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\begin{document}

\title{Roots drive oligogalacturonide-induced systemic immunity in tomato}



\author[1]{Jordi Gamir}%
\author[2]{Zhivko Minchev}%
\author[3]{Estefania Berrio}%
\author[4]{Juan M. Garcia}%
\author[5]{Giulia De Lorenzo}%
\author[4]{Maria Pozo}%
\affil[1]{Universitat Jaume I}%
\affil[2]{Koppert}%
\affil[3]{Estación Experimental del Zaidin, Consejo Superior de Investigaciones Científicas}%
\affil[4]{Estación Experimental del Zaidín}%
\affil[5]{Sapienza University of Rome}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
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 reveal the key role of roots in the coordination of the
plant responses to damage sensing.%
\end{abstract}%



\sloppy


\textbf{Keywords}

DAMPs, flavonoids, phytohormones, plant defences, metabolomics,
oligogalacturonides, roots, systemic resistance, alkaloids,

\emph{Botrytis cinerea}

\textbf{Acknowledgments}

We thank Dr. Daniela Pontiggia from Sapienza University of Rome for
providing the purified oligogalacturonides.

\textbf{ROOTS DRIVE OLIGOGALACTURONIDE-INDUCED SYSTEMIC IMMUNITY IN
TOMATO}

Jordi Gamir\textsuperscript{1, 2*}, Zhivko Minchev\textsuperscript{1},
Estefan\selectlanguage{ngerman}ía Berrio\textsuperscript{1}, Juan M. García\textsuperscript{1},
Giulia De Lorenzo\textsuperscript{3}, Maria J. Pozo\textsuperscript{1}*

\textsuperscript{1} Department of Soil Microbiology and Symbiotic
Systems, Estación Experimental del Zaidín (CSIC), Granada, Spain.

\textsuperscript{2} Current address: Metabolic Integration and Cell
Signaling Group, Plant Physiology Section, Unidad Asociada a la
EEZ-CSIC. Dept Ciencias Agrarias y del Medio Natural, Universitat Jaume
I, Castellón, Spain.

\textsuperscript{3} Dipartimento di Biologia e Biotecnologie C. Darwin,
Sapienza Università di Roma, Rome, Italy

* Maria J. Pozo (mariajose.pozo@eez.csic.es)

\textbf{Abstract}

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 \emph{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.

\textbf{Keywords}

DAMPs, flavonoids, phytohormones, plant defences, metabolomics,
oligogalacturonides, roots, systemic resistance, alkaloids,

\emph{Botrytis cinerea}

\textbf{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 and 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 et al., 2017).

DAMPs are damaged-self molecules released from host tissue disruption
that act as endogenous danger signals in both animals and plants (Heil
and Land 2014). DAMPs comprise a mixture of molecules from diverse
origin such as extracellular ATP, extracellular DNA, inducible proteins
and fragments of the cell wall (Heil and Land 2014). In plants, DAMPs
are released from disintegrated plant 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 and Ryan
1999). Local responses to DAMPs involve the generation of
H\textsubscript{2}O\textsubscript{2}, MAPKs activation, increased flux
of calcium, production of phenylpropanoids and hypomethylation in CpG
sites (Barbero et al., 2016, Duran-Flores et al., 2017, Vega-Muñoz et
al., 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 et al., 2011). Generation of
hydrogen peroxide, accumulation of proteinase inhibitors and other
defence-related proteins are produced in distal leaves upon wounding or
application of the peptidic, wound-related hormone systemin in tomato
(Orozco-Cardenas and 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 (Ferrari et al., 2013, Savatin et al., 2014; De Lorenzo et
al.,2018). OGs are oligomers of \selectlanguage{greek}α-\selectlanguage{english}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 et al., 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 et al., 1984, Doares et al., 1995, Ferrari et al., 2007; Denoux
et al., 2008; Gravino et al., 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 \emph{Botrytis cinerea} (Aziz et al.,
2004), and protection was also achieved in Arabidopsis by
spray-application of OGs (Ferrari et al., 2007; Galletti et al., 2008).
Moreover, \emph{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 \emph{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 et al. 2011; Gravino et al., 2015). Instead, it requires a
functional \emph{PAD3,} which encodes the last step of camalexin
biosynthesis (Ferrari et al., 2007). In addition, Botrytis success in
colonizing the host plant depends, in part, on the methylesterification
degree of the pectin that is tightly regulated by OGs (Lionetti et al.,
2017).

