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14TH INTERNAT IONAL SYMPOS IUM ON INSECT-PLANT INTERACT IONS
From shoots to roots: transport and metabolic changesin tomato after simulated feeding by a specialistlepidopteranSara Gómez1,2*, Adam D. Steinbrenner3, Sonia Osorio4, Michael Schueller5, Richard A.Ferrieri5, Alisdair R. Fernie4 & Colin M. Orians21Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, USA, 2Department of Biology, Tufts
University, Medford, MA 02155, USA, 3Department of Plant and Microbial Biology, University of California, Berkeley, CA
94704, USA, 4Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Potsdam-Golm, D-14476, Germany, and 5Medical
Department, Brookhaven National Laboratory, Upton, NY 11973, USA
Accepted: 30March 2012
Keywords: carbohydrates, resource allocation, primary metabolism, resource sequestration,
tolerance, induced responses, Manduca sexta, Sphingidae, oral secretion, Solanaceae, tobacco
hornworm
Abstract Upon herbivory, plants can swiftly reallocate newly acquired resources to different tissues within a
plant. Although the herbivore-induced movement of resources is apparent, the movement direction
and the role of the remobilized resources are not well understood. Here, we used a two-pronged
approach combining radioisotope and metabolomic techniques to shed light on whole-plant
resource reallocation and changes in primary metabolism within the tomato, Solanum lycopersicum
(L.) (Solanaceae), model in response to simulated herbivory by the specialist Manduca sexta (L.)
(Lepidoptera: Sphingidae).Manduca sexta regurgitant applied to damaged leaves, but not mechani-
cal damage alone, increased 11C-photosynthate allocation to roots but did not affect 11CO2 fixation
and leaf export. Changes in primary metabolite concentrations occurred mostly in sink tissues (apex
and roots) as well as in damaged leaves. Both damage treatments (with and withoutM. sexta regurgit-
ant application) resulted in increased concentrations of primary metabolites relative to undamaged
plants in the apex and decreased concentrations in the roots, but there were also extensive changes
specific to each damage treatment. Mechanical damage alone led to changes consistent with water
stress caused by tissue damage.Manduca sexta led to metabolite increases in the apex consistent with
an increase in glucose breakdown, metabolite increases in damaged leaves consistent with starch
degradation, and metabolite decreases in roots suggesting a high use of metabolites. A possible expla-
nation for the observed patterns in the aboveground tissues might be an increase in carbohydrate
degradation to support defense production in attacked leaves and vulnerable developing leaves, and/
or subsequent remobilization to belowground tissues to support high carbohydrate demand for
respiration, enhanced nutrient uptake, and storage.
Introduction
Plants are in a constant arms race with their attackers,
resulting in the evolution of a wide range of responses to
cope with herbivory. This includes resistance mechanisms,
such as the production of chemical and morphological
changes to deter or reduce herbivores’ preference and per-
formance. In addition, plants can also use tolerance mech-
anisms, which have no negative effects on the herbivore’s
fitness, but help sustaining tissue regrowth after damage
(Karban & Baldwin, 1997). The latter can include
increased photosynthesis, compensatory growth, and utili-
zation of stored reserves (reviewed in Tiffin, 2000).
Primary metabolism plays a central role in plant resis-
tance and tolerance. Carbohydrates and other primary
metabolites synthesized in source leaves are allocated to
structural and metabolic processes within the leaves, and
*Correspondence: Sara Gomez, Department of Biological Sciences,
University of Rhode Island, 9 E. Alumni Avenue, Kingston, RI 02881,
USA. E-mail: [email protected]
© 2012 The Authors Entomologia Experimentalis et Applicata 144: 101–111, 2012
Entomologia Experimentalis et Applicata © 2012 The Netherlands Entomological Society 101
DOI: 10.1111/j.1570-7458.2012.01268.x
can also be exported to support developing foliage and
fruits, stems, and roots, or they can be stored away for later
use (Gifford & Evans, 1981). These storage compounds are
critical to plant performance following stress (Trumble
et al., 1993).
The production of defense compounds is also tightly
linked to primary metabolism as compounds, such as
amino acids, sugars, and organic acids can act as precur-
sors, carbon skeletons, and as substrates to produce energy
necessary for the biosynthesis of defensive metabolites
(Arnold & Schultz, 2002; Broeckling et al., 2005; Bolton,
2009; Hanik et al., 2010a,b). Thus, it is not surprising that
herbivory could result in a reconfiguration of resource
partitioning among primary and secondary metabolism
(Schwachtje & Baldwin, 2008). The reliance of both
growth and defense on primary metabolites has led to the
suggestion of allocation trade-offs between resistance and
tolerance responses (van der Meijden et al., 2000),
although recent evidence suggests that both can be
induced by herbivory (Nunez-Farfan et al., 2007).
