Article

Ecological trade-offs between jasmonic acid-dependent direct and indirect plant defences in tritrophic interactions

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
New Phytologist (Impact Factor: 7.67). 10/2010; 189(2):557-67. DOI: 10.1111/j.1469-8137.2010.03491.x
Source: PubMed

ABSTRACT

Recent studies on plants genetically modified in jasmonic acid (JA) signalling support the hypothesis that the jasmonate family of oxylipins plays an important role in mediating direct and indirect plant defences. However, the interaction of two modes of defence in tritrophic systems is largely unknown.

In this study, we examined the preference and performance of a herbivorous leafminer (Liriomyza huidobrensis) and its parasitic wasp (Opius dissitus) on three tomato genotypes: a wild-type (WT) plant, a JA biosynthesis (spr2) mutant, and a JA-overexpression 35S::prosys plant. Their proteinase inhibitor production and volatile emission were used as direct and indirect defence factors to evaluate the responses of leafminers and parasitoids.

Here, we show that although spr2 mutant plants are compromised in direct defence against the larval leafminers and in attracting parasitoids, they are less attractive to adult flies compared with WT plants. Moreover, in comparison to other genotypes, the 35S::prosys plant displays greater direct and constitutive indirect defences, but reduced success of parasitism by parasitoids.

Taken together, these results suggest that there are distinguished ecological trade-offs between JA-dependent direct and indirect defences in genetically modified plants whose fitness should be assessed in tritrophic systems and under natural conditions.

Full-text

Available from: Chuanyou Li
Ecological trade-offs between jasmonic acid-dependent
direct and indirect plant defences in tritrophic
interactions
Jianing Wei
1
, Lizhong Wang
1
, Jiuhai Zhao
2
, Chuanyou Li
2
, Feng Ge
1
and Le Kang
1
1
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China;
2
State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology of the Chinese Academy of
Sciences, Beijing 100101, China
Author for correspondence:
Le Kang
Tel: +86 10 64807219
Email: lkang@ioz.ac.cn
Received: 15 April 2010
Accepted: 24 August 2010
New Phytologist (2011) 189: 557–567
doi: 10.1111/j.1469-8137.2010.03491.x
Key words: ecological trade-off, genetically
modified tomato plants, jasmonic acid,
Liriomyza huidobrensis, Opius dissitus, plant
defences, Solanum lycopersicum, tritrophic
interactions.
Summary
Recent studies on plants genetically modified in jasmonic acid (JA) signalling
support the hypothesis that the jasmonate family of oxylipins plays an important
role in mediating direct and indirect plant defences. However, the interaction of
two modes of defence in tritrophic systems is largely unknown.
In this study, we examined the preference and performance of a herbivorous
leafminer (Liriomyza huidobrensis) and its parasitic wasp (Opius dissitus) on three
tomato genotypes: a wild-type (WT) plant, a JA biosynthesis (spr2) mutant, and a
JA-overexpression 35S::prosys plant. Their proteinase inhibitor production and
volatile emission were used as direct and indirect defence factors to evaluate the
responses of leafminers and parasitoids.
Here, we show that although spr2 mutant plants are compromised in direct
defence against the larval leafminers and in attracting parasitoids, they are less
attractive to adult flies compared with WT plants. Moreover, in comparison to
other genotypes, the 35S::prosys plant displays greater direct and constitutive
indirect defences, but reduced success of parasitism by parasitoids.
Taken together, these results suggest that there are distinguished ecological
trade-offs between JA-dependent direct and indirect defences in genetically modi-
fied plants whose fitness should be assessed in tritrophic systems and under natural
conditions.
Introduction
The bottom-up effects of plants against herbivores indicat ed
that plants have evolved a wide range of direct and indirect
defensive strategies (Gatehouse, 2002). Plants defend them-
selves against insects directly through feeding (e.g. physical
barriers or toxins) (Kessler & Baldwin, 2002; Peiffer et al.,
2009) and indirectly through attracting natural enemies of
herbivores (Turlings et al., 1995; Heil, 2008; Dicke et al.,
2009; Kang et al., 2009). Both direct and indirect plant
defences are constitutive and inducible, while induced
strategies may be favoured over constitutive ones because of
the low cost (Kessler & Baldwin, 2002). Some studies
support a trade-off between direct and indirect defences
because of resource limitation (Ballhorn et al., 2008). For
example, plant species with a strong direct defence may not
invest in indirect defence through the emission of spec ific
volatiles (Van Den Boom et al., 2004). In addition, direct
defence traits, for example, secondary plant compounds and
physical barriers, may also have negative impacts on natural
enemies of insect herbivores. However, a recent study revealed
that there is no conflict between direct and indirect plant
defences in a highly specialized herbivore–natural enemy
system involving a brassicaceous plant species (Gols et al., 2008).
To enhance plant resistance against insect pests, defence
genes fr om plants and nonplant origins have been intro-
duced into major crops (Schuler et al., 1998; Turlings &
Ton, 2006; Kos et al., 2009). Although transgenic plants,
such as Bacillus thuringiensis (Bt)-expressing crops, can
effectively suppress population densities of target insect
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pests, concerns are often raised about the long-term and
wider ecological risks associated with the release of geneti-
cally modified plants (O’Callaghan et al., 2005). Rarely
considered are the ecological consequences of transgenic
plants on tritrophic interactions among plants, herbivorous
insects and natural enemies (Groot & Dicke, 2002; Kos
et al., 2009). In addition, the potential effects of direct
defence of genetically modified plants on the efficiency of
their indirect defence, and vice versa, are not well character-
ized, which is probably because of the lack of genotypes that
differ exclusively in the same defensive trait (Dicke et al., 2004).
