Discussion
DAMPs recognition and signalling is one of the earliest events of the
plant and animal immune system (Heil and Land 2014). The relevance of
DAMPs signalling in the immune responses has received increasing
attention in the last years (Gust et al., 2017; De Lorenzo et al., 2018)
but our mechanistic and functional understanding of the process in
plants is still very limited. Oligogalacturonides are DAMPs derived from
the plant cell wall, and the response they trigger ha been mainly
studied in Arabidopsis, at the local level (Gravino et al., 2017,
Davidsson et al., 2017). In this study we investigated how tomato plants
respond, both locally and systemically, to the perception of OGs in
roots and shoots, and whether the systemic response to OGs confers
resistance against B. cinerea .
Plant responses to OGs have been previously shown to be mediated by
hormone signalling. For example, JA mediates some responses to OGs in
tomato (Doares et al., 1995) and in Arabidopsis (Ferrari et al., 2003;
Denoux et al., 2008; Davidsson et al., 2017). Our time-course analysis
of hormone in tomato plants after OG recognition reveals a complex
regulation pattern local and systemic during the 24 hours following OG
application in roots or shoots. Hormone quantification complemented with
gene expression analysis of related biosynthetic genes show the
involvement of the JA, ABA and ET signalling pathways in the response to
OGs. The activation of these pathways in response to these damage
signals is in agreement with their reported regulation during the tomato
wound responses (Tian et al., 2014). JA signalling was activated in
leaves and roots upon leaf treatment, and ET biosynthesis was activated
as a local response to OGs in both roots and leaves. The ABA pathway was
also altered, but changes occurred mostly in roots. The speed and
magnitude of the responses depended on the organ that perceived OGs.
Fast and transient hormone changes locally in the OGtreated leaves, with
a maximum at 1 hpt, while systemic hormone changesin both leaves and
roots of leatreated plants were more pronounced at 6 hpt. Notably,
systemic hormone changes appeared stronger than local ones, suggesting
that, after a fast and transient local response, the plant allocates its
resources to defend the undamaged distal tissues.
Remarkably, the most conspicuous response was observed in the roots of
leaf-treated plants. Roots have been shown to be key regulators of plant
defence responses to aboveground challenges: for example, fast changes
occur in roots upon foliar herbivory leading to the synthesis of
antiherbivore compounds as alkaloids (Erb et al., 2009, 2012; Agut et
al., 2016). Our untargeted metabolomic analysis 6 hpt also revealed
stronger metabolic responses to OGs in roots than in leaves, as the
proportion of identified compounds that are more accumulated after OG
treatment was higher in roots than in leaves both as a local or systemic
response.
A more detailed analysis of the changes in the metabolomics profile
showed accumulation of phenylpropanoid compounds such as lignans and
flavonoids in response to OGs. Lignans accumulated only in roots both as
a local or systemic response. In contrast, flavonoids accumulated in
roots only as a local response to OGs, whereas they accumulated also in
leaves as both a local and a systemic response to leaf treatment.
Flavonoids were already shown to be synthesized in Arabidopsis roots
after OG perception (Hernandez-Mata et al. 2010). Since flavonoids are
transported through the plants (Petrussa et al., 2013), their presence
in the leaves in the absence of significant induction of the flavonoid
biosynthetic genes (Table 3) may be explained by their biosynthesis in
roots, followed by vascular transport to the distal parts of the plants.
This hypothesis is also supported by the strong induction ofCHI1 , CHS1 and PAL -all of them involved in the
biosynthesis of phenylpropanoids- in the roots after local or distal OG
treatments.
We also found accumulation of tropane alkaloids as a local response in
roots and a systemic response in leaves, but only upon root treatment.