Despite this knowledge on OG-mediated local responses in the model plant
Arabidopsis, little is known in other plant species. Even more
unexplored are the responses induced by OGs at the systemic level,
despite the well-established relevance of systemic defence responses in
plants (Hilleary and Gilroy, 2018). The induction of systemic resistance
to pathogens upon OG treatment has been so far reported only in
Arabidopsis (Ferrari et al., 2007), but the molecular mechanisms behind
this response are unexplored. Moreover, responses to OGs in roots have
been mostly studied in relation to morphogenesis but not to defence
(Hernandez Mata et al., 2010; Bellincampi et al., 1993, Savatin et al.
2011).

Tomato was one of the model plants for the pioneer studies addressing
systemic wound responses (ODonell et al., 1996; Birkenmeier and Ryan,
1998; Schilmiller and Howe, 2005). In fact, the first observations of
the biological activity of OGs as proteinase inhibitor-inducing factors
and their ability to depolarize membranes, regulate protein
phosphorylation and hormone biosynthesis were obtained in tomato (Bishop
et al., 1981, Sympson et al., 1998; Thain et al., 1995; Reymond et al.,
1995). However, while several reports support a regulatory role of JA,
SA, ET and abscisic acid (ABA) in the tomato immune responses
against\emph{B. cinerea} (El Oirdi et al., 2011, D\selectlanguage{ngerman}íaz et al., 2002,
Achuo et al., 2002, Asselbergh et al., 2007, Curvers et al., 2010), the
role of OGs in the process remains unexplored.

In this study we examined how t plant 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 \emph{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 \emph{B. cinerea} in tomato plants. The results
highlight the differences among local and systemic responses and their
dependence on the site of signal perception.

\textbf{Material and Methods}

\textbf{Plant material and growth conditions}

Tomato seeds (\emph{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.

\textbf{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\emph{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.
Oligoalacturonides 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 1000 Da (Spectra/Por®) and lyophilized.

Four-week-old tomato plants were treated with aqueous oligogalacturonide
solution (50 \selectlanguage{greek}µ\selectlanguage{english}g/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 24h 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 \selectlanguage{greek}µ\selectlanguage{english}g/mL
oligogalacturonide solution for one hour, water was used as control
treatment. As for leaf treatments, different plant parts were harvested
at 1,6 and 24h 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). (Sup. Fig. 1)

\textbf{Phytohormones quantification. LC-ESI tandem mass spectrometry}

Plant samples were stored at -80 \selectlanguage{ngerman}°C, 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\selectlanguage{english}-d6
(ABA\selectlanguage{english}-d6), salicylic acid\selectlanguage{english}-d5 (SA\selectlanguage{english}-d5) and JA\selectlanguage{english}-Ile\selectlanguage{english}-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 degC in order to hydrate the plant sample. Five glass beads (2
mm O) 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\selectlanguage{english}', 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\textsubscript{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
\selectlanguage{greek}μ\selectlanguage{english}l on an UPLC Kinetex 2.6 \selectlanguage{greek}μ\selectlanguage{english}m particle size EVO C18 100 A, 50 x 2.1 mm
(Phenomenex). The quantification of the plant hormones was done in an
Acquity ultraperformance liquid chromatography system (UPLC; Waters,
Mildford, MA, USA), which was connected to a triple quadrupole mass
spectrometer (TQD, Waters, Manchester, UK). The chromatographic and mass
spectrometry conditions were those published by Gamir et al. (2012).
Masslynx v 4. 1(Waters, Manchester, UK) software was used to process the
quantitative data obtained from calibration standards and samples.

