Roots drive oligogalacturonide-induced systemic immunity in tomato

Oligogalacturonides (OGs) are fragments of pectin released from the plant cell wall during insect or pathogen attack. They can be perceived by the plant as damage signals, triggering local and systemic defence responses. Here, we analyse the dynamics of local and systemic responses to OG perception in tomato roots or shoots, exploring their impact across the plant and their relevance in pathogen resistance. Targeted and untargeted metabolomics and gene expression analysis in plants treated with purified OGs revealed that local responses were transient, while distal responses were stronger and more sustained. Remarkably, changes were more conspicuous in roots, even upon foliar application of the OGs. The treatments differentially activated the synthesis of defence-related hormones and secondary metabolites including flavonoids, alkaloids and lignans, some of them exclusively synthetized in roots. Finally, the biological relevance of the systemic defence responses activated upon OG perception was confirmed, as the treatment induced systemic resistance to Botrytis cinerea . Overall, this study shows the differential regulation of tomato defences upon OGs perception in roots and shoots and reveals the key role of roots in the coordination of the plant responses to damage sensing. examined how we , We show that regardless Untargeted metabolomic analysis highlights supporting uncovers Finally, we show that or The results highlight the differences among and systemic responses and their dependence site of signal perception.


| INTRODUCTION
Plants respond to invading organisms triggering fast signalling processes leading to the activation of diverse defence mechanisms. They have adapted their immune system to rely on an early molecular recognition of the potential aggressor, crucial for an efficient defence reaction (Jones & Dangl, 2006). Immune responses are controlled by pattern recognition receptors, and defence signalling starts with the perception of conserved molecules associated to the damaging organism, such as pathogen (or microbe)-associated molecular patterns (PAMPs or MAMPs). Moreover, they can also recognize self-molecules associated to damage, the so-called damage-associated molecular patterns (DAMPs) (Zipfel & Oldroyd, 2017).
DAMPs are damaged-self molecules released from host tissue disruption that act as endogenous danger signals in both animals and plants (Heil & Land, 2014). DAMPs comprise a mixture of molecules from diverse origin such as extracellular nucleotides (eATP, eDNA and eNAD[P]), inducible proteins and fragments of the cell wall (Heil & Land, 2014;Li, Wang, & Mou, 2020). In plants, DAMPs are released from disintegrated cells and are sensed by the pattern recognition receptors of adjacent cells. After the recognition, plants go into an "alarm state" activating signalling cascades and triggering defence responses not only locally, at the damaged tissue, but also in distal tissues that will then be prepared to respond more efficiently to a potential upcoming aggression (Orozco-Cardenas & Ryan, 1999). Local responses to DAMPs involve the generation of H 2 O 2 , MAPKs activation, increased flux of calcium, production of phenylpropanoids and hypomethylation in CpG sites (Barbero, Guglielmotto, Capuzzo, & Maffei, 2016;Duran-Flores & Heil, 2017;Pétriacq, Ton, Patrit, Tcherkez, & Gakière, 2016;Vega-Muñoz, Feregrino-Pérez, Torres-Pacheco, & Guevara-González,-2018). Damage perception also involves cell-to-cell communication to prime distal parts of the plant. Consequently, plants activate a myriad of mobile signals that transmit the alarm state and activate defence responses over long distances. It has been reported that jasmonic acid (JA) signalling mediates some of the systemic responses in tomato plants after DAMPs perception (Sun, Jiang, & Li, 2011). Generation of hydrogen peroxide, accumulation of proteinase inhibitors and other defencerelated proteins are produced in distal leaves upon wounding or application of the peptidic, wound-related hormone systemin in tomato (Orozco-Cardenas & Ryan, 1999;Sun et al., 2011).
Oligogalacturonides (OGs) are among the best characterized plant DAMPs. They are pectin fragments hydrolysed from the cell wall that act as danger signals, triggering a signalling cascade that activates plant immunity (De Lorenzo, Ferrari, Cervone, & Okun, 2018;Ferrari et al., 2013;Savatin, Gramegna, Modesti, & Cervone, 2014). OGs are oligomers of α-1,4-galacturonic acid that are released to the extracellular cell space through the action of polygalacturonases, usually generated during pathogens or insects attack (Benedetti et al., 2015). Exogenous application of OGs induces defence responses in plants when they have a degree of polymerization between 10 and 15 and they have acquired an egg-box conformational state dependent on calcium and sodium (Benedetti et al., 2015;Cabrera, Boland, Messiaen, Cambier, & Van Cutsem, 2008). Short oligomers have been also shown to trigger plant defences, although to a lesser extent than long OGs (Davidsson et al., 2017).