Rapid herbivore-induced changes in resource allocation
are gaining attention as important defense responses, com-
plementing long-standing knowledge on induction of
defensive secondary metabolites. Traditionally, resource
mobilization from storage tissues, such as roots and stems
was viewed as the primary strategy employed post-dam-
age, whereby stored reserves are remobilized to sustain the
regrowth of aboveground tissue (Trumble et al., 1993).
Recent studies, however, have demonstrated increased car-
bohydrate transport away from the damage site and into
storage organs in response to herbivory (Holland et al.,
1996; Babst et al., 2008; Kaplan et al., 2008) and this
applies to nitrogen-based resources as well (Newingham
et al., 2007; Frost & Hunter, 2008; Gomez et al., 2010).
This indicates that herbivores can induce the sequestration
of resources into inaccessible tissues and might increase
plant tolerance to herbivores (Schwachtje et al., 2006;
reviewed in Orians et al., 2011). This could be particularly
effective against specialist herbivores that are adapted to
their host’s defenses, such as the tobacco hornworm,
Manduca sexta (L.) (Lepidoptera: Sphingidae), which is
able to tolerate nicotine produced by tobacco plants, a
potent neurotoxin effective against most herbivores. In a
recent study, M. sexta feeding on tomato led to metabolic
changes related to carbon and nitrogen mobilization as
opposed to damage by the generalist Helicoverpa zea
(Boddie), which was characterized by changes in defense-
related metabolites (Steinbrenner et al., 2011). Alterna-
tively, rapidly remobilized resources might be used to
support defense production as shown in poplar saplings
(Arnold & Schultz, 2002; Arnold et al., 2004). Thus, it is
apparent that plants can rapidly alter resource dynamics in
response to herbivore attack. Themagnitude and direction
of remobilized resources and how those changes in trans-
port affect primary and secondarymetabolic pool sizes will
likely depend on intrinsic factors like plant life-history,
ontogeny, and phenology, as well as extrinsic factors such
as biotic and abiotic conditions (Orians et al., 2011).
To shed light on the potential role of herbivore-
induced resource mobilization, we designed the present
study to assess resource transport and subsequent general
changes in primary metabolism in response to mechani-
cal damage and simulated herbivory by the specialist M.
sexta on tomato 4 h after damage. That time point was
chosen because a previous study on tomato showed
induced remobilization in response to methyl jasmonate
(MeJA), a chemical defense elicitor, at 4 h (Gomez et al.,
2010). In the first experiment, we administered 11C, a
short-lived (t1/2 = 20.4 min) carbon radioisotope to
study short-term carbon fixation, leaf export, and alloca-
tion to roots in response to damage. In a second experi-
ment, we used a metabolomic approach to characterize
chemical profiles (60 primary metabolites including
amino acids, sugars, and organic acids) in various tissues
(apex, an undamaged leaf, damaged leaves, stem, and
roots) for each damage treatment and measured metabo-
lite concentrations relative to undamaged plants. Study-
ing local and systemic changes provides a whole-plant
perspective that can help understanding the documented
changes in resource reallocation. We hypothesized that
M. sexta regurgitant application on mechanically dam-
aged leaves would favor allocation of 11C-photosynthate
toward stems and roots, as shown for another species in
the Solanaceae family, Nicotiana attenuata Torr. ex S.
Watson (Schwachtje et al., 2006), and thus lead to an
increase in sugars and amino acids in those tissues.
Mechanical damage is also present during herbivore feed-
ing and it is known to be involved in the initial steps of
the defense signaling cascade although it does not usually
trigger the same responses as herbivore feeding (McCloud
& Baldwin, 1997; Reymond et al., 2000; von Dahl &
Baldwin, 2004; Babst et al., 2009). Therefore, we hypothe-
sized that mechanical damage alone would have no effect
on resource transport, but would lead to similar (but not
identical) patterns compared with regurgitant-treated
plants regarding primary metabolite concentrations.
Materials and methods
Plant material
In the ‘carbon dynamics’ study, tomato seeds (S. lycopersi-
cum cv. First Lady II F1; Hazzard’s seeds, Deford, MI,
USA) were sown in potting soil (Pro-mix; Premier Horti-
culture, Quakertown, PA, USA) with slow release fertilizer
102 Gomez et al.
(Osmocote plus 15-9-12; The Scotts Company,Marysville,
OH, USA). Plants used in the experiments had on average
six fully expanded and four developing leaves. Plants were
grown under metal-halide lamps (350 lmol m�2 s�1) at
24 °C and L16:D8 photoperiod.
In the ‘metabolomics study’, seeds from tomato plants
were sown and grown in SunGro Metro-mix soil for
20 days and seedlings were repotted into a 1:1 sand:zeolite
mix so the roots could be easily rinsed and separated dur-
ing plant harvest. The sand:zeolite medium leads to P defi-
ciency in tomato, so to maintain plant health, they were
fertilized with 100 ml of Hoagland’s solution with a dou-
ble amount of phosphorus (1 306 lMNO3�, 603 lM Ca2+,
380 lM PO43�, 993 lM K+, 270 lM Mg2+, 272 lM SO4
2�,2 lM Mn2+, 0.0085 lM Zn2+, 0.15 lM Cu2+, 20 lM B,
1.44 lM Mo2+, and 40.5 lM Fe2+) three times weekly until
harvest. The damage treatments (described below) were
applied 54 days post-sowing when the plants reached the
6-7 leaves stage.