\textbf{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 \selectlanguage{ngerman}°C with 1 ml of MeOH:H2O (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 \selectlanguage{greek}μ\selectlanguage{english}m cellulose filters (Regenerated
Cellulose Filter, 0.20 \selectlanguage{greek}μ\selectlanguage{english}m, 13 mm D. pk/100; Teknokroma). 20 \selectlanguage{greek}μ\selectlanguage{english}l were
injected into an Acquity UPLC system (Waters, Mildford, MA, USA)
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 et al., 2014). Positive
and negative electrospray signals were analysed independently to obtain
a global view of the data conduct. To elute analytes, a gradient of
methanol and water containing 0.01\% HCOOH was used. Three independent
biological replicates per treatment, each with three technical
replicates, were randomly injected. The LC separation was performed
using an UPLC Kinetex 2.6 \selectlanguage{greek}μ\selectlanguage{english}m particle size EVO C18 100 A, 50 x 2.1 mm
(Phenomenex). Chromatographic conditions and solvent gradients and
further were established as described by Gamir et al. (2014).

\textbf{Full scan data analysis}

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
(\emph{http://www.r-project.org/}). The XCMS algorithm
(www.bioconductor.org; Smith et al., 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 \textless{} 0.01) was done.
Partial least square discriminant analysis and heat map analysis were
performed with the metaboAnalyst 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 (Kaever et al., 2014). 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).

\textbf{Quantitative RT-PCR analysis}

The expression of marker genes for the different defence related
pathways was analysed by real-time quantitative polymerase chain
reaction (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 concentratorTM (Zymo Research, USA) and
stored at -80 \selectlanguage{ngerman}°C until use. The first-strand cDNA was synthesized with 1
\selectlanguage{greek}μ\selectlanguage{english}g of purified total RNA using the PrimescriptTM 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. Expression values
were normalized using the housekeeping gene Actin (Solyc03g078400) and
relative quantification of specific mRNA levels was performed using the
comparative 2--\selectlanguage{greek}Δ\selectlanguage{english}(\selectlanguage{greek}Δ\selectlanguage{english}Ct) method (Livak and Schmittgen, 2001).

\textbf{Leucyl-aminopeptidase (LAP) and ?-1,3-glucanase activity assays}

For protein extraction, fifty milligrams of fresh plant material were
extracted in the extraction buffer (50 mM TRIS-HCl, 0.5 mM
MnCl\textsubscript{2}, pH 8). One mL of the extraction buffer was added
to each sample and were centrifuge for 20 minutes at 10,000 \emph{g} ,
4\selectlanguage{ngerman}ºC. The supernatant was recovered and stored at -20ºC. 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\textsubscript{2}. The stock solution was previously
prepared at 150 mM in ethanol and stored at -20ºC. To carry out the
analysis, 40 \selectlanguage{greek}µ\selectlanguage{english}L of the protein sample and 200 \selectlanguage{greek}µ\selectlanguage{english}L of the substrate were
incubated for 15 minutes at 37\selectlanguage{ngerman}ºC and absorbance was measured at 410 nm
as described in (Chao et al., 2000). \selectlanguage{greek}β-\selectlanguage{english}1,3 glucanase activity was
measured by the Somogy-Nelson method as described by Roman and
co-workers (de Roman et al., 2011).

\emph{Botrytis cinerea} \textbf{infection}

The fungus was cultivated in potato dextrose agar plates, supplemented
with freeze-dried tomato leaves. Three weeks later, \emph{B.
cinerea}spores were collected from plates in 0.5X potato dextrose broth
as previously described (San Martin et al., 2020). Leaf inoculation was
performed in detached leaves using two 6 \selectlanguage{greek}µ\selectlanguage{english}L drops containing a conidia
concentration of 5 x 10\textsuperscript{6} spores/mL per leaflet. Leaves
were maintained in hermetically sealed boxes with 100\% of humidity at
21\selectlanguage{ngerman}ºC in darkness. Necrotic lesions were evaluated after 5 days.

\textbf{Statistical analysis}

For the hormonal analysis, gene expression, enzymatic activity and
infection assays data a t-test was conducted using Microsoft office
Excel. All the metabolome profiling data were analysed using a
Kruskal-Wallis analysis provided in MarVis suite 2.0.



\textbf{Results}

\textbf{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 et al., 2000) (sixth true
leaf. In the case of root treatment, the equivalent sixth true leaf was
harvested to study systemic responses (suppl. Fig. S1).

