Research
Elevated CO2 decreases the response of the ethylene signaling
pathway in Medicago truncatula and increases the abundance of
the pea aphid
Huijuan Guo1,2, Yucheng Sun1, Yuefei Li1, Xianghui Liu1, Wenhao Zhang3,4 and Feng Ge1,4
1
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; 2Graduate School, Chinese Academy
of Sciences, Beijing, 100039, China; 3State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; 4Research
Network of Global Change Biology, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Summary
Authors for correspondence:
Yucheng Sun
Tel: +86 10 6480 7130
Email: [email protected]
Feng Ge
Tel: +86 10 6480 7123
Email: [email protected]
Received: 15 May 2013
Accepted: 6 August 2013
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doi: 10.1111/nph.12484
Key words: Acyrthosiphon pisum (pea
aphid), elevated CO2, ethylene, Medicago
truncatula, nitrogen (N) metabolism, resistance.
The performance of herbivorous insects is greatly affected by plant nutritional quality and
resistance, which are likely to be altered by rising concentrations of atmospheric CO2.
We previously reported that elevated CO2 enhanced biological nitrogen (N) fixation of
Medicago truncatula, which could result in an increased supply of amino acids to the pea
aphid (Acyrthosiphon pisum). The current study examined the N nutritional quality and aphid
resistance of sickle, an ethylene-insensitive mutant of M. truncatula with supernodulation,
and its wild-type control A17 under elevated CO2 in open-top field chambers.
Regardless of CO2 concentration, growth and amino acid content were greater and aphid
resistance was lower in sickle than in A17. Elevated CO2 up-regulated N assimilation and
transamination-related enzymes activities and increased phloem amino acids in both genotypes. Furthermore, elevated CO2 down-regulated expression of 1-amino-cyclopropanecarboxylic acid (ACC), sickle gene (SKL) and ethylene response transcription factors (ERF)
genes in the ethylene signaling pathway of A17 when infested by aphids and decreased resistance against aphids in terms of lower activities of superoxide dismutase (SOD), peroxidase
(POD), and polyphenol oxidase (PPO).
Our results suggest that elevated CO2 suppresses the ethylene signaling pathway in
M. truncatula, which results in an increase in plant nutritional quality for aphids and a
decrease in plant resistance against aphids.
Introduction
Global atmospheric CO2 concentrations have been increasing at
an accelerating rate from 280 ppm before industrialization to
396 ppm in Feburary 2013 (Mauna Loa Observatory: NOAAESRL), and are anticipated to reach at least 550 ppm by the year
2050 (IPCC, 2007). Elevated CO2 is expected to enhance crop
yields by increasing photosynthetic rates and water-use efficiencies, particularly in C3 crops. Observed increases in yield,
however, have not always matched theoretical expectations in
CO2-enrichment experiments (Ainsworth & Long, 2005; Long
et al., 2005), perhaps because the theory does not include interactions between plants and herbivorous insects.
The performance of aphids and other herbivorous insects is
affected by bottom-up effects of host plants in terms of nutritional status and chemical and physical defenses (Awmack &
Leather, 2002). With respect to nutritional status, aphids feed
exclusively on phloem (Douglas, 2003), which provides a protein : carbohydrate ratio (mainly amino acids : sugars) as low as
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1 : 10 (w/w) (Nowak & Komo, 2010). Although aphids have
evolved to adapt to this nutrient-poor substrate, they are still able
to discriminate among host plants with low and high nitrogen
(N) concentrations and tend to prefer plants with higher N concentrations (Nowak & Komo, 2010). Moreover, N-fertilized
plants enhance aphid population growth because of the increased
concentration of amino acids in the phloem (Honek, 1991; Petitt
et al., 1994; Ponder et al., 2000). Thus, it seems that the N nutritional status of the host plant is an important determinant of
aphid development and fecundity.
With respect to plant defenses, once aphid stylets penetrate the
epidermis, the plant triggers a common defensive response based
on reactive oxygen species (ROS) by activating superoxide dismutase
(SOD) and peroxidase (POD) (Moloi & van der Westhuizen,
2006). A further line of defense involves the rapid synthesis and
polymerization of phenolic compounds in the cell wall (Matern
& Kneusel, 1988). During this process, polyphenol oxidase
(PPO) and phenylalanine ammonia lyase (PAL) are key
secondary metabolism enzymes that mediate plant resistance
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against aphids (He et al., 2011). Effects of elevated CO2 on crop
yields should consider how elevated CO2 alters host nutrition
and host defenses relative to aphids and other herbivores.
Elevated CO2 reduces the nutritional quality of some nonleguminous C3 plants by decreasing the N concentration (Ainsworth
& Long, 2005; Ainsworth & Rogers, 2007), which may consequently increase the developmental time and reduce the fecundity
and fitness of leaf-chewing insects (Coll & Hughes, 2008). However, N concentrations in legumes were rarely affected by elevated
CO2 because of the enhancement of biological N fixation (BNF),
which counteracts the adverse effect of elevated CO2 on leafchewing insects (Karowe, 2007; Taub & Wang, 2008; Karowe &
Migliaccio, 2011). For a sap-sucking insect like the pea aphid
(Acyrthosiphon pisum), the increased BNF in legumes under elevated CO2 increases available N and thereby increases aphid
numbers (Guo et al., 2013). When BNF is suppressed by artificial
mutation, however, BNF cannot satisfy the increased demand for
N that occurs under elevated CO2, and aphid numbers do not
increase (Guo et al., 2013). Thus, it appears that enhanced BNF
is necessary for the positive response of the pea aphid to elevated
CO2.
In legumes, BNF is regulated by several hormone signaling
pathways, including the ethylene signaling pathway. The involvement of the phytohormone ethylene in nodulation was initially
proposed based on studies showing that the application of exogenous ethylene or its biosynthetic precursor 1-amino-cyclopropane-carboxylic acid (ACC) suppresses nodulation, and,
conversely, that application of chemical inhibitors of ethylene
perception (i.e. Ag+) or biosynthesis (i.e. the amino ethoxyvinyl
glycine, AVG) increases nodule numbers (Ma et al., 2002;
Penmetsa et al., 2003, 2008). Once the key gene Mtskl in the ethylene-perception pathway was mutated in Medicago truncatula,
the resulting ethylene-insensitive mutant, sickle, produced more
nodules than the wild-type, and its nitrogenase activity was
increased about two times (Penmetsa & Cook, 1997).