Tomato, Solanum lycopersicum, is an economically
important vegetable worldwide and a commonly used
model plant for biologists. It has long been used to study
defence-related signalling path ways and gene expressions
(Browse, 2005; Schilmiller & Howe, 2005). Accumulating
evidence from tomato and other model systems supports
the hypothesis that the jasmonate family of oxylipins plays
an important role in mediating herbivore-trig gered direct
and indirect plant defen ces (Kessler & Baldwin, 2002;
Browse, 2005). For example, impairing or silencing genes
related to oxylipin signalling pathways render the mutants
or transformants more susceptible to herbivores (Thaler
et al., 2002; Ament et al., 2004; Kessler et al., 2004; Li
et al., 2005; Chehab et al., 2008; Halitschke et al., 2008)
and less attractive to natural enemies under both laboratory
and field conditions (Thaler et al., 2002; Shiojiri
et al.,2006; Bruce et al., 2008; Chehab et al., 2008;
Halitschke et al., 2008). Similarly, genetic engineering of
terpenoid metabolism or overexpression of a single gene
involved in terpenoid production enables the plant to
attract more natural enemies (predator, parasitic wasp or
entomopathogenic nematode) (Kappers et al., 2005;
Schnee et al., 2006; Degenhardt et al., 2009). However, in
most of these studies, the direct and indirect defences were
investigated independently, and only a few examined their
relationship in a tritrophic system, for example, a tomato,
spider mite and predator mite system (Thaler et al., 2002).
Furthermore, the ecological consequences of genetically
modified plants were rarely characterized under natural
conditions (Halitschke et al., 2008).
Here, we investigated the roles of the jasmonic acid (JA)
pathway in regulating plant–insect interaction in a tritroph-
ic system, including the behavioural responses of herbivore
and parasitoid and the defensive signals in wild-type (WT)
plants and JA mutants; in addition, the interaction of direct
and indirect plant defences was analysed. We used a trans-
genic tomato line (35S::prosys) with constitutive JA signal-
ling, and a tomato mutant with defective JA biosynthesis
(spr2). Our study showed that, although spr2 mutant plants
are compromised in direct defence against the larval leaf-
miners and in attracting parasitoids, they are less attractive
to adult flies compared with WT plants. Moreover, in
comparison to other genotypes, the 35S::prosys plant dis-
plays greater direct and constitutive indirect defences, but
reduced success of parasitism by parasitoids. These results
indicate that there are distinguished ecological trade-offs
between JA-dependent direct and indirect defences in
tritrophic interactions under more natural conditions.
Materials and Methods
Plants and insects
Tomato (Solanum lycopersicum) line cv Castlemart was used
as the WT parent for all experiments. Tomato mutant line
spr2 (Li et al., 2003) was derived from cv Castlemart. Seeds
for the 35S:: prosystemin transgenic plants were collected
from a 35S::prosys 35S::prosys homozygous line (Howe &
Ryan, 1999) that was backcrossed five times using tomato
cv Castlemart as the recurrent parent. Tomato seedlings
were grown in 500 ml pots containing a mixture of peat
and vermiculite (4 : 1), in environmental chambers
(Conviron Co., Winnipeg, MB, Canada) under 16 h light
at 28C and 8 h dark at 18C, with irradiance of
150 lEm
)2
s
)1
during photophase, and 60% relative
humidity. Plants with four to six fully expanded leaves were
used for experiments. In the choice experiments, the age
and leaf area of each genotype were normalized by sowing
in parallel under the same culture conditions.
Colonies of the pea leafminer, Liriomyza huidobrensis ,
and the parasitoid, Opius dissitus, were maintained as
described previously (Wei & Kang, 2006). Briefly, 2-wk-
old kidney bean plants (Phaseolus vulgaris L. cv Naibai) with
two fully developed true leaves were used to culture leaf-
miners. O. dissitus females emerging from pupae were
mated within 24 h. All O. dissitus used in the behavioural
assays and parasitism experiments were 2- to 4-d-old adult
females without previous exposure to their host, L.
huidobrensis, or host plants. Each female was used only once
in the experiments.
Adult leafminer preference for tomato genotypes
Feeding and oviposition preference of adult flies to intact
tomato plants were monitored in a cage of 40 ·
40 · 40 cm. This experiment was designed as a dual-choice
test. The mutants and WT plants were of similar age and
shape. For the combination of WT plant vs spr2 mutant,
two plants of each genotype were paired in the cage. Then,
plants were exposed to 150–200 adult flies (m ale :
female = 1 : 1) for 5 h under the same conditions as used
for rearing the pea leafminer. For the combination of JA-
overexpressing line 35S::prosys vs WT plant, the number of
adult leafminers or the exposure time was increased to 300
files or 7 h to obtain the comparable density of feeding and
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oviposition punctures on the most resistant line, the
35S::prosys plant.
Adult parasitoid preferences for tomato genotypes
Y-tube experiments A Y-tube ol factometer was used to
investigate the behavioural responses of female O. dissitus to
the volatile blends from the various genotypes (Wei et al.,
2007). Each female parasitoid was placed in the olfacto-
meter for 5 min. A ‘no choice’ outcome was recorded when
the adults remained inactive during the testing period. A
‘first choice’ outcome was recorded when the adults moved
> 5 cm onto eithe r arm (visually assessed by a line marked
on each arm). Each experiment involved at least 30 females
making a choice. Each odour source (Fig. 2a–c) was pre-
pared by mixing three to four volatile collections (see the
Plant volatile collection section for details) and concentrat-
ing to 1000 ll in a nitrogen (purity 99.999%) atmosphere.
In the dual-choice tests, each solution (10 ll) of paired
genotype or treatment was applied to one piece of filter
paper (1 · 2 cm), which was placed inside one of the two
arms of the Y-tube olfactometer. The mean dosage of each
extract was equivalent to 0.2–0.4 h entrainment of volatiles.
Filter paper with plant odour was refreshed after each test.
Cage experiments Landing preferences of parasitoids to
the larval-damaged tomato genotypes were observed in a
cage of 40 · 40 · 40 cm. This experiment was designed as
a dual-choice test. Plants containing second-instar larvae
were prepared as for the larval leafminer performance
experiment. The mutants and WT plants used in this study
were of similar age and shape, and had similar leafminer
larvae numbers (Supporting Information, Table S1). Two
WT tomato plants and two mutant plants, either spr2 or
35S::prosys containing leafminer larvae, were paired and
enclosed in the cage. An individual naive female parasitoid
was released at the midpoint between two groups of tomato
plants and its behaviour was monitored for 10 min. The
plant on which parasitoid landed and its subsequent search-
ing attempts (e.g. walking and touching mines) were
recorded as a choice. If the parasitoid did not show any
oviposition attempts when landing or did not land on any
plant during the test period, it was recorded as ‘no choice’.