First, we studied the changes in the main plant defence hormones and
some precursors and derivatives {[}SA, abscisic acid (ABA), ABA
glucoside (ABA-Gluc), jasmonate isoleucine (JA-Ile) and
12-oxo-phytodienoic acid (OPDA; JA precursor){]}. The levels of these
compounds were measured at 1, 6 and 24 hours post treatment (hpt) and
the data are presented in suppl. Figs. S2 a,b,c). A summary of the
changes, expressed as fold changes compared to the corresponding
mock-treated controls is shown in Figure 1.

Local responses to OGs appear to be very different in leaves and roots
(Figure 1). In treated leaves, changes were observed mainly at 1 hpt,
consisting of a slight accumulation of ABA, an important accumulation of
JA-Ile {[}3.7 fold compared to mock-treated leaves{]} and a decrease of
SA. A decrease of OPDA at 6 h was the only significant later response.
In OG-treated roots only SA showed a change at 1 hpt: a slight decrease,
followed by a moderate increase at later time points. ABA transiently
increased at 6 hpt, dropping below control levels at 24 h.

Systemic responses of leaves to OGs were also different depending on the
type of treatment (root or leaf) (Figure 1). A decrease of SA at all
time-points and an accumulation of OPDA and ABA-Gluc levels at 6 hpt
were observed only upon leaf treatment, whereas a transient decrease of
JA-Ile was observed exclusively in RT-SL at 1 hpt. Levels of ABA-Gluc
increased in systemic leaves with both treatments, with a delay in the
case of root treatment.

Notably, systemic changes in hormone levels in the roots of leaf-treated
plants were more pronounced (Figure 1). A strong accumulation of JA-Ile
occurred at 1 hpt that was maintained up to 6 hpt and paralleled with a
reduction of the JA precursor OPDA, revealing an upregulation of the
oxylipin pathway in the roots upon leaf treatment. ABA and its
derivative ABA-Gluc also showed a systemic transient increase in roots,
and SA levels increased at the latest time point.

These data show that tomato plants respond systemically to OG treatment
in leaves, with the most conspicuous hormone changes occurring in the
root. Regardless of the site of OG treatment {[}leaf or root{]}, an
early and transient increase of ABA and a late increase in SA occur
mainly in roots. Instead, induction of the oxylipin pathway, shown as
early increases in JA-Ile levels, occurs in both leaves and roots only
upon leaf treatments, although the effect is more durable in roots.

This hormone analysis was complemented with the expression analysis of
genes involved in the JA, ABA and ET biosynthesis pathways (Table 1). In
the local response, we observed an early and transient upregulation of
the JA biosynthetic gene \emph{LOXD} in both leaves and roots. In the
systemic response, \emph{LOXD} was induced only upon leaf treatment,
with a stronger and more sustained induction in the roots than in upper
leaves, confirming a general activation of the oxylipin pathway upon
leaf treatment. Induction of the ABA biosynthetic gene \emph{NCED} was
found mostly in roots as both a local and a systemic response to leaf
treatment. In addition, we examined the expression of the
gene\emph{ACO1} , encoding the ACC oxidase 1, responsible of the
limiting step in ethylene biosynthesis. \emph{ACO1} was mostly
upregulated 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. 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
faster 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
treatment impacts hormone signalling both at a local and a systemic
level with induction of the oxylipins, ABA and ET pathways. Local
hormone-related responses to OGs in leaves occur early and transiently,
while systemic responses are delayed compared to local ones, with the
stronger effects detected at 6 hpt. Remarkably, changes in hormone
levels and expression of hormone biosynthetic genes were, in general,
stronger and more sustained during time in roots.

\textbf{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 (4\textsuperscript{th} leaf, chosen for OG local
treatment and younger/upper leaves (true six\textsuperscript{th} 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.

\textbf{Flavonoids and alkaloids biosynthetic genes are upregulated 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 (Treutter 2005; Mithöfer and Boland 2012). 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 6 h after leaf or root treatment (Table
3). For the analysis, we chose \emph{PHENYLALANINE AMMONIA
LYASE}(\emph{PAL),} involved in the first step in the phenylpropanoid
biosynthetic pathway, \emph{CHALCONE SYNTHASE 1} (\emph{CHS} .\emph{1}
), encoding the first enzyme of the flavonoid biosynthetic pathway that
produces naringenin chalcone, \emph{CHALCONE ISOMERASE} 1 (\emph{CHI1}
), responsible for the downstream reaction that converts naringenin
chalcone to naringenin, and \emph{PUTRESCINE N-METHYLTRANSFERASE (PMT)}
, encoding a key enzyme in the biosynthesis of tropane alkaloids
(Biastoff et al., 2009; Petrussa et al., 2013).