In addition to having a key role in the regulation of BNF, ethylene is the most important hormone involved in plant resistance
against pathogens and pests. The expression of genes involved in
ethylene production and ethylene signaling (ACC oxidase and
ethylene-responsive elements) are up-regulated in response to
aphid infestation (Moran et al., 2002; Divol et al., 2005). Ethylene is also responsible for the regulation of ROS and downstream
defensive enzymes against aphids (Jung et al., 2009). The sickle
mutant showed increased sensitivity to Rhizoctonia solani and
other pathogens on legumes and cereals (Penmetsa et al., 2008).
Thus, it is reasonable to speculate that the ethylene-insensitive
mutant sickle, which produces more nodules and has enhanced
BNF as well as reduced resistance to aphids relative to the wildtype, will supply more N nutrition to aphids and be less resistant
to aphids.
The results of recent studies indicate that elevated CO2 finetunes phytohormone signaling pathways when plants encounter
biotic stress, as indicated by enhanced induced defenses derived
from the salicylic acid signaling pathway and reduced jasmonic
acid-dependent defense (Zavala et al., 2008; Sun et al., 2011;
Guo et al., 2012). Furthermore, elevated CO2 tends to increase
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ethylene production in healthy plants but down-regulates the
expression of downstream genes in the ethylene signaling pathway when plants are attacked by Japanese beetles (Seneweera
et al., 2003; Casteel et al., 2008). Although it is well established
that the ethylene signaling pathway mediates plant resistance
against pathogens and aphids, it is unclear whether elevated CO2,
by modulating the ethylene signaling pathway, simultaneously
alters ethylene-dependent defense as well plant nutrition for
aphids and other herbivores.
Here, we hypothesized that elevated CO2 would decrease the
responses of the ethylene signaling pathway, which would directly
reduce ethylene-dependent plant defenses while indirectly
enhancing N availability for aphids via an increase in nodulation,
such that pea aphid abundance would be greater under elevated
CO2 than under ambient CO2. To test this hypothesis, we used
sickle (a supernodulating mutant of M. truncatula that is insensitive to ethylene) and the wild-type A17 to determine how elevated CO2 affects the interaction between M. truncatula and the
pea aphid via the ethylene signaling pathway. The specific goals
were to determine whether the supernodulating mutant sickle
grows better and has higher N metabolism than the wild-type
under elevated CO2; whether elevated CO2 affects the resistance
of the two genotypes against the pea aphid and consequently
affects pea aphid feeding behavior and abundance; and whether
the ethylene signaling pathway is involved in the regulation of
the plant–aphid interaction under elevated CO2.
Materials and Methods
Atmospheric CO2 concentration treatments
The research described in the following sections was performed
in eight octagonal, open-top field chambers (OTCs; 4.2 m in
diameter and 2.4 m high) at the Observation Station of the
Global Change Biology Group, Institute of Zoology, Chinese
Academy of Science in Xiaotangshan County, Beijing, China
(40°11′N, 116°24′E). The atmospheric CO2 concentration treatments were as follows: current atmospheric CO2 concentrations
(c. 390 ll l1); and elevated CO2 concentrations (750 ll l1, predicted concentration at the end of this century) (IPCC, 2007).
Four blocks were used for CO2 treatment, and each block contained paired OTCs, one with ambient and one with elevated
CO2. CO2 concentration in each OTC was monitored and
adjusted with an infrared CO2 analyzer (Ventostat 8102; Telaire
Company, Goleta, CA, USA) once every min to maintain relatively stable CO2 concentrations. The measured CO2 concentrations throughout the experiment (mean SD d–1) were
395 22 ppm in the ambient CO2 chambers and 752 33 ppm
in the elevated CO2 chambers. The auto-control system for
maintaining the CO2 concentrations, as well as specifications for
the OTCs, is detailed in Chen et al. (2005). The tops of the
OTCs were covered with nylon net to exclude insects. Air temperatures were measured three times per d throughout the studies
and did not differ significantly between the two treatments
(22.7 1.9°C in OTCs with ambient CO2 vs 24.2 2.0°C in
OTCs with elevated CO2).
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Aphids
The pea aphid Acyrthosiphon pisum Harris. was obtained from
the laboratory of Dr Feng Cui (Institute of Zoology, Chinese
Academy of Science). The nymphal instars from the same parthenogenetic pea aphid female were reared on Vicia faba with 14 h
light (25°C) : 10 h dark (22°C) in photoclimate chambers (Safe
PRX-450C, Ningbo, China).
Host plants and rhizobium inoculation
The supernodulating mutant sickle of M. truncatula Gaertn. and
its wild-type background (cv Jemalong A17) were kindly provided
by Professor Douglas Cook (Department of Plant Pathology and
Microbiology, Texas A&M University, TX, USA). Visual assessments for delayed petal senescence and leaf abscission were
conducted on glasshouse-grown plants (Penmetsa & Cook, 1997).
After seeds were chemically scarified and surface-sterilized by
immersion in concentrated H2SO4 for 5 min, they were rinsed
with sterilized water several times. The seeds were placed in Petri
dishes containing 0.75% agar, kept in the dark at 4°C for 2 d,
and then moved to 25°C for 2 d to germinate. The germinated
seeds were sown on sterilized soil and inoculated 2 d later with
the bacterium Sinorhizobium meliloti 1021, which was provided
by Professor Xinhua Sui (Department of Microbiology, College
of Biological Sciences, Chinese Agricultural University).