At least 30 female wasps were tested in a cage with the same
set of plants. Based on the number of landing parasitoids on
each tomato genotype, a landing rate (%) was calculated as
the preference for this genotype. Each assay was replicated
at least six times with newly prepared plants.
Parasitism of leafminer larvae on tomato genotypes
The plant treatment and experimental design were the same
as for the preference assays of parasitoid wasps. Two WT
tomato plants and two mutant plants, each infested with
similar numbers of leafminer larvae (Table S1), were
enclosed in a cage. The number of parasitoids released into
each cage was calculated to achieve a ratio of one parasitoid
for every 10 larvae. Our preliminary experiments showed
that no superparasitism would occur under this ratio within
24 h. Parasitism was determined by dissecting leafminer
larvae 3 d after exposure to adult parasitoids when larval
parasitoids had developed into the first-instar larval stage.
Larval leafminer performance on each tomato geno-
type
A no-choice experiment was designed to examine larval
performance on each genotype. Plant preparation and
treatments were similar to experiments regarding adult leaf-
miner preferences discussed earlier. For the JA-overexpressing
line, 35S::prosys, the exposure time and the number of adult
leafminers were doubled to obtain similar number of larvae as
other genotypes (Table S1). Subsequen tly, the exposed
plants loaded with leafminer eggs were moved into growth
chambers for development.
To determine larval survival on each genotype, plants
with leafminer eggs were monitored daily until the larval
mines became visible. Then the old and new mines (viable
eggs) were counted and marked by a black marker to avoid
recounting, usually on day 3–4 after oviposition. When lar-
vae completed development and were ready to pupate,
plants were laid down on Styrofoam trays and all larvae
emerging from mines were collected. Puparia from each
genotype were collected and counted daily, and were kept
in glass vials (60 · 15 mm). Larval survival (%) was calcu-
lated as the ratio of total pupae number to viable eggs.
To investigate leafminer development on each genotype,
plants with leafminer eggs were monitored twice daily (at c.
08:00 and 16:00 h) from egg hatching to pupation, and the
time from pupation to adult eclosion was recorded daily.
Each day, 10–15 randomly selected larvae were removed
from the leaves of each plant genotype using a pin. They
were preserved in 70% ethanol and the developmental stage
was determined as described by Petitt (1990). Larvae num-
bers at each developmental stage were recorded. An estimate
of larval area (length · width) was used to represent host
size (Ode & Heinz, 2002). We photographed and measured
larvae with a microscope (Leica DFC490; Leica, Wetzlar,
Germany) and processed the images using the
QWIN PLUS
software (Leica). More than 20 larvae were measured for
each genotype and developmental stage. When larvae com-
pleted development and were ready to pupate, plants were
laid down on Styrofoam trays and the numbers of collected
larvae or pupae at each time were recorded. Puparia were
collected from each genotype and placed in glass vials
(60 · 15 mm), and adult emergence was monitored daily.
The developmental time, defined as the required time for
50% of the population to reach a certain developmental
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stage, was recorded for each larval stage and tomato geno-
type in h, and then converted into d.
Quantification of proteinase inhibitor (PI)-II proteins
Using a radial immunodiffusion assay described by Ryan
(1967), we quantified the amounts of PI-II protein in the
leaflets of each genotype. Briefly, an agar plate was prepared
using 2% Noble agar (Sigma A-5431) in 0.1 M sodium ve-
ronal, 0.9% NaCl, at pH 8.5, to which 1% goat polyclonal
tomato PI-II antiserum was added.
Samples in each genotype were taken from undamaged
and larval leafminer-damaged tomato plants, respectively.
For each und amaged tomato genotype, leaflets were
removed from lower, middle and upper layers of two to
three randomly selected plants and pooled into a mortar,
and the leaf contents were extracted. For each leafminer-
infested genotype, PI-II was measured on days 4, 5 and 7,
respectively, after oviposition by adult flies as described
earlier. For each replication per genotype, leaflets were
removed from lower, middle and upper layers of two to
three randomly selected plants, with or without leafminer
damage, and pooled into a mortar, and the leaf contents
were extracted. Five millilitres of leaf extract from each
treatment were placed into a well (0.5 mm diameter) of
agar plate. After 24 h, the diameter of the immunoprecipi-
tate ring was measured to calculate the amount of PI-II
based on the following formula: ((diameter · diameter )
625) · 0.016) (Ryan, 1967). Both local and systemic PI-II
amounts were determined for leafminer-damaged plants.
There were at lea st six replications per genotype and
treatment.
Plant volatile collection
Tomato plants for volatile collection were treated as
described in Larval leafminer performance. For each treat-
ment of leafminer-damaged plant, tomato plants with 4–6
expanded leaves were transferred to environmental growth
chambers as described in Plants section. To determine the
difference of JA-induced volatile emission in various tomato
genotypes, tomato plant roots were placed in vials contain-
ing 50 ml of aqueous JA (Sigma-Aldrich) solution (1 mM)
with 0.5% alcohol for 48 h (Hopke et al., 1994), and the
vials were sealed with Parafilm. Control plants were placed
in vials with 50 ml of 0.5% alcohol for 48 h. JA-treated
plants or controls were transferred to individual glass tubes
filled with 50 ml of tap water, and then were subjected to
the volatile collection system.
The headspace volatile collection system was designed as
described by Wei et al. (2006, 2007) with minor modifica-
tions. Briefly, two plants were placed in a plastic oven bag
(40 · 44 cm; Reynolds
, Richmond, Virginia, USA), into
which a stream of filtered and moisturized air was pumped
through two freshly activated charcoal traps. The air with
emitted plant volatiles was withdrawn through a glass
collector containing 100 mg Porapak Q (80–100 mesh size;
Supelco, Bellefonte, PA, USA) by a membrane pump
(Beijing Institute of Labor Instruments, Beijing, China) at a
rate of 400 ml min
)1
for 10 h. Five to six collections were
made simultaneously, with one blank bag as control, at
24 ± 2C and 180 lEm
)2
s
)1
irradiance. The absorbed
volatiles from Porapak Q collectors were then extracted with
700 ll of high-performance liquid chromatography
(HPLC)-grade dichloromethane (Tedia Company, Fairfield,
Ohio, USA). All aeration extracts were stored at )20C until
used in chemical analyses or behavioural experiments. To
quantify volatiles, plants were weighed immediately after
volatile collection using an electronic balance (AE 240;
Mettler, Toledo, Switzerland). The larvae numbers in
tomato leaflets were recorded by carefully examining leaves
under a stereo microscope (Wild, Heerbrugg, Switzerland).