In leaves, none of those genes showed altered expression levels in
response to OGs at 6 hpt regardless the OG application site. No
expression of \emph{PMT} was detected in leaves. In contrast, the
expression of these genes showed significant changes in roots both as a
local and a systemic response to the treatment. All these marker genes
except \emph{CHS1} were induced in roots as a systemic response to OG
treatment in leaves (LT-Root). Remarkably, all four genes were strongly
upregulated locally upon root treatment, with a particularly strong
effect for \emph{CHS1} (75 fold). These results are in agreement with
the metabolic analyses and collectively evidence the key role of roots
in flavonoid and alkaloid biosynthesis following OG perception (Table
3).

\textbf{OGs induce systemic resistance to \emph{Botrytis cinerea}}

The strong impact of OG treatment in systemic tissues at very early
time-points prompted us to investigate whether OGs induce systemic
resistance in tomato plants against the necrotrophic pathogen \emph{B.
cinerea} .

Systemic protection was examined by treating tomato roots or leaves with
50 \selectlanguage{greek}µ\selectlanguage{english}g/mL of OGs and infecting at 6 hpt (the untreated true sixth leaf)
with \emph{B. cinerea} . Both root and leaf treatments (LT and RT) led
to a significant reduction of the necrotic lesions compared to the
corresponding control treated leaves (CT-SL), confirming that systemic
responses to OG treatment are associated to systemic resistance against
this pathogen in tomato. Remarkably, the protection level was similar
regardless of the site of OGs application (roots or leaves) (Figure 4a).

Local OG-induced resistance was also examined, by inoculating the
OG-treated leaves (LT-TL) at 6 hpt. No reduction of the necrotic
symptoms was observed compared to control-treated leaves (CT-TL) at
either 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL or 200 \selectlanguage{greek}µ\selectlanguage{english}g/mL OGs (Figure 4b). Thus, under our
experimental conditions and at the time point examined (6 hpt) OG
treatment in tomato induced systemic but not local resistance to
\emph{B. cinerea.}

\textbf{Antifungal defences are upregulated as a systemic response to
OGs}

We further explored antifungal defence responses induced by OGs that may
contribute to the enhanced pathogen resistance observed. Leucyl
aminopeptidase (LAP) is a JA regulated, wound-responsive protein,
displaying a dual role as aminopeptidase but also as a chaperone (Fowler
et al., 2009, Scranton et al., 2012), and \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases, are
inducible enzymes with antimicrobial properties for their action on the
\selectlanguage{greek}β-\selectlanguage{english}glucans in fungal cell walls. LAP activity was induced only in distal
leaves in response to leaf OG treatment (LT-SL), whereas no changes were
observed locally in treated nor upon root treatment (Figure 5a). Total
\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase activity did not vary among the different treatments
(suppl. Fig. S5). Several \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases are present in tomato with
differential regulation patterns, and total enzymatic activities may
mask enhancement of specific glucanase isoforms. Thus, we examined the
expression levels of \emph{GluB,} encoding a \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase, known to
be inducible by JA, ET and pathogens (van Kan et al., 1992).
While\emph{GluB} expression was not induced in OG-treated leaves (Local
response), it was boosted in leaves as a systemic response to OG
treatments in both leaves or roots treatments (Fig. 5b). Note that the
enhanced \emph{GluB} expression correlates with the tissues showing OG
induced resistance in the pathogen bioassay.

Taken together, our results suggest that the accumulation of defensive
metabolites and the induction of pathogenesis-related proteins may
underlie the efficient systemic protection against \emph{B.
cinerea}induced by OGs in tomato (Figure. 6).