S. meliloti had been cultured on YM medium (H2O, 1000 ml;
yeast, 3 g; mannitol, 10 g; KH2PO4, 0.25 g; K2HPO4, 0.25 g;
MgSO47H2O, 0.1 g; NaCl, 0.1 g, pH 7.0–7.2) for 3 d at 28°C
to obtain an approximate cell density of 108 ml1. At sowing,
each seedling was inoculated with 0.5 ml of this suspension. After
they had grown in sterilized soil for 2 wk, the M. truncatula seedlings were individually transplanted into plastic pots (35 cm
diameter and 28 cm height) containing sterilized loamy field soil
(organic carbon, 75 g kg1; N, 500 mg kg1; P, 200 mg kg1; K,
300 mg kg1) and placed in OTCs on 27 March 2012. Each
OTC contained 40 plants.
Medicago truncatula plants were maintained in the OTCs for
75 d from seedling emergence to the end of the experiment (27
March to 7 June 2012). Pot placement was re-randomized within
each OTC once every wk. No chemical fertilizers or insecticides
were used. Water was added to each pot every 2 d. On 18 May
2012, after the plants had been in the OTCs for 6 wk, they were
used for the three groups of assays described in the following
sections.
Aphid reproduction and feeding behavior as affected by plant
genotype and CO2 concentration (group 1) Six plants of each
genotype per OTC were randomly selected, and each was
infested with five apterous, fourth-instar nymphs. The plants
with these nymphs were individually caged (80 mesh gauze). Six
other plants of each genotype per OTC were not infested but
were caged and served as controls. Aphids on each plant were
counted 7, 11, 15, 19, and 23 d after inoculation.
From 19 to 25 May 2012, another six plants of each genotype
per OTC (96 plants in total) were randomly selected as host
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plant to evaluate the feeding behavior of aphids using an electrical
penetration graph (EPG) technique. EPG signals have been correlated with aphid activities as well as with tissue locations of the
stylet tips (Tjallingii & Hogen-Esch, 1993). Eight plants were
placed in a Faraday cage to avoid noise and interference. Each
plant was infested with one apterous adult pea aphid for 8 h to
record feeding behavior using the EPG method. The EPG
method is a powerful tool for determining, in real time, the
locations and activities of the aphid stylet, including probing,
salivation into sieve elements, and passive uptake of phloem sap
(Walker, 2000). The feeding behavior of pea aphids on the plants
was studied as described in Gao et al. (2008) with some modifications. Twenty-four biological replicates (six plants in each of four
OTCs) were included for each genotype (A17 and sickle) under
each CO2 concentration (96 plants in total). As noted, one apterous adult pea aphid was placed on a single trifoliate leaf, and its
feeding behavior was monitored. An eight-channel amplifier
simultaneously recorded eight individual aphids on separate
plants (on two A17 plants and two sickle plants under ambient
CO2, and on two A17 plants and two sickle plants under elevated
CO2) for 8 h and this experiment was replicated for 12 times.
Waveform patterns in this study were scored according to categories described by Tjallingii & Hogen-Esch (1993): nonpenetration (np); pooled pathway phase activities (C); salivary secretion
into sieve elements (E1); phloem ingestion (E2); derailed styled
(F); and xylem ingestion (G).
Plant growth trait and N fixation-related genes as affected by
plant genotype and CO2 concentration (group 2) On 7 June
2012, six undamaged plants were harvested for measurement of
Chl content, biomass, pod number, nodule number and gene
expression. Leaves and roots (0.05 g) of each plant were harvested
and immediately stored in liquid nitrogen for measurement of
gene expression. The quantification of gene expression is
described in the following paragraphs.
Leaf Chl content was determined with a Minolta SPAD502DL (Konica Minolta Sensing Inc., Osaka, Japan), which
measures leaf transmittance at two wavelengths: red (c. 660 nm)
and near-infrared (c. 940 nm). SPAD readings were taken on
the fourth terminal mature trifoliate leaf from the base of the
shoot. The SPAD sensor was placed randomly on leaf mesophyll
tissue.
Roots of each plant were carefully removed from the soil and
washed. A stereomicroscope was used to count the nodules on
the entire root system of each plant. The pod numbers per plant
were also determined. The shoots and roots of each plant were
collected, oven-dried (65°C) for 72 h, and weighed.
Plant enzyme activity, free amino acid concentration, and gene
expression as affected by plant genotype, CO2 concentration,
and aphid infestation (group 3) On 19 May 2012, two plants
of each genotype per OTC were randomly selected, and each was
infested with 50 apterous fourth-instar nymphs; a fine paintbrush
was used to transfer the nymphs to these plants, which were individually caged (80 mesh gauze). Two other uninfested control
plants of each genotype per OTC were caged in the same way.
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After 48 h, leaves of infested plants and leaves of the same number of uninfested control plants were harvested and immediately
stored in liquid nitrogen for chemical analysis. The quantification
of enzyme activities, free amino acid concentration, and ethylene
signaling pathway-related gene expression are described in the
following paragraphs.
The activities of key enzymes involved in N assimilation and
transamination, including glutamine synthetase/glutamate synthase (GS/GOGAT), glutamate oxalate transaminase (GOT),
and glutamine phenylpyruvate transaminase (GPT), were quantified (Schoenbeck et al., 2000; Andrews et al., 2004) using frozen leaf tissue (c. 0.5 g per plant). Once the tissue was ground to
a fine powder, leaves from three plants of the same treatment
were combined to form one sample from each OTC. The unit
of replication for statistical analyses was the OTC (n = 4). An
extract was obtained by grinding each leaf sample in 50 mM
Tris HCl buffer (pH 7.8, 3 ml g1 of leaf tissue) containing
1 mM MgCl2, 1 mM EDTA, 1 lM b-mercaptoethanol, and 1%
(w/v) polyvinylpolypyrro-lidone. This extract was immediately
frozen for later use. For assays, the thawed extract was centrifuged at 13 000 g for 10 min, and the enzymatic activities were
measured in the supernatant as described by Glevarec et al.
(2004) for GS, by Suzuki et al. (2001) for GOGAT, and by
Asthir & Bhatia (2011) for GOT and GPT. Protein concentrations of leaves and roots were measured using BSA as a standard
(Bradford, 1976).