Chemical identification and quantification of collected
volatiles
The chemical structures of collected volatile compounds
were identified as described by Wei et al. (2007) with small
modifications. Briefly, an Agilent gas chromatographer
(GC) (6890N) coupled with a mass spectrometry (MS) sys-
tem (5973 MSD; Agilent Technologies, Inc., Palo Alto,
CA, USA) was equipped with either a DB-WAX column
(60 m · 0.25 mm ID, 0.15 lm film thickness) or a DB5-
MS column (60 m · 0.25 mm ID · 0.15 lm film thick-
ness; Agilent Technologies) for chemical identifications. On
the DB-WAX column, the oven temperature was initially
kept at 40C for 4 min and then increased by a rate of
10C min
)1
to 200C, followed by a rate of 10C min
)1
to 230C. The inlet was operated in the splitless injection
mode, and the injector temperature was maintained at
250C with a constant flow rate of 1.0 ml min
)1
. The GC-
MS electron impact source was operated in the scan mode
with the MS source temperature at 230C and the MS
Quad at 150C. Volatile compounds were identified by
comparing their retention time and spectra with synthetic
standards(seeWeiet al.,2006;plus2-carene,97%;p-cymene,
97%; (Z)-3-hexenyl butyrate, 98%; Aldrich).
Referenced mass spectra were from the NIST02 library
(Scientific Instrument Services, Inc., Ringoes, NJ, USA).
To quantify collected volatiles, an Agilent GC (7890A)
coupled with an auto-injector (7683 autoinjector module,
cataloge number G2913A; Agilent Technologies, Inc.) was
equipped with the DB-WAX column described earlier, and
the same thermal progra mme was adopted. Mixed samples
consisting of heptanoic acid, ethyl ester and dodecanoic
acid, ethyl ester in different concentrations (1, 5, 20, 50, or
100 ng ll
)1
) were used as external standards for developing
standard curves to quantify volatiles.
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Data analysis
Data were analysed using the
SPSS software package (version
15.0; SPSS Inc., Chicago, IL, USA). In dual-choice tests,
the oviposition preference of adult leafminer and parasitism
and the landin g rates of par asitic wasps were compared by
paired t-test. For parametric analysis, the percentage data
(survival and landing rates) were ar csin(x
1 2
)-transformed,
while the absolute quantity data were log(x + 1)-trans-
formed to correct heterogeneity of variances. Each experi-
ment was replicated four to six times. The chi-squared test
was used to determine the significance of diff erence between
the numbers of parasitoids choosing each olfactometer arm
(Wei & Kang, 2006; Wei et al., 2007). Parasitoids that did
not make a choice were excluded from statistical analysis.
Larval survival (%) and body size were compared among
three genotypes using ANOVA and Tukey’s honestly signif-
icant difference (HSD) test. Developmental time of L.
huidobrensis wa s compared using a Kruskal–Wallis test fol-
lowed by a Mann–Whitney U-test. The temporal difference
of JA-regulated PI-II accumulation in three genotypes was
analysed using repeated-measures ANOVA with plant
genotype as an independent factor, and the PI-II amount at
each observation period was compared by Kruska l–Wallis
test followed by Mann–Whitney U-test. The amounts of
volatiles released from each genotype and treatmen t were
normalized to ng h
–1
(10 g)
–1
plant FW and compared by
Student’s t-test. The amounts of leafminer-induced volatiles
emitted by each genotype were normalized to ng h
–1
per
100 larvae (10 g)
–1
plant FW and compared by ANOVA
and HSD tests (Wei et al., 2006).
Results
Adult leafminer preferences for tomato genotypes
To determine the preference of Liriomyza huidobrensis adult
flies for different tomato genotypes, feeding and oviposition
punctures (FOPs) on WT plants paired with each mutant
were monitored in cages. Our results showed that the adults
preferred WT plants over the JA-deficient mutant spr2
(paired samples t-test, t=5.36, df = 5, P = 0.003; Fig. 1a)
and the JA-overexpre ssing geno type 35S::prosys (paired
samples t-test, t=11.21, df = 5, P < 0.0001; Fig. 1b).
Adult parasitoid preferences for tomato genotypes
To determine the preference of O. dissitu s female wasps for
volatiles emitted by different tomato genotypes, behavioural
responses and parasitism of larval leafminers by fem ale
wasps were investigated in a dual-choice olfactometer
and or in a cage. Our results showed no difference in
behavioural preference of female wasps between the unda-
maged WT and JA-deficient mutant plants (Fig. 2a), but
the undamaged 35S::prosys line was significantly preferred
by the parasitoids over the other genotypes in the Y-tube
tests (35S::prosys vs WT, v
2
= 5.12, P = 0.024; 35S::prosys
vs spr2, v
2
= 5.44, P = 0.02). In addition, leafminer-
damaged WT and 35S::prosys plants were significantly more
attractive to parasitoids than the leafminer-damaged JA-
deficient mutant (WT vs spr2, v
2
= 4.50, P = 0.034; WT
vs 35S::prosys, v
2
= 0.125, P = 0.724; 35S::prosys vs spr2,
v
2
= 5.12, P = 0.024; Fig. 2b). JA-treated spr2, 35S::prosys,
and WT plants were all more attractive to female wasps
than the controls (JA-spr2 vs spr2, v
2
= 11.55, P = 0.0098;
JA-35S::prosys vs 35S::prosys, v
2
= 7.04, P = 0.0079;
JA-WT vs WT, v
2
= 5.90, P = 0.015; Fig. 2c). In cage
experiments, the number of first landings for naive female
parasitoids on WT plants was significantly higher than on
the JA-deficient genotype (paired samples t-test, WT vs
spr2, t=8.52, df = 5, P < 0.001, Fig. 2d), while it was
similar to that on 35S::prosys plants (paired samples t-test;
WT vs 35S::prosys, t=0.54, df = 5, P = 0.614; Fig. 2d).