Herbivores
Manduca sexta is a specialist caterpillar of the Solanaceae
family, which includes tomato. Caterpillar’s oral secretion
is often used to elicit plant defense because it mimics the
response of real herbivores (Kessler & Baldwin, 2002), but
it allows standardization for tissue loss and control of the
induced response timing. To simulate herbivory in our
experiments,M. sexta regurgitant from fifth instars reared
on tomato plants for at least 48 h was used. The extracted
regurgitant from several individuals was combined and
frozen (�80 °C) until further use.
Damage treatments
Plants were randomly divided into three experimental
groups. There were two damage treatments and an
untreated group. In the carbon dynamics study, seven
plants were used in each damage treatment and four in the
control group. In the metabolomics study, six plants were
used per treatment. The damage treatments were applied
on source leaves one, three, and four counting from the
apex (L1, L3, and L4, respectively). L1 was at least 50%
expanded and L3 and L4 were fully expanded. The damage
treatment consisted of running a fabric wheel on the edge
of every leaflet in L1, L3, and L4, creating homogeneous
punctures on which a total of 80 ll of either distilled water(mechanical damage treatment, hereafter referred to as
W-plants) or caterpillar regurgitant (M. sexta treatment;
hereafter referred to as R-plants) were applied with a brush
on the lower and upper side of each leaf. The regurgitant
was centrifuged prior to application on the leaves to
precipitate plant matter. Plants received damage twice (at
10:00 and 11:30 hours) as in Schwachtje et al. (2006). The
purpose of the ‘mechanical treatment’ was to control for
the effects of mechanical damage alone caused by the fab-
ric wheel that are also present in theManduca regurgitant
treatment. The plants in the untreated group received no
damage, but were handled and touched as in the other two
treatments.
Radioisotope administration
The radioisotope 11CO2 (t1/2 = 20.4 min) was used to fol-
low recently assimilated carbon by an undamaged leaf
throughout untreated and damaged plants [see Ferrieri
et al. (2005) for full details on isotope related methodol-
ogy]. Due to the short half-life of 11C, the same plants
could be labeled pre- and post-treatment on two consecu-
tive days so that each plant served as its own control. Base-
line values were measured on Day 1 (pre-treatment) and
on Day 2 (post-treatment), damage was applied 4 h prior
to radiotracer administration. Radiotracer administration
took place at the same time of the day on both days
(Gomez et al., 2010). Briefly, each plant was individually
placed in a lighted hood for radioisotope administration 30–60 min prior to labeling. An air-tight leaf cell (5 9 10 cm)
was sealed over two lateral leaflets of L2, into which a pulse
of 11CO2 was administered. A light source was placed
directly on top of the leaf cell (920 lmol m�2 s�1).
Radioisotope measurements
The movement of the labeled photosynthate was tracked
in vivo for 2 h by a PIN diode radiation detector (Bioscan,
Washington, DC, USA) on the leaf cell and two sodium-
iodide scintillation detectors (Ortec, Oak Ridge, TN, USA)
directly above the apex area and at the root area, respec-
tively. The detectors recorded (1) 11C radiotracer uptake
by L2 (11CO2 fixation), calculated as the percentage of11CO2 fixed by the end of the radiotracer pulse; (2) radio-
tracer export from L2, which was calculated as the percent-
age of activity that left the labeled leaf after 2 h; and (3)
relative allocation of the radiotracer to the apex and roots,
calculated as the percentage of the total activity allocated
to either apex or root tissues. All the calculations were per-
formed using activity decay corrected values to account
for radioisotope decay over time.
Plant harvest, sample preparation, and metabolomic analysis
Plants were harvested at 14:00 hours, 4 h after the first
damage event and each plant was divided in five tissue
types: developing apex leaves, damaged leaves (L1, L3, and
L4 pooled together), undamaged leaf (L2), stems, and
roots. Each tissue was flash-frozen in liquid nitrogen and
stored at�80 °C until sample preparation.
Tissue extraction and preparation for the metabolite
analysis was done as described in Lisec et al. (2006).
Whole-plant changes after simulated herbivory 103
Briefly, frozen samples were ground under liquid nitro-
gen and a subsample of 100 mg fresh weight was
extracted in 1.4 ml 100% methanol with 60 ll of ribitolas internal standard. The extracts were incubated at 70 °Cfor 15 min and centrifuged at 18 067 g for 10 min. The
supernatant was mixed with 750 ll chloroform and
1.5 ml water to separate polar and non-polar compounds.