The defensive enzymes, including lipoxygenase (LOX), proteinase inhibitors (PIs), PPO, POD, and PAL, were extracted
from 0.1 g frozen leaf tissue by grinding them in a 50 mM Tris
HCl buffer (pH 7.8, 3 ml g1 of leaf tissue) containing 7% polyvinylpolypyrrolidine, 1.67 mM phenylthiourea, 0.3 M KCl, and
0.4 mM ascorbic acid. For assays, the thawed extract was centrifuged at 13 000 g for 10 min, and enzyme activity was measured
in the supernatant. The activities of these defense enzymes were
determined according to Guo et al. (2012).
For quantification of amino acid concentrations in phloem,
phloem exudates were obtained from three trifoliate leaves per
plant using the EDTA exudation technique of Douglas (1993).
The amino acids in each sample were analyzed by reverse-phase
high-performance liquid chromatography (HPLC) with precolumn derivatization using o-phthaldialdehyde (OPA) and
9-fluorenylmethyloxycarbonyl (FMOC). Amino acids were
quantified by comparison with the AA-S-17 (PN: 5061-3331;
Agilent Technologies, Palo Alto, CA, USA) reference amino acid
mixture, supplemented with asparagine, glutamine, and tryptophan (Sigma-Aldrich). Standard solutions were prepared from a
stock solution by diluting with 0.1 M HCl. Free amino acid concentrations of each of the five standard solutions were 250, 100,
50, 25, and 10 pmol ll1. Before each sample was injected into
the HPLC, 10 ll of the amino acid sample was mixed with 20 ll
of sodium borate buffer (0.4 N, pH 10.4), 10 ll of OPA, 10 ll
of FMOC, and 50 ll of water. The analysis was performed using
an Agilent 1100 HPLC system (Agilent). A reverse-phase Agilent
Zorbax Eclipse C18 column AAA (5 lm, 250 mm 9 4.6 mm)
and fluorescence detector were used for the chromatographic separation. The column was maintained at 35°C with a gradient
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(1 ml min1 flow) programmed as follows: 98/2 (1 min) to 43/
57 (25 min) to 0/100 (34 min) to 98/2 (42 min hold) of eluent
A/eluent B. Eluent A was a 40 mM disodium phenyl phosphate
buffer (pH 7.8 adjusted with sodium hydroxide). Eluent B was
45% acetonitrile, 45% methanol, and 10% water. Chemstation
Plus Family for LC software was used for data acquisition and
analysis. Amino acid concentrations were quantified by comparison of sample peak areas to standard curves of 20 reference amino
acids (Agilent).
The expressions of three genes involved in N fixation (ENOD,
nifH, and nodF) and three genes in the ethylene signaling pathway (ACC, SKL and ERF) were measured by quantitative reverse
transcription polymerase chain reaction. Each combination was
replicated four times for biological repeats, and each biological
repeat had three technical repeats. The RNA easy Mini Kit
(Qiagen) was used to isolate total RNAs from M. truncatula
leaves or roots (0.05 g from samples stored at 70°C), and 1 lg
of RNAs was used to generate the cDNAs. The mRNA levels of
the six target genes were quantified by real-time quantitative
PCR (qPCR). Specific primers for each gene were designed from
the M. truncatula expressed sequence tag sequences using
PRIMER5 software (Supporting Information Table S1). The
PCR reactions were performed in a 20 ll total reaction volume
including 10 ll of 2 9 SYBRs Premix EX Taq™ (Qiagen) master
mix, 5 mM of each gene-specific primer, and 1 ll of cDNA template. Reactions were carried out on the Mx 3500P detection system (Stratagene), and the parameters were as follows: 2 min at
94°C; followed by 40 cycles of 20 s at 95°C, 30 s at 56°C, and
20 s at 68°C; and finally one cycle of 30 s at 95°C, 30 s at 56°C,
and 30 s at 95°C. This PCR protocol produced the melting
curves, which were used to judge the specificity of PCR products.
A standard curve was derived from the serial dilutions to quantify
the copy numbers of target mRNAs. b-actin and pnp served as
internal qPCR standards for the analysis of plant and bacterial
gene expression, respectively (Vernie et al., 2008). The relative
level of each target gene was standardized by comparing the copy
numbers of target mRNA with b-actin or pnp (the housekeeping
gene) copy numbers, which remain constant under different
treatment conditions. The b-actin mRNAs of the control were
examined in every PCR plate to eliminate systematic error. The
fold-changes of target genes were calculated using the 2DDCt
method.
Statistical analysis
All data were checked for normality and equality of residual error
variances and were appropriately transformed (log or square root)
as needed to satisfy the assumptions of the ANOVA. For the
amino acids, we performed a principal components analysis
(PCA) on the correlations among the 20 response variables and
then performed factor rotation using the Varimax method
(Rasmussen et al., 2008). A split-split plot design was used to
analyze the univariate responses of the growth traits, enzyme
activities, and rotated factors of amino acids in plants (ANOVA,
PASW, 2009). In the following ANOVA model, CO2 and block
(a pair of OTCs with ambient and elevated CO2) were the main
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effects, M. truncatula genotype was the subplot effect, and aphid
infestation was the sub-subplot effect:
X ijklm ¼ l þ Ci þ BðCÞjðiÞ þ Gk þ CGik þ GBðCÞkjðiÞ þ Hl
þ CHil þ HBðCÞljðiÞ þ GHBðCÞkljðiÞ þ emðijkl Þ
where C is the CO2 treatment (i = 2), B is the block (j = 4), G is
the M. truncatula genotype (k = 2), and H is the aphid infestation
treatment (l = 2). Xijklm represents the error because of the smaller
scale differences between samples and variability within blocks
(ANOVA, SAS Institute, Cary, NC, USA). Effects were considered significant if P < 0.05. The effect of block and the interactive
effects of block and other factors were not significant (P > 0.45),
and the effect of block and its interactive effects with other factors
were not presented so as to simplify the presentation. Tukey’s
multiple range tests were used to separate means when ANOVAs
were significant. For quantifying the feeding behavior of pea
aphids on different M. truncatula genotypes under two CO2 concentrations, a split-plot design was also applied, with CO2 and
block as the main effects and M. truncatula genotype as the subplot effect. Aphid abundance was analyzed by repeated-measures
ANOVA.