(a)
(b)
Fig. 1 Feeding and oviposition preferences of adult leafminer
Liriomyza huidobrensis for tomato genotypes. Mean (N =6)
number of feeding and oviposition punctures (FOPs) of adult
leafminers on paired wild-type (WT) and spr2 plants (a), or on
paired WT and 35S::prosys (35S) plants (b). FOPs were compared
between the plants of each pair by a paired samples t-test (two-
tailed). Data were arcsin(x
1 2
)-transformed. Significant differences
between two tomato genotypes are indicated by different letters on
each bar (P < 0.05).
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To determine the impact of this odour-driven preference
on parasitism, we measured the parasitism rates in dual-
choice experiments in cages. Indee d, the parasitoid’s prefer-
ence for WT plants over the JA-deficient mutant spr2
caused a higher degree of parasitism (paired samples t-test,
t=6.92, df = 11, P < 0.0001; Fig. 2e). However, com-
pared with 35S::prosys, larvae on WT plants were parasitized
by the parasitoids at a higher rate (t=5.84, df = 5,
P = 0.002; Fig. 2e).
Direct defence: pea leafminer performance on three
tomato genotypes
The performance of leafminers differed significantly among
three genotypes (Fig. 3a–d; larval body size on the fifth day:
ANOVA, F
2,63
= 37.60, P < 0.001; also see Fig. S1; devel-
opmental time from egg to adult eclosion: Kruskal–W allis
test, v
2
= 14.7, df = 2, P < 0.001; also see Fig. S2a; pupal
weight: ANOVA, F
2,26
= 122.15, P < 0.001; proportion of
adult eclosion was similar for the three genotypes: ANOVA,
F
2,12
= 1.02, P = 0.335; see Fig. S2b). Larval survival (%)
on WT plants was significa ntly lower than on the JA-defec-
tive mutant spr2, but it was much higher than on the
JA-overexpressing genotype 35S::prosys (ANOVA, F
2,12
=
89.16, P < 0.001; Fig. 3b). Therefore, the performance of
leafminers on WT plants was poorer than on spr2 plants
but better than on 35S::prosys plants.
PI-II in three tomato genotypes
Accumulation of PI-II differed significantly among the
three tomato genotypes (repeated measures ANOVA,
F
genotype, 2,62
= 127.64, P < 0.0001; Fig. 4) and varied with
time (F
time, 2,62
= 58.83, P < 0.0001; F
genotype · time
=
16.92, P < 0.001). Compared with the other two geno-
types, undamaged 35S::prosys plants constitutively expressed
higher amounts of PI-II protein (Fig. 4). Leafminer infesta-
tion triggered PI-II accumulation in spr2 plants, but the
amounts were significantly lower than the accumulation in
WT plants (Fig. 4). In contrast, PI-II accumulation in leaf-
miner-treated 35S::prosys plants was dramatically higher
than in WT plants (Fig. 4). Thus, PI-II accumulation was
negatively correlated with leafminer performance on the
three tomato genotypes (F
PI-survival%, 1,12
= 17.12, r
2
=
0.633, P = 0.0019; F
PI-larval body size, 1,12
= 29.20, r
2
=
0.745, P < 0.0001; F
PI-development times, 1,12
= 31.04,
r
2
= 0.756, P < 0.0001; Figs 3, 4).
Indirect defence signals in three tomato genotypes
To investigate the role of the JA signalling pathway in regu-
lating volatile emissions, volatile profile, an indicator of
indirect defence, was examined in three tomato genotypes.
Undamaged, leafminer-damaged, 0.5% alcohol-treated
(JA control) and JA-treated tomato plants released five
(a) (b)
(d) (e)
(c)
Fig. 2 Behavioural responses and parasitism rates of larval leafminers by female Opius dissitus wasps. Behavioural responses of naı¨ve female
wasps to volatile blends emitted by paired undamaged tomato genotypes (a), leafminer (Liriomyza huidobrensis)-damaged genotypes (b) and
jasmonic acid (JA)-treated genotypes (c). Bars represent the percentages of parasitoids choosing either odour source offered in a Y-tube
olfactometer. Numbers in bars are the numbers of parasitoids choosing the indicated odour source. (d) Landing preference of O. dissitus naı¨ve
females determined as the percentage of parasitoids landing on each of the two offered tomato plant genotypes (N = 6). (e) Larval parasitism
rates (%) based on dissection of leafminer larvae 3 d after exposure to adult parasitoids (wild-type (WT) vs spr2 plants, N = 12; WT vs
35S::prosys plants (35S), N = 6). In each panel, *, P<0.05; **, P<0.01; n.c., no choice; NS, nonsignificant.
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monoterpenes (MTs) and one aromatic compoun d (AR,
p-cymene) (Fig. 5; for compound list see Tables S2, S3).
Interestingly, the typically inducible compounds, (Z)-3-
hexenol (Z3Hol) and (3E,7 E)-4,8,12-trimethyl-1,3,7,11-
tridecatetraene (TMTT), were constitutively released by the
undamaged and alcohol-treated 35S::prosys plants.
However, when plants were damaged by leafminer, the JA-
deficient mutant emitted significantly lower amoun ts of
inducible volatiles [Z3Hol, TMTT and other leafminer-
induced volatile com pounds (LIVOCs )] in comparison to
WT and 35S::prosys plants (ANOVA, F
2,12
= 14.38,
P < 0.001) and, qualitatively, WT plants released higher
numbers of inducible compounds than other genotypes
(Fig. 5a). Upon JA treatment, the three genotypes emitted
similar amounts of inducible volatiles [Z3Hol, TMTT, and
other JA-induced volatile compounds (JAIVOCs)]
(ANOVA, F
2,9
= 2.08, P = 0.11; Fig. 5b). In addition, the
emission of (Z)-3-hexe nol was substantially higher in leaf-
miner-damaged WT plants than in the other two mutants
(ANOVA, F
2,12
= 6.27, P = 0.033; Fig. 5a), with spr2
plants and the JA-overexpressing 35S::prosys line emitting
28.6 and 40%, respectively, of the amount emitted by WT
plants. The release rate of TMTT from 35S::prosys mutants
was comparable to WT plants (t=1.86, df = 8, P =
0.465). In contrast, the amounts of (Z)-3-hexenol and
TMTT were significantly higher in JA-treated WT plants
than in other genotypes (ANOVA, F
2,9
= 18.9, P < 0.001).
