J Chem Ecol (2010) 36:1058–1067
DOI 10.1007/s10886-010-9835-x
Partitioning of New Carbon as 11C in Nicotiana tabacum
Reveals Insight into Methyl Jasmonate Induced Changes
in Metabolism
Nils Hanik & Sara Gómez & Marcel Best &
Michael Schueller & Colin M. Orians &
Richard A. Ferrieri
Received: 26 May 2010 / Revised: 8 July 2010 / Accepted: 19 July 2010 / Published online: 15 September 2010
# Springer Science+Business Media, LLC 2010
Abstract We examined the timeline by which methyl
jasmonate (MeJA) reprograms new carbon partitioning into
key metabolite pools. The radioactive isotope 11C (t1/2
20.4 min), administered to intact leaves of Nicotiana
tabacum L. (cv Samsun) as 11CO2 gas enabled us to
measure changes in new carbon partitioning into soluble
sugar and amino acid pools of [11C]photosynthate. A
500 μM MeJA treatment resulted in a decrease in the
[11C]soluble sugar pool and an increase in the [11C]amino
acid pool after 4 h. This pattern was more pronounced 15 h
after treatment. We also examined the timeline for
11
C-partitioning into aromatic amino acid metabolites of
the shikimate pathway. [11C]Tyrosine, [11C]phenylalanine
and [11C]tryptophan were elevated 1.5-fold, 12-fold and
12-fold, respectively, relative to controls, 4 h after MeJA
treatment, while endogeneous pools were unchanged. This
suggests that only new carbon is utilized during early stages
of defense induction. By 15 h, [11C]tyrosine and [11C]
phenylalanine returned to baseline while [11C]tryptophan
N. Hanik : M. Best
Fachbereich Chemie, Johannes Gutenberg Universität,
55099 Mainz, Germany
S. Gómez
Department of Biological Sciences, University of Rhode Island,
Kingston, RI 02881, USA
M. Schueller : R. A. Ferrieri (*)
Medical Department, Brookhaven National Laboratory,
Upton, NY 11973, USA
e-mail: [email protected]
S. Gómez : C. M. Orians
Department of Biology, Tufts University,
Medford, MA 02155, USA
was elevated 30-fold, suggesting that MeJA exerts selective
control over the shikimate pathway. Finally, we measured
trans-cinnamic acid levels as a gauge of downstream
phenolic metabolism. Levels were unchanged 4 h after
MeJA treatment relative to controls, but were increased
2-fold by 15 h, indicating a lag in response of secondary
metabolism.
Key Words Methyl jasmonate . Plant defenses .
Metabolic partitioning . Chemical signaling .
Short-lived radiotracers . 11C . Nicotiana tabacum .
Shikimate pathway
Introduction
The ability of plants to withstand the stresses imposed on
them depends on a highly coordinated timeline of events,
much of which still is poorly understood. Early events that
occur from within milliseconds to minutes of damage can
include perturbations to the plasma membrane potential,
variations in cytosolic Ca+2, and variation in levels of
reactive oxygen species (Maffei et al., 2007). These events
allow for early recognition of the nature and severity of
damage, and can trigger downstream networks involving
protein kinases (Wu et al., 2007) and phytohormones
(Devoto and Turner, 2005) such as jasmonic acid (JA),
and its methyl ester (MeJA) involved in signal transduction.