Results
Fig. 2 The percentage of time pea aphids (Acyrthosiphon pisum) spent in
various feeding activities on two Medicago truncatula genotypes (A17 and
sickle) during an 8 h exposure to ambient CO2 (ACO2) and elevated CO2
(ECO2). ‘Nonpenetration’, stylets are outside the plant; ‘pathway’, mostly
intramural probing activities between mesophyll or parenchyma cells;
‘salivation’, aphids are injecting watery saliva into the sieve element;
‘phloem ingestion’, aphids are ingesting the phloem sap; ‘xylem’, stylet
penetration of tracheary elements; ‘derailed stylet’, stylets are exhibiting
penetration difficulties. Values are the means ( SE) of 24 biological
replicates. Different lowercase letters indicate significant differences
among treatments (Tukey’s multiple range test, P < 0.05).
Aphid abundance
Elevated CO2 enhanced pea aphid population growth on A17
and sickle plants (Fig. 1). Regardless of CO2 concentration,
aphids were more abundant on sickle than on A17 plants (Fig. 1).
on sickle than on A17 plants under ambient CO2. Under elevated
CO2, however, aphid feeding was not significantly affected by
plant genotype (Fig. 2).
Aphid feeding behavior
Plant growth traits and expression of N-fixation genes
Elevated CO2 decreased the percentage of time aphids spent salivating into sieve elements (E1 phase) on A17 plants but increased
phloem sap ingestion (E2 phase) for both plant genotypes
(Fig. 2). The aphids had a shorter E1 phase and a longer E2 phase
Carbon dioxide concentration and genotype had significant
effects on plant Chl content, nodule numbers, biomass and pod
numbers (Table S2) In the absence of pea aphids, elevated CO2
significantly increased Chl content by 18.6 and 12.6%, nodule
numbers by 68.5 and 26.8%, biomass by 25.9 and 33.1%, and
pod numbers by 31.6 and 21.9% for A17 and sickle plants,
respectively (Fig. 3). Regardless of CO2 concentration, Chl content, nodule number, biomass, and pod number were higher for
sickle than for A17 plants (Fig. 3).
Elevated CO2 increased the expression of ENOD, nifH, and
nodF in both genotypes (Fig. 4). Regardless of CO2 concentration, the expression of these three N-fixation genes was higher in
sickle than in A17 plants (Fig. 4).
N assimilation and transamination enzymes of plants
Fig. 1 Abundance of pea aphids (Acyrthosiphon pisum; number per plant)
when fed on two Medicago truncatula genotypes (wild-type A17 and
ethylene-insensitive sickle mutants) grown under ambient CO2 (ACO2)
and elevated CO2 (ECO2). Each value represents the mean ( SE) of four
replicates. Significant differences: *, P < 0.05.
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Carbon dioxide concentration, genotype, aphid infestation and
the interaction between CO2 and aphid infestation significantly
affected the activity of GS and GOT in plant leaves (Table S3).
CO2 concentration, genotype, aphid infestation, the interaction
between genotype and aphid infestation, as well as the interaction
among CO2, genotype and aphid infestation, significantly
affected the activity of GOGAT (Table S3). CO2 concentration,
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(a)
(c)
(b)
(d)
Fig. 3 Growth traits of two Medicago
truncatula genotypes (A17 and sickle) grown
under ambient CO2 (ACO2; open bars) and
elevated CO2 (ECO2; closed bars) without
pea aphid (Acyrthosiphon pisum) infestation.
(a) Chl content; (b) nodule numbers per
plant; (c) biomass; and (d) pod numbers.
Each value represents the mean ( SE) of
four replicates. Different lowercase letters
indicate significant differences between
ACO2 and ECO2 within the same genotype.
Different uppercase letters indicate
significant differences between genotypes
within the same CO2 treatment as
determined by Tukey’s multiple range test at
P < 0.05.
in sickle (Fig. 5). After a 48 h period of aphid infestation, elevated
CO2 increased the activities of all the N assimilation and transamination enzymes measured in A17 leaves and increased the
activity of GS, GOGAT, and GOT in sickle leaves (Fig. 5).
Regardless of aphid infestation, GS and GOGAT activities were
higher in sickle than in A17 leaves at both CO2 concentrations.
GOT activity was higher in sickle than in A17 leaves under elevated CO2 regardless of aphid infestation (Fig. 5).
Amino acid concentration in plants
Fig. 4 Expression of genes involved in nodulation and biological N fixation
(ENOD, nifH, nodF) in two Medicago truncatula genotypes (A17 and
sickle) grown under ambient CO2 (ACO2; open bars) and elevated CO2
(ECO2; closed bars). Values indicate fold-change in expression based on
quantitative PCR, and each value represents the mean ( SE) of four
replicates. Different lowercase letters indicate significant differences
between ACO2 and ECO2 within the same genotype. Different uppercase
letters indicate significant differences between genotypes within the same
CO2 treatment as determined by Tukey’s multiple range test at P < 0.05.
genotype, aphid infestation, the interaction between CO2 and
aphid infestation, as well as the interaction among CO2, genotype and aphid infestation, significantly affected the activity of
GOT (Table S3). Furthermore, all factors, with the exception of
the interaction between CO2 concentration and aphid infestation, significantly influenced the activity of GPT in plant leaves
(Table S3).
Without aphid infestation, elevated CO2 significantly
increased the activities of GOGAT and GPT in A17 and of GS
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The EDTA method and HPLC technique were used to measure
the relative composition of amino acids in the phloem sap of
M. truncatula. Because the responses of the 20 amino acids that
were measured are not independent, we then used a PCA to
reduce the number of phloem amino acid response variables to a
new set of composite variables (Rasmussen et al., 2008). To facilitate interpretation of principal components, we subjected the first
three principal components to factor rotation with the most common form of factor rotation, varimax rotation, and we retained
three rotated factors (RF1, RF2, and RF3, which accounted for
77% of the total variance) (Fig. 6). As the values of the rotated
factor increase, those variables that load heavily and positively
(loading ≥ + 0.5) also increase, while those variables that load
heavily but negatively (loading ≤ 0.5) decrease. The standardized univariate responses of these variables are shown in Fig. 7 to
facilitate the interpretation of the multivariate responses and to
allow a closer inspection of those variables loading heavily onto
RF1, RF2, and RF3.