Discussion
Ecological trade-off between direct and indirect plant
defences in JA-overexpression plants
Our data showed that although the amounts of inducible
compounds were significantly lower in 35S::prosys plants
(a) (b)
(c) (d)
Fig. 3 Effects of direct defence on the
performance of pea leafminer, Liriomyza
huidobrensis, in different tomato genotypes.
(a) Larval mining damage on the leaflet of
each genotype at day 7 (the third-instar larva
stage) after female leafminer ovipositions.
(b) Larval survival (%) on each genotype.
The bar represents the mean values for five
replicates of each genotype (N = 5). (c)
Larval body size (length · width, mm
2
)on
each genotype. The bar represents five
replicates of each genotype (N =5)( , wild-
type (WT);
, spr2; , 35S::prosys (35S)).
Days 4, 5 and 7 after oviposition correspond
to the developmental stage of first-, second-,
and third-instar larvae, respectively. (d)
Developmental time (d) from egg hatch to
pupation was recorded for each genotype.
There were at least six replications per
genotype per developmental stage (N = 6).
Bars indicate means ± SEM, and significant
differences among three tomato genotypes
are indicated by letters on each bar.
Fig. 4 Proteinase inhibitor II (PI-II) accumulates in the leaflets of
three tomato genotypes in response to leafminer damage (N = 6).
Bars indicate means ± SEM; significant differences among three
tomato genotypes are indicated by letters on each bar. Days 4, 5
and 7 after oviposition correspond to the developmental stage of
first-, second-, and third-instar larvae, respectively. WT, wild-type;
35S, 35S::prosys (
, WT; , spr2; , 35S).
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than in WT plants after leafminer damage (see Fig. 5),
naive female wasps did not discriminate between leafminer-
damaged 35S::prosys and WT plants, suggesting that the
induced indirect defence was not affecte d in the JA-overex-
pressing line. One question is whether constitutive emission
of attractants by resistant genotypes poses a conflict between
direct and indirect plant defences (but see Turlings & Ton,
2006 and Gols et al., 2009). Interestingly, we demonstrated
in a cage experiment that larvae feeding on the JA-overex-
pressing line were parasitized at a lower rate than those feed-
ing on WT plants. It is possible that plant secondary
chemicals, such as toxins and anti-digestive proteins, have a
negative impact on the development of herbivorous insects
as well as on the fitness and or foraging behaviour of their
natural enemies (Gols et al., 2009). We found that after 5 d
of development, PI-I I accumulation in the leaflets of
35S::prosys plants was 2.3-fold higher than that in WT
plants, and the average size of larvae on 35S::prosys was
threefold smaller than on WT plants (see Figs 3d, S1). In
addition, female O. dissitus wasps preferred to frequently
parasitize in bigger larvae than in smaller ones (Bordat
et al., 1995). These data imply that there is a potential eco-
logical trade-off between direct and indirect plant defences
in the JA-overexpressing tomato genotype, and the reduced
parasitism on 35S::prosys plants may be partially the result
of the poor quality of host larvae (Gols et al., 2009).
Another study showed that JA treatment of a tomato culti-
var (Lycopersicon esculentum var. Ace) reduced the perfor-
mance of a caterpillar (Spodoptera exigua) and its parasitoid
wasp (Hyposoter exiguae) (Thaler, 1999). However, this
study revealed increased rates of parasitism in JA-induced
plants, which were not attributed to longer parasitoid expo-
sure or lower herbivore quality. The author postulated that
increased volat ile production from JA-treated plants
attracted more parasitoids and resulted in a higher parasitism
rate, which was inconsistent with our results using a
prosystemin overexpression line. Based on the slow growth
high mortality hypothesis (Benrey & Denno, 1997) and the
fact that the larval development was about 4 d longer
(Fig. 3) on 35S::prosys plants than on WT plants, one might
predict a higher overall vulnerability to parasitism of larvae
feeding on 35S::prosys than on WT plants. Nevertheless,
Benrey & Denno (1997) and Williams (1999) also indi-
cated that slow growth does not always result in increased
parasitism, especially when different host plant species with
various defence traits were compared or when parasitoids
are involved.
Inducible volatiles, such as (Z)-3-hexenol and TMTT,
play important role in host habitat location by
parasitoids
Our data also demonstrated that transgenic 35S::prosys
plants have a higher degree of direct defence against the leaf-
miners than WT plants. Since constitutive and induced
expression of PI-II was enhanced in 35S::prosys plants, it is
not surprising that this genotype exhibited the high est resist-
ance to leafminers. Interestingly, undamaged 35S::prosys
plants also constitutively released th e typical defence-related
volatiles, HIPVs (herbivore-induced plant volatiles), (Z)-3-
hexenol and TMTT, all of which were undetectable in other
genotypes when undamaged. Transgenic tomato overex-
pressing the prosystemin gene exhibits enhanced expression of
TomLOX-C (lipoxygenase C in tomato), TomLOX-D and
hydroperoxide lyase (HPL) genes (Corrado et al., 2007),
which could explain our finding that this JA-overex pressing
mutant constitutively released (Z)-3-hexenol and TMTT.
Behavioural assays in the Y-tube olfactometer showed that
female parasitic wasps preferred the odour of undamaged
35S::prosys plants to that of the other two genotypes
(a) (b)
Fig. 5 Leafminer (Liriomyza huidobrensis)- and jasmonic acid (JA)-
induced volatile compounds released from three tomato genotypes.