It has been demonstrated that OGs perception stimulates antioxidant systems in plants (Camejo et al., 2012) and the biosynthesis of different antimicrobial enzymes through responses regulated by the main defence related phytohormones: JA, salicylic acid (SA) and ethylene (ET) (Bishop, Pearce, Bryant, & Ryan, 1984;Denoux et al., 2008;Doares, Syrovets, Weiler, & Ryan, 1995;Ferrari et al., 2007;Gravino, Savatin, Macone, & De Lorenzo, 2015). These hormonal signalling pathways play a key regulatory function in the interaction of plants with potential aggressors as pathogens and herbivores (Pieterse et al., 2014). Therefore, the modulation of these pathways by OGs would likely have a relevant impact in these biotic interactions.
The ability of OGs to induce defence responses in plants stimulated the scientific community to study the potential of OGs for plant protection. In grape, pre-incubation of excised leaves with OGs leads to protection against the necrotrophic pathogen Botrytis cinerea (Aziz, Heyraud, & Lambert, 2004), and protection was also achieved in Arabidopsis by spray-application of OGs (Ferrari et al., 2007;Galletti et al., 2008). Moreover, in-vivo production of bioactive OGs oligomers in Arabidopsis boosts plant defences and induces resistance to necrotrophic and biotrophic pathogens (Benedetti et al., 2015). Some research efforts have been devoted to analyse the plant responses to OGs that mediate this locally induced enhanced resistance. In Arabidopsis, OG-induced resistance against B. cinerea does not require JA and SA signalling, nor the oxidative burst generated in plants by OG perception (Aziz et al., 2004;Ferrari et al., 2007;Galletti et al., 2008;Galletti, Ferrari, & De Lorenzo, 2011;Gravino et al., 2015). Instead, it requires a functional PAD3, which encodes the last step of camalexin biosynthesis (Ferrari et al., 2007. Based on previous evidences, formulations combining OGs with chitosan oligomers are already available for plant protection against pathogens (van Aubel, Cambier, Dieu, & Van Cutsem, 2016).
Little is known about the responses induced by OGs at the systemic level, despite the well-established relevance of systemic defence responses in plants (Hilleary & Gilroy, 2018). The first observations of the function of OGs as an elicitor of systemic responses were obtained in tomato (Bishop, Makus, Pearce, & Ryan, 1981;Reymond, Grünberger, Paul, Müller, & Farmer, 1995;Simpson, Ashford, Harvey, & Bowles, 1998;Thain, 1995). However, the induction of systemic resistance to pathogens upon OG treatment has been so far reported only in Arabidopsis (Ferrari et al., 2007), although the molecular mechanisms behind this response are unexplored. Tomato was one of the model plants for the pioneer studies addressing systemic wound responses (Birkenmeier & Ryan, 1998;O'Donnell et al., 1996;Schilmiller & Howe, 2005) and tomato defence responses against B. cinerea are known to involve the wound related hormones JA, SA, ET and abscisic acid (ABA) (Achuo, Audenaert, Meziane, & Höfte, 2002;Asselbergh et al., 2007;Curvers et al., 2010;Díaz, ten Have, & van Kan, 2002;El Oirdi et al., 2011). Hence, an important question is how tomato plants respond not only locally but also systemically to OGs recognition and if these responses are able to trigger induced resistance (IR) against pathogens.
In this study we examined how tomato plants coordinate local and systemic responses to OG perception in different organs. In addition, we addressed the biological relevance of these responses by testing their efficacy in enhancing plant resistance against B. cinerea, a common and polyphagous necrotrophic pathogen. We show that changes in hormone levels induced by OGs are fast and transient at the local level and more sustained at the systemic level, and notably, that OGs have a stronger impact in roots than in leaves, regardless of the application site. Untargeted metabolomic analysis highlights the differential response to OGs in local and systemic tissues, supporting the notion of a precise fine-tuning of plant defences in response to this class of DAMPs, and uncovers the major pathways targeted by OG signalling. Finally, we show that root or leaf treatment with OGs induces systemic resistance against B. cinerea in tomato plants. The results highlight the differences among local and systemic responses and their dependence on the site of signal perception.