Jasmonic acids are ubiquitous in all plants, and they fill
many roles in plant growth and development. For example,
they are involved in the regulation of numerous developmentrelated physiological processes (Sembdner and Parthier, 1993;
Creelman and Mullet, 1997; Wasternack, 2007) including
embryogenesis, seed germination, flowering, pollen devel-
J Chem Ecol (2010) 36:1058–1067
opment (Creelman and Mullet, 1997; Wasternack and
Parthier, 1997; Feussner and Wasternack, 2002), drought
stress (Moons et al., 1997), accumulation of storage proteins
(Beardmore et al., 2000) and leaf senescence (He et al.,
2002) although the latter effect is controversial (see
Seltmann et al., 2010). Jasmonates are especially well
known for their roles played in defense against tissue
damage, pathogen infection, and herbivory (van Kleunen
et al., 2004; Arnold and Schultz, 2002; Howe, 2004;
Babst et al., 2005; Truman et al., 2007), and serving as
local and systemic signal molecules (Zhang and Baldwin,
1997; Stratmann, 2003; Ferrieri et al., 2005; Thorpe et al.,
2007). They promote the transcriptional modification of
numerous genes (Jung et al., 2007) that can impact wholeplant resource allocation of both nitrogen- and carboncontaining substrates (Meuriot et al., 2004; Babst et al.,
2005; Gómez et al., 2010), as well as manifest in
responses at cellular levels (Creelman and Mullet, 1997;
Rickauer et al., 1997; Xie et al., 1998; Reymond and
Farmer, 1998; Weber, 2002; Cheong et al., 2002; Farmer
et al., 2003; Pauwels et al., 2008).
Such responses can manifest in the up-regulation of
specialized biochemical pathways that have evolved as
defenses against abiotic and biotic stressors (Osbourn, 1996;
Farmer et al., 2003; Zhao et al., 2005). For example, the
pathway of phenolic acid metabolism in plants requires the
initial steps of general phenylpropanoid metabolism providing precursors for the synthesis of lignin, tannins, flavonoids,
and other phenolics that serve to build physical barriers and/
or reserves of alleochemicals in plant defense (Arnold and
Schultz, 2002). It is well known that JAs will elevate the
activity of key enzymes, including phenylalanine ammonialyase (Hudgins et al., 2004; Bower et al., 2005) and caffeoyl
CoA O-methyltransferase (Lee et al., 1997), that promote the
deamination of phenylalanine, the primary substrate of this
pathway, as well as catalyze the O-methylation of key
intermediates leading to increased lignification (Lee et al.,
1997; Caño-Delgado et al., 2003; Hudgins et al., 2004).
Upstream from the phenylpropanoid pathway, the biosynthesis of aromatic compounds proceeds via the seven-step
shikimate pathway to a branch point intermediate chorismate
(Kloosterman et al., 2003). Chorismate subsequently is
converted to three aromatic amino acids, tryptophan, tyrosine,
and phenylalanine, as well as other aromatic compounds, via
specific terminal pathways. This process is coupled tightly
via metabolite feedback control mechanisms (see dashed lines
in Fig. 1) impacting a subset of three key regulatory enzymes
in the pathway: 3-deoxy-D-arabino-heptulosonate 7phosphate (DAHP) synthase, chorismate mutase, and prephenate dehydratase (Bickel and Schultz, 1979; Herrmann
and Weaver, 1999). Yet, little is known about the regulation
of the shikimate pathway in concert with downstream
phenolic metabolism.
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Current “omics” research on plant defense responses has
provided many new insights into induced plant responses
and when coupled with physiological and biochemical
studies at the whole plant level, can be illuminating. For
example, Naoumkina et al. (2007), using cell cultures from
Medicago truncatula, observed a 24 h delay in transcriptional modification of the early metabolic steps of the
phenylpropanoid pathway after MeJA treatment, but a rapid
(within hours) increase in medicarpin, an isoflavonoid
produced from this pathway. This highlights that transcriptional up-regulation is not necessarily coupled with biochemical up-regulation.
Understanding the latent manifestations of highly integrated and interactive response networks can be difficult
(Meuriot et al., 2004; Jung et al., 2007; Pauwels et al.,
2008). In a recent prospectus, Schwachtje and Baldwin
(2008) suggest that a key link in the defense network is
reprogramming of primary metabolism, where such metabolites may themselves serve as signals that subsequently
activate downstream pathways. Thus, primary metabolites
may serve dual roles as signal and substrate for downstream
secondary metabolism.