The volatile profiles include undamaged ( ) and leafminer-damaged
(
) tomato plants (a), and 0.5% alcohol-treated ( ) and JA-treated
(
) plants (b). MT, monoterpenes (a-pinene, 2-carene, a-
phellandrene, limonene, b-phellandrene); SQT, sesquiterpene (b-
caryophyllene); AR, aromatic (p-cymene); Z3Hol, (Z)-3-hexenol;
TMTT, (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene; LIVOCs,
other leafminer-induced volatile organic compounds ((Z)-3-hexenyl
butyrate, (Z)-3-hexenyl acetate); ALIVOC, alcohol-induced volatile
compounds (butanoic acid, ethyl ester); JAIVOCs, other JA-induced
volatile compounds ((E)-b-ocimene, (E)-2-hexenal, (Z)-3-hexenyl
acetate, propanoic acid, ethyl ester). Compound lists of tomato
genotypes and treatments are presented in the Supporting
Information (Tables S2, S3); comparisons among genotypes or
between treatments are presented in Table S4. Bars indicate
means ± SEM; *, P<0.05. In (a) there were five replications per
genotype per treatments (N = 5). In (b) there were four replications
per genotype per treatments (N = 4).
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(Fig. 3a), confirming that the inducible volatiles, such as
(Z)-3-hexenol and TMTT, can effectively attract parasitic
wasps to the leafminers’ habitat (We i et al., 2007). The
production of saturated hexanol and hexanal was higher in
spr2 mutants than in WT and 35S::prosys plants (Sanchez-
Hernandez et al., 2006), but the preference of parasitoids
was not interfered with by these saturated C6 compounds.
Ecological trade-off in herbivore attraction in
JA-deficient mutants
We found that the spr2 mutant displayed markedly reduced
direct and indirect defences against larval leafminers.
Susceptibility to a larval leafminer was correlated with
decreased production of PI-II protein and reduced attrac-
tion and parasitism success by a parasitic wasp, which was
consistent with the decreased inducible volatile emission. In
herbivore-damaged plants, the reduced PI-II accumulation
and volatile emission in the JA biosynthesis mutant spr2
could be the result of the loss of function of x-3 chloroplast
fatty acid desaturase, which inhibits LA (the lipid-derived
fatty acid 18:3) content in leaflets to < 10% of the amount
in WT plants (Li et al., 2003). Meanwhile a corresponding
increase in LA proportion (18:2) resulted in augmented pro-
duction of saturated hexanol and hexanal in this mutant (Li
et al., 2003; Sanchez-He rnandez et al., 2006). Moreover,
compared with WT and 35S::prosys plants, reduced volatile
emission was constitutively detected in unwounded and
wounded spr 2 plants (see Table S4), which was consistent
with a previous study using spr2 plants (Sanchez-Hernandez
et al., 2006). The observed reduction of volatile emission in
this mutant could be the result of impaired MT synthesis as
a consequence of decreased expression of 1-deoxy-
D-xylulose
5-phosphate synthase (DXS2), which is a key gene involved
in volatile synthesis in chloroplast (Sanchez-Hernandez
et al., 2006). A green leaf volatile, (Z)-3-hexenol, was
decreased in spr2 plants to 28.6% of the amount in WT
plants after leafminer damage. Interestingly, we found that
this mutant is less attractive to adult flies in a cage experi-
ment compared with WT plants (Fig. 1a), which was
consistent with a study using a transgenic Nicotiana
attenuata plant in the field (Halitsch ke et al., 2008), suggest-
ing that there is an ecological trade-off in terms of herbivore
attraction in JA-regulated defence and that the lower
attraction to foraging herbivores might be the result of the
reduced emission of green leaf volatiles. However, it was also
reported that Manduca sexta adults preferred to oviposit on
spr2 in the cage experiment (Sanchez-Hernandez et al.,
2006). Therefore, these plants should be examined under
natural conditions to elucidate the underlying mechanisms
and the corresponding ecological consequences.
In summary, based on our studies and other reports
(Thaler et al., 2002; Ament et al., 2004; Kessler et al.,
2004; Halitschke et al., 2008), we hypothesize that silencing
of the jasmonate cascade simultaneously attenuates direct
and indirect defence traits, but also makes plants less attrac-
tive to herbivores. In addition, using a transgenic plant, our
study provides the first evidence that enhanced direct
defence may compromise the efficiency of indirect defence.
Parasitism on silenced and overexpressing genotypes was less
successful than on the WT plants, irrespective of whether
the genetic manipulations resulted in improved or impaired
direct defence in modified plants. Therefore, there are
remarkable ecological trade-offs between JA-dependen t
direct and indirect defences in terms of herbivore attraction
and paras itoid acceptance in genetically modified plants.
We do not intend to discourage efforts to enhance plant
defences by using plant- and or nonplant-originated resist-
ance genes against agricultural insect pests. Instead, as a
promising and vital component of integrated pest manage-
ment, genetic engineering should consider the traits for both
direct and indirect defen ce (Turlings & Ton, 2006;
Degenhardt et al., 2009; Kos et al., 2009), and vice versa. In
particular, the effects of direct defence on the performan ce
of the third trophic level should not be ignored and the
plant fitness should be examined under natural conditions.
Acknowledgements
We thank Prof . Turlings (Uni versity of Neuchatel,
Switzerland), Dr Gols and Dr Joop van Loon (Wageningen
University, the Netherlands), Dr Salzman (Texas A&M
University), Dr Owain Edwards (CSIRO Entomology,
Australia) and five an onymous reviewers for critical com-
ments, reviews and English editing of this manuscript. We
also thank Dr L.Y. Jiang and Dr G.X. Qiao for assistance
with leafminer larvae photography and measurement, and
we thank Ms S. Zhang and Y. Luo for experiment assistance
and plant and insect maintenance. The research was sup-
ported by the National Basic Research Program of China
(973 Program) (no. 2006CB102006), the Chinese
Academy of Sciences (project no: KSCX2-YW-N-045), and
the National Nature Science Foundation of China
(30670356, 30621003 and 30970434).
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Supporting Information
Additional supporting information may be found in the
online version of this article.
Fig. S1 Photographs of larval leafminers collected from
three tomato geno types.
Fig. S2 Liriomyza huidobrensis performance on three
tomato genotypes.