| Plant material and growth conditions
Tomato seeds (Solanum lycopersicum, cv Castelmart) were surface sterilized in 4% sodium hypochlorite, rinsed thoroughly with sterile water and germinated for 3 days on moistened filter paper at 25 C in darkness.
Subsequently, seedlings were transferred into 3 L plastic containers and grown hydroponically with water during the first week and with 0.5x Long Ashton nutrient solution (Hewitt, 1966) until the end of the experiment. The nutrient solution was replaced by fresh solution once a week.

| Oligogalacturonide treatments
Oligogalacturonides (DP 10-15) were prepared as previously described (Benedetti et al., 2017): A PGA solution (2% [w/v; Alfa Aesar]) was incubated with endo-polygalacturonase II (0.1 RGU/ml), purified from Aspergillus niger Pectinase (Sigma), for 180 min at 30 C in a water bath under gentle shaking. The digest was boiled for 10 min in a water bath to inactivate the enzyme and cooled at 4 C on ice. Oligogalacturonides were precipitated by diluting the solution with cold 50 mM sodium acetate and ice-cold ethanol to a final concentration of 0.5% (w/v) PGA and 17% (v/v) ethanol. The solution was incubated overnight at 4 C and centrifuged at 30,000 ×g for 30 min to recover the pellet. This was solubilized and centrifuged at 30,000 ×g for 30 min. The supernatant containing the oligogalacturonides was recovered, dialyzed against ultrapure water in a dialysis tube with a molecular mass cut-off of 1,000 Da (Spectra/Por®) and lyophilized.
Four-week-old tomato plants were treated with aqueous oligogalacturonide solution (50 μg/ml in milliQ water) either in leaves or roots. A time course analysis of the response was performed by harvesting leaf material at 1, 6 and 24 h after treatments. For leaf treatments (LT-), the fourth true leaf of each plant was sprayed with the oligogalacturonide solution using an aerograph until running off.
Control treatments were carried out with water (CT-). Plastic was used to cover the rest of the plant during spraying to avoid contact of the solution with other plant parts. Water was applied similarly for the control treatment. Treated leaves were harvested at the different time points after treatment for the study of local responses (LT-TL). The sixth fully developed untreated leaf of each plant was also harvested to study systemic leaf responses (LT-SL), and the untreated roots were harvested to study root systemic responses to leaf treatments (LT-Root). For the root treatments (RT-), roots were incubated in a 50 μg/ml oligogalacturonide solution for 1 hr, water was used as control treatment. As for leaf treatments, different plant parts were harvested at 1,6 and 24 h following the incubation. Treated roots were harvested for local responses (RT-Root). The sixth fully developed untreated leaves were also harvested to study systemic responses in shoots upon OG root treatments (RT-SL). ( Figure S1).