The mixture was centrifuged for 15 min at 1 475 g.
A 150 ll aliquot from the polar phase was vacuum dried
and stored until further analysis. The samples were
reconstituted in 40 ll of methoxyaminhydrochlorid and
derivatized for 2 h at 37 °C. Small metabolites were mea-
sured using gas chromatography-mass spectroscopy and
electron ionization. Samples (1 ll) were injected into a
GC–TOF–MS system (Pegasus III; Leco, St Joseph, MI,
USA). Chromatograms and mass spectra were evaluated
using CHROMA TOF 1.6 and TAGFINDER 4.0 software
(Luedemann et al., 2008). Retention time index and mass
spectrum for individual compounds were compared with
library sets, providing a list of identified compounds. The
mass spectra were cross-referenced with those in the Golm
Metabolome Database (http://gmd.mpimp-golm.mpg.de;
Kopka et al., 2005). Mass-normalized peak area values of
treatment samples were compared with samples from
undamaged plants, yielding relative metabolite concentra-
tions after each damage treatment.
Statistical analysis
Paired t-tests were used to test for treatment effects on 11C
fixation, export, and root allocation pre- and post-dam-
age. The metabolomic data set consisted of concentrations
of 60 metabolites relative to undamaged plants measured
in the five different tissues. Not all 60 metabolites were
present in detectable levels in all tissues, but metabolites
present in one tissue were always detected across treat-
ments. Principal component analysis (PCA) was per-
formed to visualize patterns in different treatments and
tissues. One-way multivariate analysis of variance
(MANOVA) was performed on PCs 1-3 as response
variables for each tissue to test for treatment effect. Mean
peak areas for individual metabolites from six plants per
treatment were compared with means from undamaged
control plants using a Student’s t-test. All analyses were
performed using JMP 9.0 (SAS Institute, Cary, NC, USA)
and SPSS 19 (IBM, Armonk, NY, USA).
Results
Carbon dynamics: 11C fixation, export, and allocation
There was no treatment effect on 11CO2 fixation or leaf
export (Figure S1, Supporting information). Leaf export
comprised on average 29% of total recently fixed carbon
across treatments. Despite the lack of treatment effect on
carbon fixation and export, application ofM. sexta regurg-
itant resulted in a significant increase in 11C allocation to
roots (Figure 1). Four hours after damage, R-plants parti-
tioned 72% of the 11C-photosynthate exported from the
undamaged load leaf (L2) to the roots, as opposed to 53%
before damage occurred. Mechanical damage alone did
not cause an increase in root allocation.
Metabolomics
Principle component analysis revealed a high clustering of
metabolic profiles by tissue (Figure 2). A one-way MA-
NOVA on the top three principal components showed a
significant effect of treatment on the metabolite profile of
damaged leaves (L1, L3, L4) (F6,26 = 9.617, P<0.0001) androots (F6,26 = 2.687, P = 0.036), but not of apex, undam-
aged leaves (L2), or stem tissue (Table 1). Interestingly,
the extent of variation within treatments was extremely
low for roots. This low variation, along with the fact that
the MANOVA included three principal components,
explains the significant treatment effect despite the similar
clustering evident in Figure 2.
Most metabolite changes occurred in the sink tissues
(apex and roots) and damaged leaves (Figure 3). Individ-
ual metabolites that significantly changed in the apex,
undamaged leaves, and the stem generally increased in
both damage treatments compared with control plants
(especially amino acids; Table 2). In roots, the general
trend was a decrease in concentrations of individual
metabolites (especially sugars) and damaged leaves
showed a mixed pattern – mechanical damage alone
decreased sugars, whereas addition ofM. sexta regurgitant
increased sugars and amino acids (particularly glycine,
0
20
40
60
80
100
Control (4) Water (7) Regurgitant (7)
% 11
C ro
ot p
artit
ioni
ng ns ns **
Figure 1 Mean (+ SE) 11C-photosynthate export allocated toroots (%) pre-damage (white bars) and 4 h post-damage (black
bars). The number of plants tested is indicated in parentheses.
Significant changes between pre- and post-damage values are
marked by asterisks (Paired t-test: **, 0.001>P>0.01; ns, notsignificant).
104 Gomez et al.
which increased 10-fold after regurgitant application;
Table 2). Although both damage treatments tended to
follow the same general pattern in the various tissues, there
was no complete overlap in individual compounds
increasing or decreasing (Figure 3).