Table S1 The mean number of larvae (mean ± SEM) per
plant used in different experiments
Table S2 Presence of volatile compounds released from
undamaged plants (UDPs) and L. huidobrensis larvae-
damaged plants (Lh-LDPs) of three tomato genotypes
Table S3 Presence of volatile compounds released from
0.5% alcohol-tr eated plants (ALPs) and jasmonic acid-
treated plants (JAPs) of three tomato genotypes
Table S4 Statistical analysis of total volatile emissions
among genotypes or between the undamaged and leafminer-
infested plants, or between 0.5% alcohol-treated (jasmonic
acid (JA) controls) and JA-treated tomato plants
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the New Phytologist Central
Office.
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    • "However, the phytohormones JA and SA are also known to regulate the production of plant volatiles (Dicke et al., 1999; Ozawa et al., 2000; Lou et al., 2005). Herbivoryinduced plant volatiles (HIPVs) play vital roles in enabling herbivores and their natural enemies to locate their food from a distance (Dicke et al., 1990; Turlings et al., 1995; Bruce et al., 2005; Wei et al., 2007; Dicke and Baldwin, 2010; Bruce and Pickett, 2011). Although a few studies have explored such negative SA–JA crosstalk in plant–herbivore–natural enemy interactions (Zhang et al., 2009; Thaler et al., 2010), to date it is largely unknown how SA–JA negative crosstalk affects host-plant selection behaviour of herbivores. "
    [Show abstract] [Hide abstract] ABSTRACT: The jasmonic acid (JA) and salicylic acid (SA) signalling pathways, which mediate induced plant defence responses, can express negative crosstalk. Limited knowledge is available on the effects of this crosstalk on host-plant selection behaviour of herbivores. We report on temporal and dosage effects of such crosstalk on host preference and oviposition-site selection behaviour of the herbivorous spider mite Tetranychus urticae towards Lima bean (Phaseolus lunatus) plants, including underlying mechanisms. Behavioural observations reveal a dynamic temporal response of mites to single or combined applications of JA and SA to the plant, including attraction and repellence, and an antagonistic interaction between SA- and JA-mediated plant responses. Dose-response experiments show that concentrations of 0.001mM and higher of one phytohormone can neutralize the repellent effect of a 1mM application of the other phytohormone on herbivore behaviour. Moreover, antagonism between the two signal-transduction pathways affects phytohormone-induced volatile emission. Our multidisciplinary study reveals the dynamic plant phenotype that is modulated by subtle changes in relative phytohormonal titres and consequences for the dynamic host-plant selection by an herbivore. The longer-term effects on plant–herbivore interactions deserve further investigation.
    Full-text · Article · Jun 2014 · Journal of Experimental Botany
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    • "Previous research indicated that several JA-overexpression mutants exhibit greater resistance against insects than wild-type plants424344. Wei et al. (2011) showed that there are ecological trade-offs between JA-dependent direct and indirect defences in genetically modified plants22. Our previous research indicated that the JA-overexpression tomato mutant 35S was resistant to B. tabaci under a high O3 concentration and whitefly infestation and there was a reduction in the fitness of conspecific B. tabaci that fed on three previously infested tomato genotypes that differed in the JA pathway4546. "
    [Show abstract] [Hide abstract] ABSTRACT: We experimentally examined the effects of elevated O3 and whitefly herbivory on tomato volatiles, feeding and oviposition preferences of whiteflies and behavioural responses of Encarsia formosa to these emissions on two tomato genotypes, a wild-type (Wt) and a jasmonic acid (JA) defence-enhanced genotype (JA-OE, 35S). The O3 level and whitefly herbivory significantly increased the total amount of volatile organic compounds (VOCs), monoterpenes, green leaf volatiles (GLVs), and aldehyde volatiles produced by tomato plants. The 35S plants released higher amount of total VOCs and monoterpene volatiles than Wt plants under O3+herbivory treatments. The feeding and oviposition bioassays showed that control plants were preferred by adult whiteflies whereas the 35S plants were not preferred by whiteflies. In the Y-tube tests, O3+herbivory treatment genotypes were preferred by adult E. Formosa. The 35S plants were preferred by adult E. formosa under O3, herbivory and O3+herbivory treatments. Our results demonstrated that elevated O3 and whitefly herbivory significantly increased tomato volatiles, which attracted E. formosa and reduced whitefly feeding. The 35S plants had a higher resistance to B. tabaci than Wt plant. Such changes suggest that the direct and indirect defences of resistant genotypes, such as 35S, could strengthen as the atmospheric O3 concentration increases.
    Full-text · Article · Jun 2014 · Scientific Reports
    • "Investment of resources in defence can be costly, and therefore , allocation constraints are predicted when multiple defence strategies are employed (Koricheva et al. 2004). Few studies have examined so-called direct and indirect defences simultaneously (Ballhorn et al. 2008; Rodriguez-Saona et al. 2011; Wei et al. 2011; Kos et al. 2012). Ballhorn et al. (2008) measured in wild and cultivated Lima bean plants (Phaseolus lunatus L.) the effect of JA treatment, which is often used to simulate herbivory by biting-chewing herbivores, and measured foliar levels of hydrogen cyanide (HCN) and volatile emissions as proxies for direct and indirect defences, respectively. "
    [Show abstract] [Hide abstract] ABSTRACT: Plant secondary metabolites play an important role in mediating interactions with insect herbivores and their natural enemies. Metabolites stored in plant tissues are usually investigated in relation to herbivore behaviour and performance (direct defence), whereas volatile metabolites are often studied in relation to natural enemy attraction (indirect defence). However, so-called direct and indirect defences may also affect the behaviour and performance of the herbivore's natural enemies and the natural enemy's prey or hosts, respectively. This suggests that the distinction between these defence strategies may not be as black and white as is often portrayed in the literature. The ecological costs associated with direct and indirect chemical defence are often poorly understood. Chemical defence traits are often studied in two-species interactions in highly simplified experiments. However, in nature, plants and insects are often engaged in mutualistic interactions with microbes that may also affect plant secondary chemistry. Moreover, plants are challenged by threats above- and belowground and herbivory may have consequences for plant-insect multitrophic interactions in the alternative compartment mediated by changes in plant secondary chemistry. These additional associations further increase the complexity of interaction networks. Consequently, the effect of a putative defence trait may be under- or overestimated when other interactions are not considered.
    No preview · Article · Mar 2014 · Plant Cell and Environment
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