\textbf{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 \emph{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 of\emph{CHI1}
, \emph{CHS1} and \emph{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 \emph{PMT} gene expression --coding for a key
enzyme of the tropane alkaloid biosynthesis pathway- as both a local and
systemic response to OGs. Interestingly, \emph{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 \emph{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 \emph{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 \emph{B. cinerea}
. We explored the activity or gene expression levels of the
antimicrobial PR proteins \selectlanguage{greek}β-\selectlanguage{english}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 \emph{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
\selectlanguage{greek}µ\selectlanguage{english}g/mL) have been shown to induce glucanase activity in grapevine cells
(Aziz et al., 2004) and that \emph{GluB} gene expression is up-regulated
in the\emph{Solanum 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
and\emph{GLUB} 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.

\textbf{Acknowledgments}

We thank Dr. Daniela Pontiggia from Sapienza University of Rome for
providing the purified oligogalacturonides. This work was supported by
the Spanish Government through the CSIC Intramural project 201840E130,
grant RTI2018-094350-B-C31 from the Ministerio de Ciencia, Innovaci\selectlanguage{ngerman}ón y
Universidades and two Postdoctoral fellowships to J. Gamir,
FJCI-2015-23575 from Ministerio de Ciencia, Innovación y Universidades
and CDEIGENT/2018/015 from Generalitat Valenciana. None of the major
findings of the research described here has been submitted to other
journals or described elsewhere.

\par\null

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\textbf{Table 1.} \textbf{Transcriptional regulation of phytohormone
biosynthetic genes upon OG treatment.} Quantitative RT-qPCR analysis of
LOXD (lypoxigenase D, involved in JA biosynthesis), NCED
(9-cis-epoxycarotenoid dioxygenase, involved in ABA biosynthesis, and
ACO1 (ACC Oxidase 1, coding for the enzyme responsible for the limiting
step in ET biosynthesis) in leaves and roots of OG treated plants.
Changes related to local responses were evaluated in treated leaves and
roots. Systemic responses were evaluated in roots and upper leaves upon
OG treatment in leaves (LT) or in upper leaves upon OG treatment in
roots (RT). Numbers correspond to the fold induction of the gene
expression levels in treated vs control plants. Values are normalized
relative to the tomato housekeeping gene Actin. Bold numbers indicate
significant differences (t-test; p-value\textless{}0.05).\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{RESPONSE} & & \textbf{LEAVES} & \textbf{LEAVES} &
\textbf{LEAVES} & \textbf{ROOTS} & \textbf{ROOTS} &
\textbf{ROOTS}\tabularnewline
& time & LOXD & NCED & ACO1 & LOXD & NCED & ACO1\tabularnewline
\textbf{LOCAL} & 1h & \textbf{3.8} & 0.8 & \textbf{7} & \textbf{9} & 0.7
& \textbf{11}\tabularnewline
& 6h & 1.3 & 1 & 1.3 & 0.6 & \textbf{2.6} & \textbf{4.5}\tabularnewline
\textbf{SYSTEMIC (LT)} & 1h & \textbf{2.2} & 0.5 & 2 & \textbf{27} &
\textbf{4.7} & 0.7\tabularnewline
& 6h & 0.93 & 1.1 & 1 & \textbf{3.7} & \textbf{5} &
\textbf{3.6}\tabularnewline
\textbf{SYSTEMIC (RT)} & 1h & \textbf{0.5} & 0.9 & 0.8 & &
&\tabularnewline
& 6h & 0.4 & 1 & 0.5 & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} ~ \textbf{Pathways of identified metabolites
differentially accumulated locally or systemically upon OG treatment in