Discussion11C fixation, transport, and allocation
Herbivory often results in decreased photosynthesis due to
disruption in the vascular system, down-regulation of
photosynthesis, changes in sink demands, and autotoxicity
(Nabity et al., 2009). In our system, application ofM. sexta
regurgitant did not alter photosynthesis or leaf export of
recently fixed carbon from undamaged leaves in response
to any damage treatment. Interestingly, although the
amount of 11C-photosynthate exported from source
(undamaged) leaves to sink tissues (apex and roots) was
similar to undamaged plants, R-plants showed an altered
allocation pattern, having 72% of 11C-photosynthate
export partitioned to roots instead of 53% as shown
pre-damage. This suggests that cues in the regurgitant of
M. sexta (Halitschke et al., 2001) altered the sink strength
balance between apex and roots, favoring belowground
allocation. Herbivory has been shown to alter source-sink
gradients in other systems (Arnold & Schultz, 2002; Kap-
lan et al., 2011). In tobacco, simulated herbivory resulted
in increased root sink strength and subsequent accumula-
tion of sugars in the roots shortly after damage
(Schwachtje et al., 2006). Remobilized resources might be
temporarily unavailable to other plant functions such as
growth or defense, and might thus be costly for the plant
(Orians et al., 2011). If the plant uses the sequestered
resources after damage to increase fitness, however,
–8
–4
0
4
8
–8 –4 0 4 8
Prin
cipa
l com
pone
nt 2
(26.
5%)
Principal component 1 (35.4%)
L1,L3,L4 (damaged)
Apex
Root
Stem L2 (undamaged)
Figure 2 Principal component (PC) analysis of metabolic
profiles of tomato plants. Plot of top two PCs including 60
metabolites measured in various tissues. The amount of variation
explained by each PC is given in parentheses. Symbols indicate
average (± SE; n = 6 each) concentrations for control plants
(circles), mechanically damaged plants (triangles), and
regurgitant-treated plants (squares).
Table 1 Treatment effects on the metabolomic composition of
various tissues of tomato plants. Results of a one-way MANOVA
on principal component 1, 2, and 3 in individual tissues
Tissue F6,26 P
Apex 1.075 0.40
L2 (undamaged) 2.687 0.036
L1, L3, and L4 (damaged) 9.617 <0.0001Stem 1.241 0.32
Root 1.799 0.14
13 8
W R
4
4 0
W R
0
3 11
W R
3
3 0
W R
0
0 1
W R
0
0 0
W R
0
0 0
W R
0
4 4
W R
1
0 0
W R
0
8 12
W R
6
Up Down
Apex
L2 (undamaged)
L1, L3, L4 (damaged)
Stem
Root
Figure 3 Venn diagrams showing significant increasing (left
pannels) and decreasing (right pannels) metabolites compared
with control plants in apex, undamaged leaves (L2), damaged
leaves (L1, L3, L4), stem, and root. In each venn diagram, the
circle on the left represents metabolic changes in water-treated
plants (W), the circle on the right representsmetabolic changes
in regurgitant-treated plants (R), and the overlapping area
represents the number ofmetabolites that change in both
treatments.
Whole-plant changes after simulated herbivory 105
Table2
Ratiosofindividualmetaboliteconcentrationsineach
dam
agetreatm
entrelativeto
controls(W
,water;R
,regurgitant)invarioustissuesoftomatoplants
Metabolites
Apex
L2(undam
aged)
L1,L3,L4
(dam
aged)