Here, we report on the use of the short-lived radioisotope, carbon-11 (t½ 20.4 min) as 11CO2 to measure in
Nicotiana tabacum the partitioning of new carbon into
metabolite pools of photosynthate, as well as into metabolites of the shikimate pathway. The use of carbon-11 for
studying the partitioning of new carbon into metabolic
pools enabled us to retest the same plant over short periods
of time. These studies would be difficult to carry out if not
impossible by using carbon-14 or carbon-13, as the
metabolic pools would become enriched with tracer
preventing a clear delineation between new and old carbon
utilization. Furthermore, the use 11C at true tracer concentrations placed us in a unique position to explore the effects
MeJA had on [11C]photosynthate pools unimpaired by the
feedback control mechanisms described above. Most
particularly, we were able to explore how such effects
filtered down to regulation of the shikimate pathway and
its coordination with up-regulation the phenylpropanoid
pathway.
Here, we hypothesized that MeJA would cause rapid
changes in [11C]photosynthate pools, thus favoring increased partitioning of new carbon into amino acids.
Furthermore, we hypothesized that MeJA would impart
selective control over the shikimate pathway, thus giving
rise to different partitioning of new carbon into its
metabolites. We showed for the first time that defense
induction, triggered by application of MeJA, can manifest
in selective reprogramming of new carbon utilization in the
shikimate pathway. As importantly, we show that metabolic
reprogramming can be transient matching key transcript
expression patterns.
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J Chem Ecol (2010) 36:1058–1067
Fig. 1 Shikimate pathway showing known metabolite feedback
loops (dashed lines) that can
affect metabolic regulation. This
pathway is controlled by three
key regulatory enzymes: 3deoxy-D-arabino-heptulosonate
7-phosphate synthase, chorismate
mutase, and prephenate dehydratase. Metabolite feedback loops
truncate at specific branch points
within the pathway and are designated by+or—for promotion or
inhibition (as adapted from Bickel
and Schultz, 1979; Herrmann and
Weaver, 1999)
Methods and Materials
General Approach In the studies described, we devised a
protocol for measuring the effects of our treatment on the
partitioning of recent carbon (as 11C) and unlabeled
endogenous carbon (as 12C), into key metabolite pools.
Specifically we dosed leaf-3 (counting down from the apex)
of an intact plant with 11CO2 tracer, and then 4 h or 15 h
after treatment, we administered a second dose tracer to
leaf-4 of the same plant. A 2 d study cycle (Fig. 2) shows
the coordination in time between baseline and retest tracer
administrations with MeJA treatment and the plant’s light/
dark cycle. The present study also required extraction of
small amounts of tissue from the plant through leaf excision
at its petiole. Prior to beginning our MeJA studies, we
performed a proof-of-concept study to confirm that leaf
excision would not induce metabolic reprogramming. At 4
and 15 h post leaf-3 excision, the [11C]photosynthate
composition was unchanged between leaf-3 and leaf-4 as
was the 11C amino acid profiles for tyrosine, phenylalanine,
and tryptophan (data not shown). In summary, we found
that cleanly excising a study leaf from the plant at its
petiole does not induce changes in the metabolites of
interest within the remaining tissues. With this knowledge,
we designed an experimental protocol for testing same
plants by using separate doses of 11C before and after
treatment.
Plant Material Tobacco plants (Nicotiana tabacum L. cv
Samsun) were grown from seed in commercial potting mix
J Chem Ecol (2010) 36:1058–1067
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Fig. 2 Two-day study cycle of plants (Nicotiana tabacum) subjected
to either a 4 h or 15 h metabolite profiling. Changes in metabolite
profiles at either time point were always compared back to baseline
responses from the same plant. Baseline 11C tracer administration was
always at 09.45 h Leaf excision for tissue extraction always occurred
45 min after tracer administration. Note that the 4 or 15 hr metabolite
profiling time points dictated the time of day when control or MeJA
treatments were applied to the study plant. Treatments were always
applied during the light cycle
with a slow release fertilizer (Osmocote) under metal-halide
lamps at 24°C with a 16/8 h-350 umol m−2 sec−1 photoperiod, and were used for experiments at their 7-leaf stage of
development.
We administered 11C to leaf-3 and leaf-4 in series,
before, and after treatment. Leaf designation was determined by counting down from the apex, with leaf-1
designated as the first leaf that was expanded to 50% of
its full capacity.