leaves or roots.} 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\textless{}0.01 between
groups were used for subsequent identification.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{RESPONSE}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{LEAVES}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{ROOTS}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
\textbf{LOCAL}\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
\textbf{(2 \%)} Flavonoid biosynthesis (3) Porphyrin and chlorophyll
metabolism (2)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
\textbf{(5,8 \%)} Flavonoid biosynthesis (4) Lignan (3) Alkaloids
biosynthesis (3) Auxin degradation (2) Biotin metabolism (2)
Terpenoids-quinone biosynthesis (2) Lysine biosynthesis (2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{SYSTEMIC} \textbf{(LT)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{(1,8 \%)} Flavonoid Biosynthesis (3) Purine metabolism (2) Amino
acid metabolism (2) Fatty acid biosynthesis (2)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{(3,4 \%)} Amino acid metabolism (7) Dipeptide (3) Lignan
(2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{SYSTEMIC} \textbf{(RT)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{(2.2 \%)} Alkaloids biosynthesis (3) Amino acid metabolism
(3)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3. Transcriptional regulation of flavonoids and alkaloids
biosynthetic genes upon OG treatment.} Quantitative RT-qPCR analysis of
PAL, CHS1.1, CHI1.1 (Flavonoid biosynthesis genes) and PMT (Alkaloid
biosynthesis gene) confirms upregulation of such metabolic pathways in
roots in response to OGs treatments. Numbers correspond to the fold
induction of the gene expression levels in treated vs control plants (n
= 6 from six biological replicates). Values are normalized relative to
the tomato housekeeping gene Actin. Bold numbers indicate significant
differences (t-test; p-value\textless{}0.05).\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\textbf{Response} & \textbf{Leaves} & \textbf{Leaves} & \textbf{Leaves}
& \textbf{Leaves} & \textbf{Roots} & \textbf{Roots} & \textbf{Roots} &
\textbf{Roots}\tabularnewline
& \textbf{PAL} & \textbf{CHS1} & \textbf{CHI1} & \textbf{PMT} &
\textbf{PAL} & \textbf{CHS1} & \textbf{CHI1} &
\textbf{PMT}\tabularnewline
\textbf{LOCAL} & 0.6 & 0.9 & 0.7 & nd & \textbf{3} & \textbf{75} &
\textbf{3.4} & \textbf{5}\tabularnewline
\textbf{Systemic (LT)} & 0.6 & 0.6 & 0.8 & nd & \textbf{2.8} & 0.97 &
\textbf{2.8} & \textbf{3}\tabularnewline
\textbf{Systemic (RT)} & 1.4 & 0.8 & 0.8 & nd & & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure 1. Time course analysis of local and systemic changes in
hormonal levels upon OG treatment in leaves or roots of tomato
plants.}Hormonal levels were quantified by UPLC-MS/MS at 1, 6 and 24
hours in tomato plants elicited with an OGs solution of 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL. For
leaf treatments (LT), the fourth true leaf was sprayed with the OGs
solution and samples from the treated leaf (Local response-leaves-),
upper leaf and roots (systemic response) were harvested at the different
time points. For root treatments (RT) the OG solution was applied to
roots and samples at the different time points were taken from roots
(local response-roots-) and in the upper leaves for systemic responses
(Systemic, RT). Hormone contents are shown in Sup Fig1a, b and c. Here,
numbers represent fold induction in the hormone levels of treated vs
control plants. Bold numbers indicate significant differences (t-test;
p-value\textless{}0.05). Grey cells highlight significantly
overaccumulated compounds.