Stem
Root
WR
WR
WR
WR
WR
Aminoacids
Alanine
1.3
1.0
Arginine
1.1
1.0
1.0
1.0
Asparagine
0.7
0.7
Asparticacid
1.3
1.1
1.0
1.1
1.0
0.9
1.2
1.2
0.9
0.9
b-Alanine
1.2
1.3
1.0
1.3
1.5
2.0
1.0
0.8
0.9
0.8
GABA
1.2
1.4
0.6
0.7
0.7
0.6
0.8
0.8
0.6
0.7
Glutamate
1.3
1.3
1.0
1.1
1.1
1.2
1.3
1.3
1.0
0.9
Glutamine
1.8
1.7
1.9
1.4
1.0
0.9
1.9
1.3
0.8
0.7
Glycine
1.7
2.3
1.1
10.9
1.5
1.3
Isoleucine
1.5
1.5
1.4
1.3
1.1
1.7
1.1
1.0
1.0
0.8
Lysine
1.4
1.4
1.4
1.3
1.0
1.1
0.9
0.8
Methionine
1.6
1.3
1.3
1.4
1.2
1.3
Ornithine
0.8
1.0
1.8
1.5
1.5
1.5
Phenilalanine
1.2
1.1
1.3
1.1
1.3
1.6
1.1
1.0
0.9
0.8
Proline
1.9
1.1
1.2
1.6
1.6
1.7
1.3
1.0
1.2
0.9
Serine
1.4
1.3
1.3
1.3
1.1
1.2
1.1
1.1
1.1
1.1
Threonine
1.3
1.2
1.2
1.2
1.0
0.9
1.3
1.1
1.0
0.9
Tryptophan
1.8
1.7
0.9
1.0
1.2
3.1
1.5
0.9
0.8
0.7
Tyram
ine
1.3
1.4
1.2
1.1
0.9
1.3
1.0
0.9
Tyrosine
1.3
1.2
1.2
1.0
1.3
1.1
1.0
0.8
Valine
1.4
1.4
1.4
1.2
1.0
1.4
1.2
0.9
1.0
0.9
Sugars
Erythritol
0.9
1.2
0.9
1.2
Fructose
1.0
1.0
1.1
0.8
0.8
0.9
1.2
1.1
0.7
0.7
Fructose-6-phosphate
1.2
1.7
1.6
1.0
1.0
1.0
0.9
1.1
Fucose
0.8
1.0
0.8
1.1
Galactinol
1.2
1.0
1.0
1.1
1.0
1.0
1.1
1.2
1.0
1.1
Glucose
0.7
0.8
1.3
0.9
0.7
0.9
1.1
1.0
0.8
0.7
Glucose-6-phosphate
1.2
1.8
1.1
1.0
1.3
2.5
0.8
1.1
Isomaltose
1.1
1.2
0.8
0.9
Maltitol
1.1
1.2
Maltose
1.0
0.9
1.0
1.1
0.8
1.6
Maltotriose
0.6
0.7
106 Gomez et al.
Table2
Continued
Metabolites
Apex
L2(undam
aged)
L1,L3,L4
(dam
aged)
Stem
Root
WR
WR
WR
WR
WR
Myo-inositol
1.0
0.8
1.0
1.0
1.1
1.2
1.2
1.1
0.9
0.9
Raffinose
1.3
1.0
0.9
2.2
0.9
0.8
0.7
0.6
Rham
nose
1.1
0.9
0.9
1.1
0.9
0.8
1.2
1.1
0.9
0.8
Sorbitol
1.1
1.4
Sucrose
1.1
1.0
1.2
1.0
1.1
1.0
1.1
0.9
0.6
0.7
Xylose
1.0
1.0
1.1
1.1
0.7
0.8
1.0
1.0
0.9
0.8
a,a’Trehalose
1.1
1.2
1.0
1.0
1.1
1.0
0.8
0.7
Organicacids
2-Oxo-glutaricacid
0.9
0.8
1.1
1.1
0.9
1.0
Caffeicacid
1.4
1.1
0.9
0.9
Citricacid
1.6
1.6
0.9
1.0
1.0
1.1
1.3
1.0
0.7
0.7
Dehydroascorbicacid
1.4
1.2
1.0
1.1
0.9
0.8
1.2
0.9
1.0
0.9
Fumaricacid
1.6
1.5
1.4
1.3
1.2
1.1
1.2
1.2
0.8
0.8
Galactonic/glucuronicacid
1.3
1.2
1.3
1.1
0.9
0.9
Glycericacid
1.1
1.2
1.2
1.2
0.9
1.0
0.9
0.9
Malicacid
1.3
1.2
1.1
1.0
0.8
0.8
1.2
1.2
1.0
1.0
Pyruvicacid
0.9
0.4
Quinicacid
1.1
1.0
1.2
1.0
1.2
1.2
0.8
0.8
Saccharicacid
1.2
1.9
1.1
2.8
1.1
1.1
0.8
0.9
Succinicacid
1.2
1.0
0.9
1.0
1.0
1.2
1.0
1.0
Threonicacid
0.9
1.2
0.8
1.8
Miscellaneous
1-O-m
ethyl-a
glucopyranoside
1.0
1.2
0.9
0.8
3-Caffeoyl-cis-quinicacid
1.2
1.1
1.1
1.0
1.1
1.0
1.2
1.3
3-Caffeoyl-trans-quinicacid
1.2
1.1
1.1
1.1
0.7
0.9
1.3
1.1
1.2
1.2
Glycerol-3-phosphate
1.2
1.6
0.9
1.0
Nicotinicacid
0.8
0.7
Phosporicacid
1.1
2.0
0.6
1.0
Putrescine
1.6
1.5
0.8
1.2
2.0
2.3
1.0
1.0
0.9
0.9
Pyroglutamicacid
1.4
1.4
1.5
1.3
Values>1and<1indicatean
increaseanddecrease,respectively,inpoolsizeinacertaintreatm
entcompared
withtheuntreatedcontrolplants.Statisticallysignificantdifferencesfrom
undam
aged
plants(Student’st-test:P<0.05)aremarkedinboldtext.M
arginallysignificant(P<0.1)
valuesaremarkedinitalics.
Whole-plant changes after simulated herbivory 107
induced sequestration would have an adaptive value for
the plant. In contrast to R-plants, W-plants showed no dif-
ference in root allocation. Similarly in N. attenuata, only
plants treated with herbivore regurgitant exhibited
changes in carbon transport and the authors suggested that
roots of plants treated with herbivore regurgitant might
import sugars more efficiently (Schwachtje et al., 2006).