Radiotracer Production 11CO2 was produced via the 14 N
(p,α)11C nuclear transformation (Ferrieri and Wolf, 1983)
from 50 ml volume high-purity nitrogen gas target using
17 MeV protons from the TR-19 (Ebco Industries Ltd,
Richmond, BC, Canada) cyclotron at Brookhaven National
Laboratory, and captured on molecular sieve (4Å) (full
details in Ferrieri et al., 2005).
Radiotracer Administration The 11CO2 that was trapped on
the molecular sieve was desorbed and quickly released into
an air stream at 400 mlmin−1 as a discrete pulse for labeling
a leaf in a 5×10 cm lighted cell (920 umol m−2 sec−1)
(Ferrieri et al., 2005). A PIN diode radiation detector
(Bioscan Inc., Washington DC, USA) affixed to the bottom
of the cell enabled continuous measurement of radioactivity
levels within the cell. The difference between the amount of
radioactivity registered in the pulse height, and residual
radioactivity after the pulse had passed through the cell was
a reflection of leaf tracer fixation. This value was used to
normalize measurements to the same amount of carbon-11. In
past studies (Ferrieri et al., 2005), we were able to correlate
these measurements in radioactivity with direct changes in
the CO2 concentration entering and exiting the leaf cell by
using infrared gas exchange, IRGA (Li-Cor model 6162).
Tissue Extraction and Derivatization Thirty minutes after
the tracer was administered, the study leaf was cleanly
excised at its petiole. The 5×10 cm area exposed to 11CO2
was cut away from the remaining leaf tissue, weighed, and
flash frozen in liquid nitrogen (LN2). Using a mortar and
pestle, the frozen tissue was ground to a fine particulate,
and extracted in 3 ml of methanol (50% v/v in DI
water) for 10 min at 90°C. The extract was separated by
pipette and filtered through a 0.22 μM filter (Millipore
Inc., Billerica, MA, USA). The total volume of the
extract was measured and 150 μl aliquot delivered into
a 2 ml brown-glass vial (Fisher Scientific, Inc.,
Pittsburgh, PA, USA). An equal volume of o-phthalaldehyde amino acid derivatizing reagent (OPA) containing
0.1% (v/v) mercaptoethanol and 0.1% (v/v) sodium
hypochlorite (Sigma Life Science, St. Louis, MI, USA)
was delivered into the same vial. The mixture was
vortexed and then allowed to react at ambient temperature for 3 min. Primary amino acids are readily converted
into UV absorbing iso-indole derivatives by OPA
enabling their separation with reversed-phase highperformance liquid chromatography and quantification
(Chow et al., 1987). While the OPA method has been
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successfully used for measuring amino acids in purified
plant extract, some have questioned how effective the
method is for unpurified extracts (Martin et al., 1982). To
confirm the validity of this method, we tested several
amino acid standards in pure methanol/water (50% v/v),
and in a methanol/water solution containing unpurified
tissue extract, and subjected each sample to OPA
reaction. Using the chromatography method described
in the next section, we observed the same UV response
from standards (within 5% standard error) in both types
of formulations.
Analysis of Amino Acids A 50 μl volume of the derivatized
mixture was injected onto a reversed-phased analytical
HPLC column (Phenomenex, Torrance, CA, USA: Ultramex™ C18, 10 μ particle size, 250×4.6 mm i.d.) using a
pre-column gradient mixer (Isco, Lincoln, NE, USA) and a
mobile phase comprised of A (DI water), B (0.01 M
NaH2PO4-buffered at pH 6.8 using trifluoroacetic acid),
and C (methanol). At injection, the mobile phase (1.5 ml
min−1) was sustained at 75% A: 25% B for 5 min and then
programmed to attain 20% B: 80% C by 30 min. The mass
levels of the amino acid derivatives were measured with a
variable wavelength UV detector (Sonntek Inc., Upper
Saddle River, NJ, USA) at 340 nm. The outlet of the UV
mass detector was connected in series to a NaI gamma
radiation detector (Ortec Inc, Oak Ridge TN, USA) that
enabled direct measurement of the amount of radioactivity
associated with each substrate eluting the column. Analog
outputs from both the mass and radiation detectors were fed
to a chromatography data acquisition station (SRI Instruments, Torrance, CA, USA) where integrated peak areas
were measured from both inputs.