| Phytohormones quantification. LC-ESI tandem mass spectrometry
Six independent plants were harvested and stored at −80 C. The samples were freeze dried and powdered for subsequent analysis. Fifty milligrams of dry powder were used for hormonal extraction. Ultrapure water (Millipore, www.merckmillipore.com) was added containing a pool of internal standards abscisic acid-d6 (ABA-d6), salicylic acid-d5 (SA-d5) and jasmonate isoleucine-d6 (JA-Ile-d6). Precise quantification was performed by using external calibration curves with each pure chemical compound. The content of the tube was vortexed and left at 4 C in order to hydrate the plant sample. Five glass beads (2 mm Ø) were added into each microtube and the extraction was performed in a mixer mill at a frequency of 30 Hz for 3 min. Tubes were centrifuged at 13,000 rpm for 30 0 , and supernatant was recovered and placed into a new tube. A second extraction was then conducted, and the supernatant was added to the previous one. The pH was adjusted to 2.5-2.7 with acetic acid and the extraction was partitioned twice against diethyl ether. The two organic fractions were concentrated until dryness in a centrifugal evaporator (Speed vac) at room temperature. Samples were resuspended in 1 ml of H 2 O/MeOH (90:10) with 0.01% of HCOOH leading to a final concentration of internal standards of 100 ng/ml. The chromatographic separation was carried out by injection of 20 μL on an UPLC Kinetex 2.6 μm particle size EVO C18 100 A, 50 × 2.1 mm (Phenomenex). The quantification of the plant hormones was done in an Acquity ultraperformance liquid chromatography system (UPLC; Waters, Mildford, MA), which was connected to a triple quadrupole mass spectrometer (TQD, Waters, Manchester, UK). The chromatographic and mass spectrometry conditions were those published by Gamir, Pastor, Cerezo, and Flors (2012). Masslynx v 4. 1(Waters, Manchester, UK) software was used to process the quantitative data obtained from calibration standards and samples.

| LC-ESI full scan mass spectrometry
The metabolomic analysis was carried out with six biological replicates per treatment. Fifty milligrams of freeze-dried leaf or root material were extracted at 4 C with 1 ml of MeOH:H 2 O (10:90) containing 0.01% of HCOOH. After the centrifugation at full speed at 4 C for 15 min, the supernatant was filtered through 0.2 μm cellulose filters (Regenerated Cellulose Filter, 0.20 μm, 13 mm D. pk/100; Teknokroma). Twenty microlitres were injected into an Acquity UPLC system (Waters, Mildford, MA) interfaced with a hybrid quadrupole time-of-flight instrument (QTOF MS Premier). Subsequently, a second fragmentation function was introduced into the TOF analyser to identify the signals detected. This function was programmed in a t-wave ranging from 5 to 45 eV to obtain a fragmentation spectrum of each analyte (Gamir, Pastor, Kaever, Cerezo, & Flors, 2014). To elute analytes, a gradient of methanol and water containing 0.01% HCOOH was used. Six independent biological replicates per treatment were randomly injected. The LC separation was performed using an UPLC Kinetex 2.6 μm particle size EVO C18 100 A, 50 × 2.1 mm (Phenomenex). Chromatographic conditions and solvent gradients and further were established as described by Gamir et al. (2014).

| Full scan data analysis
Positive and negative electrospray ionization (ESI) signals were analysed independently to obtain a global view of the data conduct.
For ESI positive, the instrument detected 5,927 signals and, for ESI negative, 2,962 signals. The data files raw acquired with the Masslynx 4.1 software (Masslynx 4.1, Waters) were transformed into .cdf files with Databridge tool. Chromatographic data files were processed using the software R (http://www.r-project.org/). The XCMS algorithm (www.bioconductor.org; Smith, Want, O'Maille, Abagyan, & Siuzdak, 2006) was used to obtain the peak peaking, grouping and signal corrections. Metabolite amounts were analysed based on the normalized peak area units relative to the dry weight. To test the metabolomic differences between treatments, a nonparametric Kruskal-Wallis test (p < .01) was done. Partial least square discriminant analysis and heat map analysis were performed with the meta-boAnalyst 4.0 (Chong et al., 2018). Adduct and isotope correction, filtering, clustering, exact mass mapping and metabolic pathway exploration was carried out with the packages MarVis filter, MarVis cluster and MarVis pathway that are integrated in the Marvis suit 2.0 . Metabolite identification was carried out based on exact mass accuracy and fragmentation spectra matching with different online database. The database kegg (https://www.genome.jp/ kegg/) was used for exact mass identity and for fragmentation spectrum analysis, the Massbank and the Metlin databases were used (www.massbank.jp; www.masspec.scripps.edu).