Chemical profiles in source and sink tissues: herbivore-derived cuesvs. mechanical damage
Damaged leaves. Damage resulted in the local
concentration increase of several metabolites compared
with control plants. Although there was certain overlap in
the altered metabolites in both damage treatments,
herbivore-derived cues led to a distinct chemical profile in
damaged leaves by altering the concentrations of almost
four times more metabolites. In regurgitant-treated leaves,
30% of the detected metabolites were significantly
different from those of control plants, compared with only
15% of metabolites in W-plants. This shows that
mechanical damage alone leads to very different responses
(i.e., changes in various metabolites) than herbivore-
derived cues, as shown for 11C-photosynthate transport
within the plant.
Both damage treatments shared changes in somemetab-
olites likely due to the physical disruption caused by the
mechanical damage inflicted on the leaves in both treat-
ments. Mechanical damage is unavoidable during leaf
chewing and it has its own effects on the plant’s physiol-
ogy, such as an increase in water loss due to breakage of
vascular tissue (Aldea et al., 2005). This might lead to
localized water stress as suggested by the observed increase
in proline, an osmoprotectant typically elevated under
water stress conditions (Hare & Cress, 1997). Water stress
has been shown to induce nutrient recycling from stressed
parts to other tissues (White, 1984). Consistent with this,
damaged leaves in both damage treatments showed ele-
vated concentrations of b-alanine and putrescine, which
are both products of the catabolism of the polyamine sper-
midine (Rastogi & Davies, 1990). Accumulation of putres-
cine, and polyamines in general, has also been suggested to
play a role in the wound response (Walters et al., 2002;
Cowley &Walters, 2005).
Major sugars did not change in response to either treat-
ment on damaged leaves. A number of other sugar-related
compounds, however, increased in regurgitant-treated
leaves. The accumulation of maltose (a product of starch
hydrolysis) and glucose-6-phosphate (a more stable form
of glucose) in regurgitant-treated leaves could be due to an
increase in the breakdown of leaf carbon reserves (starch)
in response to herbivore-derived cues. Such local starch
degradation has been shown in Populus tremuloides
Michx., 8 h after jasmonic acid application (Babst et al.,
2005). In our study, starch degradation would represent
‘old’ carbon pools and may explain why an increased leaf
export of 11C-photosynthate was not observed. The lack of
accumulation of sucrose suggests that this metabolite is
either readily exported out of the damaged leaves or that
an increase in concentration is not evident 4 h after herbi-
vore damage. Although sucrose is the major transport
sugar in most plants, sorbitol and oligosaccharides in the
raffinose family have also been linked to transport and
storage of carbon (Philippe et al., 2010), both of which sig-
nificantly increased in regurgitant-treated leaves. Thus, it
is possible that an increase in those substrates as a conse-
quence of starch degradation may result in subsequent
carbon remobilization to other tissues and/or support
secondarymetabolism (Broeckling et al., 2005).
Manduca sexta cues resulted in a decrease in pyruvic
acid and a marginally significant increase in succinic acid,
which suggests greater utilization of substrates involved in
cellular respiration (tricarboxylic acid cycle). Higher cellu-
lar energy might be used to sustain cellular processes dur-
ing herbivore damage including secondary metabolism. In
agreement with such defensive role, there was an increase
in several amino acids involved in defense signaling and
defense production. The amino acid isoleucine forms con-
jugates with jasmonic acid and it is involved in defense sig-
naling (Staswick & Tiryaki, 2004). Isoleucine almost
doubled its concentration in damaged leaves of regurgit-
ant-treated plants, possibly providing a larger pool for
induced hormone production. Tryptophan in regurgitant-
treated plants was almost three-fold higher than control
plants, but it did not differ to mechanically damaged
plants. Phenylalanine was 1.6 times higher than control
plants in damaged leaves, but this increase was only mar-
ginally significant. Both tryptophan and phenylalanine are
end products of the shikimate pathway and can lead to the
formation of defense compounds, such as alkaloids and
phenolics, which can be toxic to many herbivores (Bennett
&Wallsgrove, 1994). Tryptophan is also an important pre-
cursor in auxin production, a hormone involved in plant
growth (Woodward & Bartel, 2005). A similar rapid tryp-
tophan and phenylalanine increase was shown in tobacco
plants after MeJA treatment (Hanik et al., 2010a) suggest-
ing that regardless of the role of these amino acids on
either regrowth or defense, it may be a common response
in the Solaneaceae family against herbivores.
Glycine concentration exhibited the most dramatic
change of all measured metabolites, increasing 10-fold in
regurgitant-treated leaves. In a study on Nicotiana taba-
cum L., MeJA application resulted in an 50% increase in
glycine and other amino acids in the treated leaves 15 h
post-treatment, but no change was observed in NH3
108 Gomez et al.
assimilation, suggesting this increase might represent a
non-photorespiratory source of nitrogen to rapidly supply
energy for defense production (Hanik et al., 2010b). Alter-
natively, it remains uncertain, but possible that this glycine
had an insect rather than a plant origin as it was observed
locally in the damaged leaves where regurgitant was
applied and only to a much lesser extent in the apex. Free
glycine is highly abundant in the midgut content of several
lepidopteran families where it counteracts plant protein
denaturing activity to maintain leaf quality (Konno et al.,
1996, 1997).