Compounds were identified by their elution times and
correlated to those of authentic standards. Changes in
endogenous substrate concentrations were correlated by
correcting each chromatogram for the gram fresh weight of
tissue that was used for a particular extraction. Samples also
were corrected for analytical dilutions. Changes in radioactive substrate levels were normalized to a standard amount
of 11C tracer that was administered to a particular leaf.
Radioactivity data was decay corrected back to a common
time point owing to the extremely short half-life of 11C
(20.4 min). An additional 50 μl aliquot was taken in order
to assay for total [11C]photosynthate radioactivity in each
tissue extract. This measurement was carried out using a
separate static gamma counter. The efficiencies of both the
flow and static gamma counters were calibrated against
standards.
We note that although a full profile of [11C]amino acids
was measured, that data went beyond the scope of the
present work and are not reported here. However, we
pooled all observed radioactive amino acids and reported
J Chem Ecol (2010) 36:1058–1067
their total distribution relative to the soluble [11C]photosynthate fraction.
Analysis of Radioactive Soluble Sugars A 50 μl volume of
the methanol:water extract was injected onto a reversedphased analytical HPLC column (Phenomenex, Torrance,
CA, USA: Luna™ NH2, 5 μm particle size, 250×4.6 mm
i.d.). At injection, the mobile phase (1.5 mlmin−1) was
sustained at 80% acetonitrile: 20% water. Elution profiles
of soluble sugars were matched against standards using a
refractive index (RI) detector (Sonntek Inc., Upper Saddle
River, NJ, USA). For radioactive extract analysis, the
outlet of the RI detector was connected in series to a NaI
gamma radiation detector (Ortec Inc, Oak Ridge TN,
USA) that enabled direct measurement of the amount of
radioactivity associated with each substrate eluting the
column. Analog outputs from both the mass and radiation
detectors were fed to a chromatography data acquisition
station (SRI Instruments, Torrance, CA, USA) where
integrated peak areas were measured from both inputs.
The same corrections were made, as described above, for
sample size and radioactive decay of the tracer. For the
purpose of this work, all radioactive sugars were pooled
and related back to their total distribution relative to the
soluble [11C]photosynthate fraction.
Measurement of Endogenous Cinnamic Acid Levels PAL
catalyzes the formation of trans-cinnamic acid from phenylalanine. Therefore, measurement of endogenous cinnamic
acid levels as a function of time after treatment should give us
insight into the coordination between downstream phenolic
metabolism and the shikimate pathway. In parallel studies,
leaves were dried for 24 h in an oven at 70°C, weighed to
obtain a dry mass, and extracted in tetrahydrofuran (Sigma
Life Science, St. Louis, MI, USA) for 3 h at 25°C according to
the procedure of Budi-Muljono et al. (1998). Gas chromatography analysis was performed on aliquots of tissue extract
using a non-polar fused-silica column (30 m×0.53 mm i.d.)
coated with methyl silicone gum (HP-1: Hewlett Packard
Co. Rockville, MD, USA) at a film thickness of 2.65 μm.
The gas chromatograph (Hewlett Packard 5890 Series II)
was equipped with a flame ionization detector operated at a
temperature of 300°C. The other instrument conditions were
as follows: injector temperature, 230°C; carrier gas-helium;
carrier gas inlet pressure, 100 kPa; oven temperature, 50–
250°C at 10°Cmin−1. Analyses were performed using the
splitless injection mode. Data was acquired using a Vision
IV chromatography station (Scientific Systems, Inc., State
College, PA, USA). Trans-cinnamic acid was identified
based on its elution time matched against that from an
authentic sample. Peaks were integrated and correlated to a
mass amount using a calibrated detector mass response
curve.
