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.