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 µg/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<0.05). Grey cells highlight significantly
overaccumulated compounds.
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 µg/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<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<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).
Figure 3: Impact of OG treatment on the root metabolic profilesa) 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 µg/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<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<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).
Figure 4. OG treatments induces systemic resistance against
Botrytis cinerea . Three weeks old tomato plants were treated in roots
with a 50 µg/mL OGs solution or in leaves with 50 µg/mL or 200 µg/mL OGs
solution. Six hours after the treatment, plants were drop inoculated in
upper or treated leaves with a Botrytis cinerea conidia suspension of
5x106 spore x mL-1. Lesion diameter
was measured 5 days after inoculation. Data presented shows the average
lesion diameter ±SE (n=10 plants). Asterisks indicate statistically
significant differences compared to control plants (* water treated;
t-test; p<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.
Figure 5. OG perception in leaves increases systemic leucyl
aminopeptidase activity and β-1,3-glucanase GLUB gene expression.Leucyl aminopeptidase activity (JA-responsive protein) and quantitative
RT-qPCR analysis of GLUB (coding for a pathogen inducible
β-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 ± SD, n = 6
from six biological replicates. Asterisks indicate statistically
significant differences compared to control plants (t-test;
p-value<0.05).
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.