J Chem Ecol (2010) 36:1058–1067
Treatments Plants were sprayed uniformly with either DI
water (as a control) or a 500 μM solution of MeJA in DI
water immediately following excision of leaf-3 to acquire
the baseline measurement of metabolic partitioning of
tracer. Because MeJA was shown to distribute rapidly
throughout the plant, we opted to treat the entire aboveground foliar tissue (Thorpe et al., 2007). After treatment,
plants were housed individually in a positive air flow
holding box illuminated using auxiliary halogen lights
(300 μmolm−2 sec−1). This ensured that interplant communication via plant volatile emissions would not compromise
retest responses in study plants. Plants then were retested
using leaf-4, and a second dose of 11C tracer, at 4 or 15 h
after treatment (Fig. 2).
Statistical Analysis One of the advantages of using shortlived radioisotopes is that they do not accumulate in the
plant, and so retests can be performed over time and after
treatment allowing the same plant to be used as its own
control. All data derived from matched plant studies
(baseline/post-treatment) and were presented as change in
response relative to baseline values. As pointed out earlier,
by using the same plant in a test-retest protocol, rather than
averaging values across individual plants at baseline
(untreated) and post-treatment (treated), we obtained a
smaller standard error. Comparisons were made in the
change of the 12C and 11C profiles from baseline levels to
post-treated levels of control and MeJA treated plants using
ANOVA single-variate analysis.
Results
The effects of MeJA on primary plant metabolism were
evident from the changes seen in the [11C]photosynthate
composition (Fig. 3) 4 and 15 h after treatment. In
unstressed plants (N=4), the typical [11C]photosynthate
comprised 90.2±2.0% soluble sugars, 3.0±0.6% amino
acids and 6.8±2.1% of other labeled substrates that were
not accountable in our sugar and amino acid analyses.
Control treatments (N=5) had no effect on this distribution.
However, MeJA (N=4) caused a significant decrease in the
[11C]sugar fraction to 83.7±2.3% (P=0.041) by 4 h posttreatment relative to controls with a commensurate increase
in the [11C]amino acid fraction to 6.3±0.9% (P=0.039) and
an increase in the other [11C]labeled substrate fraction to
10.0±2.5% (P=0.046). This trend was more significant by
15 h post-treatment using MeJA with the [11C]sugar
fraction decreasing to 59.4±6.6% (P<0.001), the [11C]
amino acid fraction increasing to 27.6±3.1% (P<0.001),
and the [11C]other fraction increasing to 13.0 ± 7.5%
(P=0.054). Alhough tissues were extracted just 30 min
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Fig. 3 Effect of treatments of Nicotiana tabacum at 4 h (control, N=
5; MeJA, N=4) and 15 h (MeJA, N=4) on the partitioning of new
carbon as 11C into soluble sugar, amino acid and unknown substrate
fractions of [11C]photosynthate. Error bars denote standard deviations
on means. Data bars denoted by * and ** indicate that change in 11Cpartitioning into metabolic pools from the untreated state to the control
or MeJA treated states was significant at the 5% and 1% confidence
levels, respectively, according to the ANOVA single variate analysis
after tracer administration, leaf export of selected components comprising the [11C]photosynthate mix might have
altered its composition in the study leaf. However, we note
that leaf export of [11C]photosynthate by this time point
was unchanged by MeJA treatment: 19.1±5.1% of fixed
11
C in untreated leaves and 19.6±2.8% 15 h after MeJA
treatment (Gómez, unpublished).
Over the same timeline 11C-partitioning into tyrosine
(Fig. 4a) was only slightly elevated by MeJA at 4 h posttreatment though significantly (P=0.28) relative to controls.
For phenylalanine (Fig. 4b) and tryptophan (Fig. 4c),
11
C-partitioning into these amino acids was much more
responsive to MeJA with each showing a 12-fold elevation
relative to baseline levels. These changes were significantly
different from those of control treatments (P<0.001 and
P=0.017, respectively). Interestingly, no change in the 12C
profiles of these compounds was noted during this time
course. By 15 h, the elevated partitioning of 11C into
phenylalanine and tyrosine returned to baseline levels, and
were not significantly different from those of controls. [11C]
Tryptophan levels, however, continued to grow in magnitude reaching a 30-fold elevation relative to baseline levels
(P<0.001). Again, no change was noted in the 12C profiles
of these metabolites by 15 h post-treatment. Like others
(e.g., Zangerl, 2003), we observed that MeJA reduced leaf
level photosynthetic activity by 40% (by 15 h posttreatment) although this result was only marginally
significant (P=0.075).