Eleven amino acids (Gln, Ile, Val, Cys, Asn, Arg, Leu, Gly,
Met, His, and Phe; seven essential amino acids and four nonessential amino acids for aphid) loaded heavily and positively onto
RF1 (Fig. 6a). CO2 concentration, genotype, aphid infestation
and the interaction between CO2 and aphid infestation
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Fig. 5 Activities of enzymes involved in
nitrogen (N) metabolism in two Medicago
truncatula genotypes (A17 and sickle) grown
under ambient CO2 (ACO2) and elevated
CO2 (ECO2) and with (+PA) and without pea
aphid (Acyrthosiphon pisum) infestation. (a)
Glutamine synthetase (GS); (b) glutamate
synthase (GOGAT); (c) glutamate oxalate
transaminase (GOT); and (d) glutamine
phenylpyruvate transaminase (GPT). Each
value represents the mean ( SE) of four
replicates. Different lowercase letters indicate
significant differences among the
combinations of aphid treatment and CO2
concentrations within the same genotype.
Different uppercase letters indicate
significant differences between genotypes
within the same CO2 treatment and aphid
treatment as determined by Tukey’s multiple
range test at P < 0.05.
(a)
(b)
(a)
(b)
(c)
(d)
(c)
and Ser (one essential amino acid and five nonessential amino
acids for aphid) loaded heavily and positively onto RF2 (Fig. 6b).
Genotype, aphid infestation, the interaction between CO2 and
genotype, as well as the interaction among CO2, genotype and
aphid infestation significantly affected the RF2 (Table S4). Elevated CO2 without aphid infestation decreased RF2 in the
phloem of both A17 and sickle plants. Elevated CO2 with aphid
infestation significantly increased RF2 in the phloem of A17
plants but decreased RF2 in the phloem of sickle plants (Fig. 7c,
d). Thr and His (both are essential amino acids for aphid) loaded
heavily and positively onto RF3 (Fig. 6c). CO2 concentration,
genotype and their interaction significantly affected RF3. Regardless of aphid infestation, elevated CO2 increased RF3 in the
phloem of A17 and sickle plants (Fig. 7e,f).
Plant defensive enzymes
Fig. 6 The loadings for each individual amino acid of Medicago truncatula
phloem sap onto the first three rotated factors (RFs). The individual amino
acids loading heavily either positively (loading ≥ 0.5) or negatively (loading
≤ 0.5) are highlighted in black. These multivariate responses can be
interpreted as increasing as the positively loading variables increase and
decreasing as the negatively loading variables increase.
significantly affected the RF1 (Table S4). Elevated CO2 without
aphid infestation increased RF1 in the phloem of sickle plants.
Elevated CO2 with aphid infestation increased RF1 in the
phloem of both genotypes (Fig. 7a,b). Trp, Tyr, Pro, Ala, Glu,
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All factors, with the exception of the interaction between CO2
concentration and genotype, significantly influenced the activity
of SOD in plant leaves (Table S3). Genotype, the interaction
between CO2 and genotype, the interaction between CO2 and
aphid infestation, and the interaction between genotype and
aphid infestation significantly affected the activity of POD in
plant leaves (Table S3). CO2 concentration, genotype, aphid
infestation, the interaction between CO2 and genotype, and the
interaction between genotype and aphid infestation significantly
affected the activity of PPO and PAL (Table S3).
Without aphid infestation, elevated CO2 significantly
increased PAL activity in A17 plants (Fig. 8). After a 48 h period
of aphid infestation, elevated CO2 decreased the activities of
SOD, POD, and PPO but increased the activity of PAL in A17
plants but not in sickle plants (Fig. 8). With aphid infestation but
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286 Research
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 7 The mean response of rotated factors (a, c, e) and the standardized univariate response (b, d, f) of individual amino acids in phloem sap of two
Medicago truncatula genotypes (A17 and sickle) to CO2 concentration (ambient CO2, ACO2; elevated CO2, ECO2), genotype, pea aphid (Acyrthosiphon
pisum) infestation (+PA), and their interactions. Different lowercase letters indicate significant differences among the combinations of aphid treatments
and CO2 concentrations within the same genotype. Different uppercase letters indicate significant differences between genotypes within the same CO2
treatment and aphid treatment as determined by Tukey’s multiple range test at P < 0.05. The underlined individual amino acids indicate these are essential
amino acids for aphids.
regardless of CO2 concentration, SOD and POD activities were
lower in sickle than in A17 plants. With aphid infestation and
ambient CO2, PPO activity was lower in sickle than in A17
plants. With aphid infestation and elevated CO2, PAL activity
was lower in sickle than in A17 plants. Aphid infestation significantly increased the activities of SOD, POD, and PAL, regardless
of CO2, and increased PPO activity under ambient CO2 in A17
plants but did not affect the activity of these enzymes in sickle
plants (Fig. 8).
Expression of ethylene signaling pathway genes
Without aphid infestation, elevated CO2 significantly down-regulated the expression of ACC, SKL, and ERF in A17 plants and
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down-regulated ACC in sickle plants (Fig. 9). After a 48 h period
of aphid infestation, elevated CO2 down-regulated expression of
ACC, SKL, and ERF in A17 plants and down-regulated expression of ACC in sickle plants (Fig. 9). Regardless of CO2 concentration, aphid infestation significantly increased the expression of
ACC, SKL, and ERF in A17 plants but only that of ACC in
sickle plants (Fig. 9).
