precursors of the tropane alkaloids biosynthesis.
In roots, the impact of OGs on the tomato metabolome is much stronger compared to that in leaves (Figure 3a). The heat-map analysis (Figure 3b) allowed us to pinpoint the cluster R1, corresponding to metabolites that strongly accumulated as a root local response to OG (RT-Root), and the cluster R2, corresponding to metabolites that accumulated in roots as a systemic response to leaf treatment (LT-Root). Tentative identification of the features in cluster R1 revealed that roots respond to OGs by accumulating flavonoids, alkaloids and lignans, all well-known antimicrobial metabolites (Table 2). In addition, auxin, biotin, lysine and terpenoids-quinone biosynthesis were also accumulated locally in treated roots. Noteworthy, biotin and lysine metabolism are related to the alkaloid biosynthetic pathways (Kegg pathway map00780). As a systemic root response to leaf treatment (cluster R2) lignans also accumulated, but mostly the amino acid metabolism was affected.
In summary, flavonoids accumulation was a common response to OGs in roots and shoots. Alkaloids accumulated only following root treatments, either locally in roots (RT-Root) or systemically in leaves (RT-SL), indicating that roots lead the responses related to alkaloids accumulation in all plant organs. As an example, the alkaloid putatively identified as anatalline accumulated in both roots and leaves of root treated plants (suppl. Fig. S4), but did not change upon leaf treatment. On the other hand, lignans exclusively increase in roots both as local and systemic response, pointing to lignan accumulation as a common root response to OGs, regardless the site of perception. However, signals showing higher changes in the systemic root response were from the primary metabolism, suggesting a reorganization of root metabolism after the perception of danger signals in leaves.