Undamaged leaves and stem. The undamaged source
leaf shares vascular connections with the damaged
leaves (Orians et al., 2000), implying that transport of
metabolites and defense signaling to L2 is possible. In
contrast with the metabolically active damaged leaves,
however, the undamaged source leaf and stem in R-plants
showed no significant metabolic changes compared with
control plants. It is possible that 4 h after damage any
changes inmetabolite concentrations had already returned
to control values or that the systemic response was still not
apparent at that time. W-plants, however, did show some
changes in metabolite concentrations in the undamaged
leaf and in the stem. Spatial studies on expression of
defense proteins in tomato have shown that different types
of damage, leaf age, and timing are important factors
influencing expression of systemic induction (Stout et al.,
1996), which might explain the lack of changes in
undamaged source leaves of R-plants.
Sink tissues: apex, roots. Apex and roots are strong sink
tissues within a plant. In fact, undamaged tomato plants
exhibited ca. 1:1 allocation of newly acquired exported
carbon among both sinks, which only changed when
herbivore regurgitant was added to the wounds. Most of
the metabolic changes occurred in those two tissues,
regardless of the type of damage. Metabolite con-
centrations tended to increase in the apex, but tended to
decrease in the roots. The two damage treatments showed
a high degree of overlap in metabolic changes in the roots
(75% of the increased metabolites in the W-plants were
the same in the R-plants), there was less overlap in the
apex (50%). This implies that changes aboveground are
muchmore specific to the type of damage.
In the apex, all significant concentration increases in
individual metabolites were stronger and more abundant
in response tomechanical damage. These included numer-
ous amino acids and some organic acids, but no sugars. In
agreement with a possible response to water stress due to
physical damage, the concentrations of two major trans-
port amino acids (glutamate and glutamine) increased,
suggesting increased breakdown of protein and/or poly-
amines. This is consistent with the observed increase in
putrescine. In the apex of R-plants, there was an increase
in glucose-6-phosphate and fructose-6-phosphate, which
was not present in W-plants. These are both products
from glycolysis and therefore important sources for energy
production. Energy derived from glycolysis might be
needed to produce defense compounds or their precursors
to protect valuable young tissues (Arnold & Schultz,
2002).
There was an increase in root sink strength as shown by
the higher allocation of 11C-photosynthate. The overall
decrease in metabolite concentrations in the roots despite
the increase in carbon allocation might be explained by
rapid conversion of soluble sugars into starch pools or
rapid use to support root respiration to increase nutrient
uptake. Increased nutrient uptake might support defense
production and/or future regrowth. In N. attenuata,
M. sexta regurgitant caused a similar increase in root allo-
cation of 11C-photosynthate and lack of subsequent sugar
increase, suggesting that carbon resources were rapidly uti-
lized (Schwachtje et al., 2006). Most altered metabolites
were responsive to both damage treatments. Several amino
acids and sugar related compounds decreased in roots of
R-plants, includingmajor sugars like sucrose and glucose.
Conclusion
We showed that tomato plants undergo a wide metabolic
reprogramming detectable soon after damage at the whole
plant level, especially affecting sink tissues. Plants treated
with regurgitant exhibited greater carbon allocation to
roots and metabolic profiles were highly tissue-specific
and damage-specific for some tissues. More specifically,
herbivore-derived cues resulted in metabolic changes that
hint at a reorganization of resources in damaged leaves,
perhaps from the breakdown of temporary leaf starch
reserves, and an increase in accumulation of primary
metabolites in the apex and damaged leaves that could
(a) supply the high sink demand in the roots as shown by
the general decrease of metabolites, and (b) fuel defense
production in young and damaged leaves. How these
short-term changes alter patterns of regrowth, storage,
and defense induction in the long-term deserves further
attention.
Acknowledgements
The authors thank B. Trimmer for providingM. sexta lar-
vae and three anonymous reviewers for valuable feed-back
on the manuscript. ADS was supported by The Neubauer
Scholars Program, The Paula Frazier Poskitt Memorial
Whole-plant changes after simulated herbivory 109
Scholarship, and the Astronaut Scholarship. This research
was supported by the National Research Initiative of
the USDA Cooperative State Research, Education and
Extension Service under USDA/CSREES grant 2007-
35302-18351 to CMO and supported by DOE’s Office of
Biological and Environmental Research under contract
DE-AC02-98CH10886 to RAF.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 (A) Mean (+ SE) percentage of 11CO2 fixed
and (B) percentage of the fixed 11C-photosynthate
exported out of undamaged source leaf 2, pre-damage
(white bars) and 4 h post-treatment (black bars). The
number of plants tested is indicated in parentheses.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
Whole-plant changes after simulated herbivory 111