Discussion
Elevated CO2 affects herbivorous insects mainly by altering host
plant nutritional quality and resistance (Awmack & Leather,
2002). Under ambient CO2, the ethylene-insensitive mutant
sickle, which produces more nodules and exhibits a stronger
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Fig. 8 Activities of resistance enzymes of two
Medicago truncatula genotypes (A17 and
sickle) grown under ambient CO2 (ACO2)
and elevated CO2 (ECO2) with (+PA) and
without pea aphid (Acyrthosiphon pisum)
infestation. (a) Superoxide dismutase (SOD);
(b) peroxidase (POD); (c) polyphenol oxidase
(PPO); (d) phenylalanine ammonia lyase
(PAL). Each value represents the mean ( SE)
of four replicates. Different lowercase letters
indicate significant differences among the
combinations of aphid treatment and CO2
concentrations within the same genotype.
Different uppercase letters indicate
significant differences between genotypes
within the same CO2 treatment and aphid
treatment as determined by Tukey’s multiple
range test at P < 0.05.
Research 287
(a)
(b)
(c)
(d)
BNF than the wild-type, grew better and was less resistant than
the wild-type A17 and consequently supported higher numbers
of pea aphids than A17. Although the increased growth under
elevated CO2 could increase plant N demand (Daepp et al.,
2000), the increased BNF in both genotypes under elevated
CO2 provided sufficient N so that the plants could produce
greater biomass and more pods than under ambient CO2. Our
results indicate that elevated CO2 tends to suppress the ethylene
signaling pathway in wild-type A17 plants, so that increased
nodulation and BNF satisfy the increased N requirement for
growth under elevated CO2. By decreasing the ethylene signaling pathway, however, elevated CO2 reduced plant resistance
against the pea aphid. In summary, impairment of the ethylene
signaling pathway by elevated CO2 has two important effects in
M. truncatula: it up-regulates amino acid metabolism but
reduces aphid resistance ability; and the increased plant growth
and reduced resistance result in increased numbers of pea aphids
per plant.
The response to elevated CO2 differs among insect feeding
guilds (Robinson et al., 2012). Typically, elevated CO2 tends to
prolong the development of chewing insects because it decreases
the N content and increases secondary metabolites in host tissues
(Coll & Hughes, 2008). By contrast, elevated CO2 has speciesspecific effects on phloem-sucking insects such as aphids, which
obtain food from phloem sieve elements (Sun & Ge, 2011).
Some aphid species exhibit increased fecundity, abundance, and
survival under elevated CO2 (Pritchard et al., 2007). Although
Newman proposed that aphid populations tend to be larger
under elevated CO2 if host plants have higher N supplementation (Newman et al., 2003), evidence concerning how elevated
CO2 affects both bottom effects on aphids (via host nutrition
and resistance) has been lacking until the current study. Our previous study revealed that increases in pea aphid numbers under
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elevated CO2 depend on an increase in BNF and thus an increase
in host plant amino acid metabolism (Guo et al., 2013). When
BNF was suppressed by mutation, host plant amino acid metabolism was not increased by aphid infestation under elevated CO2.
Because BNF is important in supporting the N nutrition of pea
aphids, we speculated that pea aphids would be able to obtain
more N from the supernodulating genotype sickle than from the
wild-type A17.
As important indices of BNF, nodules were more abundant
and expression of ENOD, nifH, and nodF was higher in sickle
than in A17 plants. The increases in N metabolism leads to
increased investment in Rubisco and other C assimilation-related
enzymes (Gleadow et al., 1998), which in turn results in greater
Chl content, biomass, and pod number in sickle than in A17
plants. Although elevated CO2 increased nodule number, gene
expression involved in nodulation and BNF, Chl content, biomass and pod number for both genotypes, it did not change the
growth advantages of sickle relative to A17. The improved growth
traits of sickle had positive bottom-up effects on the aphid, as
demonstrated by greater aphid abundance and feeding efficiency
on sickle than on A17, regardless of CO2 concentrations.
Aphids have stylet-like mouthparts and feed mainly on phloem
sap (Douglas, 2003). Most previous studies have measured aphid
response to elevated CO2 as a function of whole-leaf composition
rather than phloem sap composition (Robinson et al., 2012). The
current study indicated that M. truncatula exhibited three patterns of individual amino acid concentrations in phloem sap in
response to elevated CO2 and aphid infestation. When infested
by aphids, the concentrations of amino acids loading on RF1
(seven essential amino acids and four nonessential amino acids)
were higher in sickle than in A17 regardless of CO2 concentrations. However, elevated CO2 has a contrasting effect on the concentrations of amino acids loading on RF2 (mainly nonessential
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288 Research
Fig. 9 Expression of genes 1-amino-cyclopropane-carboxylic acid (ACC),
sickle gene (SKL) and ethylene response transcription factors (ERF) in the
ethylene signaling pathway in Medicago truncatula as affected by CO2
concentration (ambient (ACO2) vs elevated (ECO2)), plant genotype (A17,
sickle), and pea aphid (Acyrthosiphon pisum) infestation. Values indicate
fold-change in expression based on quantitative PCR, and each value
represents the mean ( SE) of four replicates. Different lowercase letters
indicate significant differences among the combinations of aphid
treatment and CO2 concentrations within the same genotype. Different
uppercase letters indicate significant differences between genotypes
within the same CO2 treatment and aphid treatment as determined by
Tukey’s multiple range test at P < 0.05.
amino acids) in which sickle was higher than A17 plant under
ambient CO2 but lower under elevated CO2. These results confirmed that sickle plants provide better N nutrition for aphids
than A17 plants, and elevated CO2 tends to decrease this nutritional advantage of sickle when compared with A17. Although
elevated CO2 increased the activities of N assimilation and
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transamination-related enzymes, and consequently increased
amino acid concentration of infested plants of both genotypes,
the pattern for this enhancement of amino acids differed between
the two genotypes. Elevated CO2 increased individual amino acid
concentrations loading onto RF1, RF2, and RF3 in A17 plants
but only increased the concentration of amino acids loading onto
RF1 in sickle plants. The relative improvement of N nutrition in
response to elevated CO2 was less in sickle than in A17 plants
because the basal N metabolism under ambient CO2 was much
higher in sickle plants.
