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.