\textbf{Figure. 2. Impact of OG treatment on the leaf metabolic
profiles} a) sPLS-DA representation of ESI- and ESI+ signals obtained
from a non-targeted analysis by UPLC-QTOF to monitor metabolomic changes
6 hours after OGs treatments. Three weeks old plants were treated in
leaves or roots with a 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL solution of OGs. Leaf samples were
harvested 6 hours 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\textless{}0.01 between groups was used for subsequent
processing. b) Heatmap analysis of leaf metabolites responding to OGs.
Signals from ESI+ and ESI- with p-value\textless{}0.01 were used to
generate the heatmap analysis. The top 250 signals with the lowest
p-value were selected to represent the heatmap. The relative amount of
the metabolites was determined in all the samples by normalizing the
chromatographic pick area of each compound with the dry weight of the
corresponding sample. Local leaf responses were studied by comparing
control-treated leaves (CT-TL) to OG-treated leaf (LT-TL). Systemic leaf
responses were analysed comparing upper untreated leaves from
water-treated plants as control (CT-SL) upper leaves from OGs treated
plants in leaves (LT-SL) and to upper leaves from OGs treated plants in
roots (RT-SL).

\textbf{Figure 3: Impact of OG treatment on the root metabolic
profiles}a) sPLS-DA representation of ESI- and ESI+ signals obtained
from a non-targeted analysis by UPLC-QTOF-MS to monitor metabolomic
changes 6 hours after OGs treatments. Three weeks old plants were
treated in leaves or roots with a solution of OGs 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL. Root samples
were harvested 6 hours 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\textless{}0.01 between groups was used for subsequent
processing. b) Heatmap analysis of root metabolites responding to OGs.
Signals from ESI+ and ESI- with p-value\textless{}0.01 were used to
generate the heatmap analysis. The top 100 signals with the lowest
p-value were selected to represent the heatmap. The relative amount of
the metabolites was determined in all the samples by normalizing the
chromatographic pick area of each compound with the dry weight of the
corresponding sample. Local root responses were studied comparing
responses from control treated plants in roots (CT-Root) to OG treated
roots (RT-roots). Systemic root responses were studied in roots of leaf
treated plants (LT-Roots).

\textbf{Figure 4. OG treatments induces systemic resistance against
Botrytis cinerea} . Three weeks old tomato plants were treated in roots
with a 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL OGs solution or in leaves with 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL or 200 \selectlanguage{greek}µ\selectlanguage{english}g/mL OGs
solution. Six hours after the treatment, plants were drop inoculated in
upper or treated leaves with a Botrytis cinerea conidia suspension of
5x10\textsuperscript{6} spore x mL\textsuperscript{-1}. Lesion diameter
was measured 5 days after inoculation. Data presented shows the average
lesion diameter \selectlanguage{ngerman}±SE (n=10 plants). Asterisks indicate statistically
significant differences compared to control plants (* water treated;
t-test; p\textless{}0.05). Systemic leaf responses were analysed
comparing upper untreated leaves from water-treated plants as control
(CT-SL) to upper leaves from OGs- leaf treated plants (LT-SL) and to
upper leaves from OGs-root treated plants in roots (RT-SL). Local leaf
responses were studied by comparing water-treated leaves (CT-TL) to OGs
treated leaf (LT-TL) at two different concentrations.

\selectlanguage{greek}\textbf{Figure 5. OG perception in leaves increases systemic leucyl
aminopeptidase activity and β-1,3-glucanase GLUB gene expression.}\selectlanguage{english}Leucyl
aminopeptidase activity (JA-responsive protein) and quantitative RT-qPCR
analysis of GLUB (coding for a pathogen inducible \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase).
Local responses were analysed in water-treated (CT-TL) or OG-treated
leaves (LT-TL). Systemic responses were analysed comparing upper
untreated leaves from water-treated plants as control (CT-SL) to upper
leaves from OG-leaf treated plants (LT-SL) and to upper leaves from
OG-root treated plants (RT-SL). Bars represent mean \selectlanguage{ngerman}± SD, n = 6 from six
biological replicates. Asterisks indicate statistically significant
differences compared to control plants (t-test; p-value\textless{}0.05).

\textbf{Figure 6. Summary of the responses activated in tomato leaves
and roots upon OG treatments.} The model displays local and systemic
tomato responses at the hormonal, enzymatic and metabolomic level
depending on the organ of the perception. (a) Plant responses to OG
treatment in leaves. Local responses in treated leaves and systemic
responses in leaves and roots are shown. (b) Plant responses to OG
application in roots. Local responses in roots and systemic response in
leaves are shown.

\textbf{Supporting Information}

\textbf{Suppl. Figure 1.} Schematic model of the leaf of root treatments
and nomenclature of the different samples harvested.

\textbf{Suppl. Figure 2.} Phytohormone levels in roots and leaves of
tomato plants upon treatment with a OG solution. a) Local responses to
OG treatment in leaves. b). Systemic responses in leaves to OG
application in leaves or roots. c) Local and systemic responses to OGs
in roots.

\textbf{Suppl. Figure 3.} Overview of the metabolic reprograming in
roots and leaves after OGs perception.

\textbf{Suppl. Figure 4.} Relative quantification of the alkaloid
anatalline in leaves and roots upon OG treatment in the roots (6hpt).

\textbf{Suppl. Figure 5.} \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase enzymatic activity in leaves
upon OGs treatment (6hpt).

\textbf{Table S1.} Primers used in this study for gene expression
analysis.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure5/Figure5}

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\includegraphics[width=0.70\columnwidth]{figures/Figure6/Figure6}

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