| Quantitative RT-PCR analysis
The expression of marker genes for the different defence related pathways was analysed by qRT-PCR using the gene specific primers shown in Table S1. Total RNA from leaves and roots was extracted using Tri-Sure (Bioline, London, UK) according to the manufacturer's instructions. The RNA was treated with NZY DNase I (NZYtech, Portugal), purified through a silica column using the RNA clean and con-centrator™ (Zymo Research, Irvine, CA) and stored at −80 C until use.
The first-strand cDNA was synthesized with 1 μg of purified total RNA using the Primescript™ RT master mix (Takara, Japan) according to the manufacturer's instructions. The qRT-PCR was conducted using the StepOnePlus™ (Applied Biosystem). Six independent biological replicates were analysed per treatment. We measured the expression of three different housekeeping genes, actin (Solyc03g078400), elongation factor 1-α (Solyc06g005060) and β-tubulin (Solyc04g081490) and, to find the optimal normalization gene among these three, we used the Normfinder software (https://moma.dk/normfindersoftware). According to the results, expression values were normalized using the housekeeping gene elongation factor 1-α (EF-1α), and relative quantification of specific mRNA levels was performed using the comparative 2-Δ(ΔCt) method (Livak and Schmittgen, 2001).

| Leucyl-aminopeptidase (LAP) and β-1,3-glucanase activity assays
For protein extraction, 50 mg of fresh plant material were extracted in the extraction buffer (50 mM TRIS-HCl, 0.5 mM MnCl 2 , pH 8). One millilitre of the extraction buffer was added to each sample and was centrifuged for 20 min at 10,000 g, 4 C. The supernatant was recovered and stored at −20 C. For LAP activity, Leu-p-nitroanilide (Sigma) was prepared from the stock solution as enzyme substrate at a concentration of 3 mM in a solution of 50 mM TRIS-MnHCl 2 . The stock solution was previously prepared at 150 mM in ethanol and stored at −20 C. To carry out the analysis, 40 μL of the protein sample and 200 μL of the substrate were incubated for 15 min at 37 C and absorbance was measured at 410 nm as described in (Chao, Pautot, Holzer, & Walling, 2000). β-1,3-glucanase activity was measured by the Somogy-Nelson method as described by Román et al. (2011).

| Botrytis cinerea infection
The fungus was cultivated in potato dextrose agar plates, supplemented with freeze-dried tomato leaves. Three weeks later, B. cinerea spores were collected from plates in 0.5X potato dextrose broth as previously described .
Four-week-old tomato plants were treated with an aqueous solution of OGs in leaves (200 and 50 μg/ml) or roots (50 μg/ml). Six hours after the treatment, treated leaves (for local responses) and the sixth fully developed untreated leaf (for systemic responses) of each plant were detached for the pathogen bioassays. Leaf inoculation was performed applying to the detached leaves 6 μL drops containing a conidia concentration of 5 × 10 6 spores/ml. In total, we used the sixth fully developed leaf of 10 tomato plants. We inoculated four leaflets per leaf and we applied two drops per leaflet. Leaves were maintained in hermetically sealed boxes with 100% of humidity at 21 C in darkness. Necrotic lesions were evaluated after 5 days.