Indeed, the putatively identified anatalline, a JA-inducible tropane,
piperidine and pyridine alkaloid (Hakkinen et al., 2004) shows elevated
levels in roots and leaves of root treated plants, but no change upon
leaf treatment. In agreement with these elevated levels, roots displayed
a strong increase in PMT gene expression –coding for a key
enzyme of the tropane alkaloid biosynthesis pathway- as both a local and
systemic response to OGs. Interestingly, PMT gene expression in
leaves was non-detectable, supporting the notion that roots are
responsible for the synthesis of tropane alkaloids, which can be later
transported systemically. The synthesis of tropane alkaloids in roots
has been reported (Kohnen-Johannsen and Kayser 2019), and reciprocal
grafting experiments show that the alkaloid patterns in leaves of
solanaceae species are determined by the rootstock rather than the
foliage (Bais et al., 2001).
Here we show that the responses observed upon OG treatments are
biologically relevant for defence in tomato, since they confer
resistance against the necrotrophic pathogen B. cinerea . So far
only one report, in Arabidopsis, describes systemic protection against
this fungus, with no further mechanistic study (Ferrari et al., 2007).
Most of the knowledge relates instead to the protection induced by local
elicitation with OGs (Aziz et al., 2004, Ferrari et al., 2007, Galletti
et al., 2008 and Galletti et al., 2011). Here we show that OG
pre-treatment in tomato leaves or roots 6 h before pathogen inoculation
confers efficient systemic protection against B. cinerea but,
unexpectedly, not local protection, even at higher doses. Thus, our
study clearly reveals striking differences between local and systemic
defence/resistance responses in tomato.
Besides the potentially fungicide compounds systemically accumulated in
OG treated plants, such as the alkaloids found in leaves of root-treated
plants, we also looked for other potential players that may contribute
to the observed OG-systemic induced resistance against B.
cinerea . We explored the activity or gene expression levels of the
antimicrobial PR proteins β-1,3-glucanase (GluB) (van Kan et al., 1992)
and leucyl aminopeptidase A (LAP A). We found that expression of was
higher in the tissues showing increased resistance to B. cinerea(RT-SL and LT-SL), supporting its possible role in OGs-induced systemic
resistance. It is worth noting that high doses of OGs (500 µg/mL) have
been shown to induce glucanase activity in grapevine cells (Aziz et al.,
2004) and that GluB gene expression is up-regulated in theSolanum lycopersicoides Botrytis interaction (Smith et al.,
2014). LAP-A is a JA-inducible enzyme that plays a key role in tomato
plant responses towards biotic attack (Fowler et al., 2009). LAP
activity increased upon leaf treatment only in systemic leaves and not
in the treated ones. Thus, the induction of both LAP activity andGLUB expression in systemic leaves, but not in the OG-treated
leaves correlates with the systemic induced resistance observed.
In summary, we show that in tomato, responses to OGs involve the
regulation of JA, ABA and ET signalling pathways, and the activation of
main metabolic pathways for the biosynthesis of antimicrobial
metabolites such as alkaloids, flavonoids and lignans. Most of them are
likely synthesized in the roots, even when OGs are applied in leaves.
Indeed, our wide analyses highlight the key role of roots in
coordinating systemic responses in plants. Finally, we show that OGs
trigger the production in distal leaves of defence-related lytic enzymes
that in the OG-triggered enhanced systemic resistance against
pathogens.
Overall, our work reveals the complexity of the plant responses to
damage perception, showing that, upon OG treatment, defence responses
are triggered throughout the plant, but they differ depending on the
site of DAMP application (summarized in Figure 6 a&b). Our results are
in agreement with the hypothesis of Tytgat and coworkers (2013) pointing
that roots and aerial organs can activate different signalling cascades,
thus contributing information about the site of induction. This would
provide plants with a mechanism to fine-tune defence responses according
to the damaged organ. The observation that systemic responses are
stronger than local ones in both roots and shoots suggests that plants
invest more resources in preparing distal tissues for efficient defence
activation against a potential upcoming attack (Gomez et al., 2010;
Steinbrenner et al., 2011; Kundu et al., 2018).
Understanding the spatio-temporal regulation of plant responses to DAMPs
is essential for our knowledge on how plants integrate danger signals
and shape the appropriate defence responses. Moreover, this research
paves the way for optimal biotechnological application of natural
elicitors such as OGs for sustainable crop protection.