The regulation of nodule formation by ethylene signaling
greatly affects the plant’s ability to adapt to elevated CO2, in that
enhanced BNF can satisfy the increased demand for N under elevated CO2 (Penmetsa & Cook, 1997). The current study showed
that elevated CO2 down-regulated the expression of the ethylene
signaling pathway genes ACC, SKL and ERF in A17 plants. Similarly, Casteel et al. (2012) found that elevated CO2 decreased the
ethylene signaling pathway when attacked by Japanese beetles
(Popillia japonica).This result suggests that, under elevated CO2,
M. truncatula suppresses the ethylene signaling pathway so as to
increase nodulation and BNF and thereby satisfy the increased
demand for N. The ethylene signaling pathway in M. truncatula,
however, has also been found to provide resistance against the
pea aphid (Gao et al., 2008).
To access the amino acids in phloem sap, aphids must overcome a number of plant defense responses. One of the early plant
responses to aphids is the release of ROS (Moloi & van der
Westhuizen, 2006). Two other important defense enzymes are
PPO and PAL, which are involved in the synthesis of phenolic
compounds that may be absorbed by the salivary sheath of the
aphid stylet. The further polymerization of phenolic compounds
causes browning of cells in contact with the saliva, which is disadvantageous to aphid feeding (Jiang & Miles, 1993). In our study,
aphid infestation increased the activities of SOD and POD
(which are involved in ROS synthesis) and of PPO and PAL in
A17 plants. In sickle plants, however, aphid infestation did not
induce SOD or POD, which is consistent with a previous finding
that the inhibition of ethylene synthesis or perception blocks the
ROS response (de Jong et al., 2002). These results indicate that
because its ethylene signaling pathway was mutated, sickle could
not trigger the downstream ethylene-dependent defense in
response to aphid infestation. This is consistent with our EPG
finding that, relative to aphids feeding on A17, aphids feeding on
sickle spend less time on salivation and more time on ingestion in
phloem. Furthermore, elevated CO2 decreased the activities of
SOD, POD, and PPO in infested A17 plants. As noted earlier,
the decreased ethylene signaling pathway of M. truncatula under
elevated CO2 affects the pea aphid in two ways, that is, by maintaining host N metablism and reducing host resistance.
Most plants are well adapted to process ‘extra’ carbon under
elevated CO2, and this allows them to grow faster and larger. To
satisfy the increased N demand under elevated CO2, legume
plants evidently decrease their ethylene sensitivity so as to
increase the formation of nodules and enhance BNF. Furthermore, considering the vital role of the ethylene signaling pathway
in regulating plant resistance against aphid infestation, our results
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suggested that the down-regulation of the ethylene signaling
pathway is accompanied by decreased plant resistance against the
pea aphid. In other words, in decreasing the ethylene signaling
pathway under elevated CO2 to match their N budgets and
growth, plants sacrifice their resistance against aphids. Furthermore, given that the ethylene pathway was mutated in sickle
plants, it is reasonable to expect that the mutated plants would
lack one of the phytohormone signaling pathways that increases
N metabolism in response to elevated CO2. Unexpectedly,
although elevated CO2 only increased amino acids in RF1 for
sickle plants infested with aphids, but increased amino acids in
RF1, RF2, and RF3 for A17 plants infested with aphids, nodule
numbers, expression of genes related to N fixation, growth traits,
and N nutrition for pea aphids were all increased in sickle by elevated CO2. This suggests that the ethylene signaling pathway
may not be the only phytohormone pathway regulating plant
response to elevated CO2. The interaction between plant and
aphid is coordinated by other interacting signaling phytohormones (such as jasmonic acid and salicylic acid pathways), except
for ethylene (Felton & Korth, 2000). These signaling pathways
are cross-talked in a complex network, which supports plants rapidly adapting to biotic and abiotic stresses by triggering an enormous regulatory mechanism. Among the three signaling
pathways, jasmonic acid and ethylene signaling pathway has an
antagonistic interaction with salicylic acid signaling pathway.
The emerging data suggest that elevated CO2 tends to modulate
these phytohormone signaling pathways, such as the jasmonic
acid and salicylic acid pathways, that affect responses to insect
herbivores (DeLucia et al., 2012; Sun et al., 2013; Zavala et al.,
2013). Thus, additional research is needed to determine how
multiple phytohormone signaling pathways are coordinately regulated by elevated CO2.
In summary, elevated CO2 increased pea aphid abundance on
M. truncatula by affecting both host plant nutritional quality and
resistance. Elevated CO2 decreased the ethylene-dependent resistance of wild-type M. truncatula against the pea aphid. On the
other hand, the decrease in the ethylene signaling pathway
increased the nodulation and BNF and thereby increased the
phloem amino acids supporting aphid reproduction. The two
effects of the ethylene signaling pathway would synergistically
increase the fitness of pea aphids under elevated CO2. Because
the supernodulating genotype sickle has higher amino acid metabolism and lower resistance, it is more suitable for the pea aphid
than the A17 plant under ambient CO2, and the greater suitability of sickle in comparison to A17 is not changed by elevated
CO2.
Acknowledgements
We thank Prof. Bruce Jaffee (University of California at Davis,
CA, USA) for reviewing a draft of the manuscript. This project
was supported by the ‘National Basic Research Program of
China’ (973 Program) (no. 2012CB114103) and the National
Nature Science Fund of China (nos. 31170390, 31000854, and
31221091).
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Table S1 Primer sequences used for real-time quantitative PCR
Table S2 F- and P-values from MANOVAs for the effect of CO2
concentration and Medicago truncatula genotype on growth traits
of two M. truncatula genotypes
Table S3 F- and P-values from MANOVAs for the effect of CO2
concentration, Medicago truncatula genotype, and pea aphid
infestation on enzyme activities in leaves of two M. truncatula
genotypes
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
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Phytologist
Table S4 F- and P-values from MANOVAs for the effect of CO2
concentration, Medicago truncatula genotype, and pea aphid
infestation on rotated factors of individual amino acids in
phloem sap of two M. truncatula genotypes
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