| Statistical analysis
For the hormonal analysis and infection assays data a t-test was conducted using Microsoft office Excel. For qPCR data and enzymatic activity, a one-way ANOVA was used to find overall differences among the expression levels. Post hoc LSD was used to find significant differences among treated and control plants (p < .05). All the metabolome profiling data were analysed using a Kruskal-Wallis analysis provided in MarVis suite 2.0.

| Treatment with OGs triggers local and systemic hormone responses in tomato roots and leaves
The spatio-temporal regulation of local and systemic responses to OGs was analysed on tomato plants grown in a hydroponic system.
An aqueous OG solution was applied to the fourth fully expanded leaf (leaf treatment; LT) or to the roots (root treatment; RT). Local responses were examined in the treated organs (local, leaves or roots).
In the case of leaf treatment, systemic responses were analysed in roots and in the non-consecutive, fully developed younger leaf, attending to the vascular connection in tomato plants (Orians, Pomerleau, & Ricco,2000) (sixth true leaf). In the case of root treatment, the equivalent sixth true leaf was harvested to study systemic responses ( Figure S1). The role of ET in DAMPs signalling has been previously described (Díaz et al., 2002;O'Donnell et al., 1996;Simpson et al., 1998). For example, OGs treatments in tomato and Arabidopsis seedlings boost local ET levels and ET biosynthetic genes (Gravino et al., 2015;Simpson et al., 1998). Here, we examined the expression of the gene ACO1, well defined marker of ET pathway encoding the ACC oxidase 1, responsible of the limiting step in ET biosynthesis (Jafari, Haddad, Hosseini, & Garoosi, 2013). ACO1 was markedly up-regulated as a local response in both leaves and roots. The induction was transient in OG-treated leaves, but stronger and more sustained in OG-treated roots. As a systemic response, ACO1 was induced in leaves only upon leaf treatment, showing a similar regulation pattern than the JA biosynthetic gene. Systemic induction in the roots was also observed upon leaf treatment. Thus, gene expression analyses confirm the activation by OG treatment of JA, ABA and ET signalling, with varying patterns according to the application site. They also support the conclusion of a strong response to OGs in roots, either as a local or a systemic response (Table 1).
As a whole, the transcriptional and metabolic data reveal that OG    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 porphyrinchlorophyll 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 F I G U R E 2 Impact of OG treatment on the leaf metabolic profiles (a) sPLS-DA representation of ESI− and ESI+ signals obtained from a nontargeted analysis by UPLC-QTOF to monitor metabolomic changes 6 hr after OGs treatments. Three-week-old plants were treated in leaves or roots with a 50 μg/ml solution of OGs. Leaf samples were harvested 6 hr post treatment. Data points represent six biological replicates injected randomly into the UPLC-QTOF. The signals corresponding to different treatments were compared using the non-parametric Kruskal-Wallis test, and only data with a p-value < .01 between groups was used for subsequent processing. 3.3 | Flavonoids and alkaloids biosynthetic genes are up-regulated in tomato roots as a local and systemic response to OGs Flavonoids and alkaloids, both accumulated in response to OGs, are phenylpropanoids derivatives that significantly contribute to plant resistance (Mithöfer & Boland, 2012;Treutter, 2005). To gain further insight into the regulation of flavonoids and alkaloids in response to OGs, we studied changes in the expression of relevant biosynthetic genes at the local and systemic level 1 and 6 hr after leaf or root treatment (Table 3).

| OGs induce systemic resistance to Botrytis cinerea
The T A B L E 2 Pathways of identified metabolites differentially accumulated locally or systemically upon OG treatment in leaves or roots

Alkaloids biosynthesis (3)
Amino acid metabolism (3) Notes: The percentage of signals showing significantly higher accumulation from the total identified signals for a given treatment is shown in brackets. Identified metabolites were assigned to their corresponding metabolic pathways, and the number in the right refers to the identified compounds within that pathway. The signals corresponding to different treatments were compared using the non-parametric Kruskal-Wallis test, and only data with a p-value < .01 between groups were used for subsequent identification.