Finally, our studies assessed changes in endogenous
cinnamic acid levels over the same timeline of treatment.
PAL catalyzes the conversion of phenylalanine to transcinnamic acid which serves as a substrate for subsequent
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J Chem Ecol (2010) 36:1058–1067
steps in the phenylpropanoid pathway in high plants. We
found that cinnamic acid levels were unchanged for control
and MeJA treatments over the 4 h timeline (Fig. 5).
However, by 15 h post-treatment the metabolite’s level
was elevated 2.4-fold in response to MeJA, and was
significantly different relative to control responses at the
1% confidence level.
Discussion
While compensatory photosynthesis is considered an
important physiological response of plants (McNaughton,
1983; Crawley, 1983; Rosenthal and Kotanen, 1994;
Strauss and Agrawal, 1999), it generally is restricted to
re-growth tissue, and is not a universal response (Zangerl et
al., 1997). A reduction in the available carbon resources
through suppression of photosynthetic activity might reduce
the endogenous pools of amino acids since there would
simply be fewer carbon skeletons available. The fact that
we see no change in the size of the endogenous aromatic
amino pools, even though metabolic partitioning of new
carbon (as 11C) into these pools is seen to increase, may be
a reflection of the balance between carbon input, metabolic
reprogramming, and even resource re-allocation (i.e.,
physical transport from source tissues). Indeed, partitioning
of new carbon (as 11C) away from soluble sugars and into
amino acids is evident with 27.6% of the [11C]photosynthate comprising amino acids by 15 h post-treatment with
MeJA. Given the short-lived nature of the isotope, we
doubt that the increased [11C]amino acid fraction after
MeJA treatment is due to increased protein turnover.
Furthermore, other studies by us have shown that MeJA
will increase radioactive 13 N (t½ 9.97 min) utilization as
amino acids, as well as increase leaf export of these
substrates (Gómez et al., 2010). Even so, there are many
conditions (e.g., pathogen attack) that will reduce photo-
Fig. 4 Effect of treatments of Nicotiana tabacum at 4 h (Control, N=
4; MeJA, N=4) and 15 h (Control, N=4; MeJA, N=3) on the
metabolic partitioning of 12C and 11C into the shikimate pathway
involving biosynthesis of tyrosine (Fig. 4a), phenylalanine (Fig. 4b),
and tryptophan (Fig. 4c) amino acids. Results presented as the change
in 12C and 11C levels within individual compounds relative to their
baseline values (untreated). Error bars denote standard deviations on
means. Data bars denoted by * and ** indicate that the change in 11Cpartitioning into specific aromatic amino acids from the untreated state
to the control or MeJA treated states was significant at the 5% and 1%
confidence levels, respectively, according to the ANOVA single
variate analysis
Fig. 5 Effect of control (N=4) and MeJA (N=4) treatments at 4 and
15 hr on endogenous levels of trans-cinnamic acid in tissue extract
J Chem Ecol (2010) 36:1058–1067
synthesis, but will increase levels of soluble sugars and
amino acids due to the higher energy demands of the plant
during stress (Baena-González, 2010). Future studies will
explore how the balance of carbon and nitrogen is
maintained during early stress induction.
Most importantly, our results suggest that defense
induction triggered by MeJA manifests in the reprogramming of only new carbon and not of existing carbon stores.
This observation correlates with observations made by
Arnold and Schultz (2002) that new carbon is incorporated
into tannins during defense induction. It remains unclear
why existing carbon stores were not utilized to up-regulate
the phenylpropanoid pathway. This could reflect the fact
that remobilization of existing carbon stores is a slower
process and was not detected during the short timeline of
this present study. We note that analyses of jasmonateresponsive genes in Arabidopsis (Jung et al., 2007) reveals
a complex profile of both up-regulated and down-regulated
transcripts differing in expression patterns over time for up
to 24 h after MeJA treatment. In future studies, we need to
explore whether longer time points give further insight into
plant responsiveness to MeJA and its ability to utilize
available carbon (both old and new) in defense.