| Antifungal defences are up-regulated as a systemic response to OGs
Plant resistance to pathogens is generally the result of a combination of different defence mechanisms. Aiming to understand why the OG treatments induced systemic-but not local-pathogen resistance, we explored other potential antifungal defence responses that may contribute to the enhanced resistance against B. cinerea. Hence, we analysed the well characterized pathogenesis related proteins Leucyl aminopeptidase (LAP) and β-1,3-glucanases. LAP is a JA regulated, wound-responsive protein, displaying a dual role as aminopeptidase but also as a chaperone (Fowler et al., 2009;Scranton, Yee, Park, & Walling, 2012). β-1,3-glucanases, are inducible enzymes with antimicrobial properties for their action on the β-glucans in fungal cell walls.
F I G U R E 3 Impact of OG treatment on the root metabolic profiles (a) sPLS-DA representation of ESI− and ESI+ signals obtained from a nontargeted analysis by UPLC-QTOF-MS to monitor metabolomic changes 6 hr after OGs treatments. Three-week-old plants were treated in leaves or roots with a solution of OGs 50 μg/ml. Root samples were harvested 6 hr post treatment. Data points represent six biological replicates injected randomly into the UPLC-QTOFMS. The signals corresponding to different treatments were compared using the non-parametric Kruskal-Wallis test, and only data with a p-value < .01 between groups was used for subsequent processing. DAMPs derived from the plant cell wall, and the responses they trigger have been mainly studied in Arabidopsis, at the local level (Davidsson et al., 2017;Gravino 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 (Davidsson et al., 2017;Denoux et al., 2008;Ferrari, Plotnikova, De Lorenzo, & Ausubel, 2003). 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 untargeted metabolomic analysis showed that the most prominent response occurs in the roots of leaf-treated plants. Roots are the key regulators of plant defence responses to aboveground challenges; for example, foliar herbivory induces fast changes in roots leading to the synthesis of antiherbivore compounds such as alkaloids (Erb, Lenk, Degenhardt, & Turlings, 2009;Erb, Meldau, & Howe, 2012;Agut, Gamir, Jaques, & Flors, 2016). Metabolic responses to OGs are stronger in roots than in leaves and, in the same line, the proportion of identified compounds over-accumulating after OG treatment is also higher in roots than in leaves. Similarly, JA accumulation in tomato leaves after the attack of the root-knot nematode Meloidogyne incognita depends on the production of electric signals and ROS accumulation in roots (Wang et al., 2019). In conclusion, our results support that damaged-self recognition impacts the root metabolic composition altering aboveground responses. Together, we suggest that lignans and flavonoids are synthetized in roots after OG recognition, and flavonoids are transported through the vascular system to the distal parts of the plants.
We also found an accumulation of tropane alkaloids as a local response in roots and as a systemic response in leaves, but only upon root treatment. Indeed, the putatively identified anatalline, a JAinducible tropane, piperidine and pyridine alkaloid (Häkkinen et al., 2004) showed elevated levels in roots and leaves of root treated plants, but no change upon leaf treatment. The role of alkaloids in plant defence has been extensively studied against herbivore insects (Agut et al., 2016;Erb et al., 2009) Here, we show that the responses observed upon OG treatments are biologically relevant for defence in tomato, since they confer systemic 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;Galletti et al., 2011). 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, we also looked for other potential players that may contribute to the observed OG-systemic induced resistance against B. cinerea. Therefore, we explored the activity or gene expression levels of leucyl aminopeptidase (LAP) and the antimicrobial PR proteins β-1,3-glucanase (GluB) (van Kan et al., 1992). GluB expression 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 upregulated in the Solanum lycopersicoides Botrytis interaction (Smith, Mengesha, Tang, Mengiste, & Bluhm, 2014). LAP is a JA-inducible enzyme that plays a key role in tomato plant responses towards biotic attack (Fowler et al., 2009)