Interestingly, 4 h after MeJA treatment, the level of [11C]
phenylalanine was transiently increased, but quickly returned
to baseline levels by 15 h after treatment. As the first step in
phenolic metabolism, this is a crucial biochemical reaction
supporting both plant development and plant defense. Such
behavior might be reasonable in light of findings by Jung
et al. (2007) showing that MeJA will transiently induce
transcript accumulation of numerous genes in Arabidopsis
(e.g., At5g67300 associated with a MYB transcription factor
in signal transduction). We note that many MYB transcription factors play essential roles in regulating phenolic
metabolism during defense induction (Stracke et al., 2007;
Malone et al., 2009). Furthermore, phenylalanine itself has
been shown to promote PAL activity in Cryptomeria and
Perilla cell suspension cultures (Ishikura et al., 1986), as did
JA up-regulate transcripts associated with the phenylalanine
ammonia lyase isoform 1 in Populus nigra L. (Babst et al.,
2009). Indeed, we observed increased endogenous levels of
cinnamic acid, but only 15 h after MeJA treatment, thus
highlighting the need for studies that explore the coordination between the expression profiles of genes and relevant
metabolism. Finally, we note that JA will up-regulate
transcripts associated with DAHP synthase in Populus
(Babst et al., 2009), as will wounding increase DAHP
activity promoting increased carbon turnover within the
shikimate pathway (Dyer et al., 1989). Our observation of
increased partitioning of new carbon into [11C]phenylalanine
and other metabolites of the shikimate pathway may be a
reflection of direct action of the phytohormone on DAHP
and possibly other branch-point enzymes of that pathway.
1065
Future studies using transformed plants should provide
additional insight here.
Like phenylalanine, a similar transient behavior was
observed in the metabolic partitioning of 11C into tyrosine,
although not as large. Since phenylalanine and tyrosine
derive from the same branch point (prephenate-to-arogenate)
within the shikimate pathway, it seems reasonable that they
exhibit similar transitory behavior.
Finally, MeJA treatment rapidly increases 11C-partitioning
into tryptophan, which continues to increase over a 15 h
window after treatment. We note that tryptophan promotes
chorismate mutase in the shikimate pathway, which may
affect carbon partitioning from chorismate to tyrosine and
phenylalanine (Bickel and Schultz, 1979). However, the
endogenous concentrations of aromatic amino acids were
unchanged over the timeline of our studies. Therefore, we
rule out the possibility that feedback loops, that either
promote or inhibit amino acid biosynthesis are likely
responsible for the changes in 11C-partitioning that we
observed.
Tryptophan plays a major role in plant metabolism as
the primary precursor in the biosynthesis of indole-3acetic acid (IAA), a growth regulator that is part of the
auxin family, key hormones that regulate growth and
development (Doerner, 2008). Jung et al. (2007) reported
a group of genes that were induced 1-2 h after MeJA
treatment, reaching a maximum accumulation by 8 h and
diminishing after 24 h. These middle-stage expression
genes included those for transcription factors as well as
tryptophan synthase subunits and the JR2 gene linked to
IAA-Ala hydrolase. Therefore, it seems reasonable that
11
C-partitoning into tryptophan would remain elevated
over the timeline of our study.
In summary, the use of 11C as described in this paper
offers unique opportunities to explore rapid temporal
changes in new carbon metabolism. As we have demonstrated, studies of this nature can provide insight into how
plants use their new carbon resources in response to stress.
Acknowledgements This research was supported in part by the U.S.
Department of Energy, Office of Biological and Environmental
Research under contract DE–AC02–98CH10886, in part by the
National Research Initiative of the USDA National Institute of Food
and Agriculture, under grant 2007-35302-18351, and by German
Academic Exchange Service (Deutscher Akademischer Austauschdienst=DAAD), Bonn, which supported N. Hanik and M. Best.
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