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A Plasma Membrane Protein from Zea mays Binds with the
Herbivore Elicitor Volicitin
Christopher L. Truitt, Han-Xun Wei, and Paul W. Paré1
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409
Volicitin (17-hydroxylinolenoyl-L-Gln) present in the regurgitant of Spodoptera exigua (beet armyworm caterpillars) activates
the emission of volatile organic compounds (VOCs) when in contact with damaged Zea mays cv Delprim (maize) leaves. VOC
emissions in turn serve as a signaling defense for the plant by attracting female parasitic wasps that prey on herbivore
larvae. A tritiated form of volicitin was synthesized and shown to induce volatiles in the same fashion as the biological form.
[3H]-L-volicitin rapidly, reversibly, and saturably bound to enriched plasma membrane fractions isolated from Z. mays leaves
with an apparent Kd of 1.3 nM and a Hill coefficient of 1.07. Analog studies showed that the L-Gln and hydroxy moieties of
volicitin play an important role in binding. Treatment of plants with methyl jasmonate (MeJA) increased the total binding of
[3H]-L-volicitin to the enriched plasma membrane more than threefold, suggesting that MeJA activates transcription of the
gene encoding the binding protein. S. exigua feeding also increased total binding fourfold. Cycloheximide pretreatment of
plants significantly decreased binding of radiolabeled volicitin to the enriched plasma membrane. These data provide the
first experimental evidence that initiation of plant defenses in response to herbivore damage can be mediated by a binding
protein–ligand interaction.
INTRODUCTION
The coevolution of plants and insects has resulted in a wide array
of chemical plant defenses that effectively reduce damage
caused by feeding herbivores. Certain of these defenses, such
as toxic alkaloids, terpenoids, and phenolics, are expressed
constitutively (Wittstock and Gershenzon, 2002), whereas others
such as proteinase inhibitors (Tamayo et al., 2000) and polyphenol oxidases (Constabel et al., 1995) are not normally
triggered unless the plant is damaged. Although these latter
defense responses are known to be wound inducible, there are
numerous studies showing that injury by insects can result in
augmented or attenuated physiological and biochemical plant
responses compared with mechanical damage alone. These
conclusions are based on measurements of a number of
parameters such as plant regrowth (Dyer et al., 1995), timing of
transcript accumulation (Korth and Dixon, 1997), and volatile
organic compound (VOC) emissions (Paré and Tumlinson, 1999).
Most recently, microarray studies that have compared mRNA
levels in mechanically wounded and caterpillar-wounded plants
have revealed very different transcript profiles. In Arabidopsis
thaliana, insect wounding resulted in a downregulation of
water stress–induced genes compared with wounding alone
1To
whom correspondence should be addressed. E-mail paul.pare@
ttu.edu; fax 806-742-1289.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Paul W. Paré
([email protected]).
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.017723.
(Reymond et al., 2000), whereas in Nicotiana tabacum (tobacco),
wound-induced transcripts are modified by insect regurgitant
either by suppressing wound-induced transcripts systemically or
by amplifying the wound response in the attacked leaves
(Hermsmeier et al., 2001).
Herbivore-specific elicitors have been isolated from two insect
fluids that regularly are exposed to plant wounds. Oviposition
fluid of Callosobruchus maculatus (cowpea weevils) contains
long-chain diols that are monoesterified and diesterified with
3-hydroxypropanoic acid, referred to as bruchins (Doss et al.,
2000). These long-chain hydrocarbons elicit neoplastic growth of
Pisum sativum (pea) pods that results in C. maculatus eggs being
lifted out of the oviposition site and impedes larval entry into the
pod. From larval oral secretions, two classes of elicitors have
been isolated that trigger defense responses in plants. The lytic
enzyme group includes b-glucosidase (Mattiacci et al., 1995),
glucose oxidase (Felton and Eichenseer, 1999), and alkaline
phosphatase (Funk, 2001), whereas the fatty acid–amino acid
conjugate group includes a series of saturated and unsaturated
fatty acids that are 16 or 18 carbons in length and coupled to
a-Glu or a-Gln through a peptide bond (Alborn et al., 1997;
Pohnert et al., 1999; Halitschke et al., 2001).
Plant VOCs released by herbivore attack have been shown to
be attractive to arthropod predators and parasitoids in the
laboratory (Dicke and van Loon, 2000) as well as outdoors in
natural field conditions (Kessler and Baldwin, 2001). The VOC
response can be highly specific, and this specificity can be used
by certain parasitic wasps to locate particular herbivore species
(De Moraes et al., 1998). In addition to attracting natural enemies
of herbivores, VOC emissions can function as a direct defense
by repelling the ovipositing herbivore (De Moraes et al., 2001).
Such plant VOCs usually originate from at least three distinct
The Plant Cell Preview, www.aspb.org ª 2004 American Society of Plant Biologists
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The Plant Cell
Figure 1. Verification of Synthetic Volicitin Activity Using a Z. mays
Bioassay.
of linoleic acid for linolenic acid or the removal of the Gln or
hydroxyl substituents resulted in loss-of-elicitor activity. This
suggests a rigid set of chemical requirements to confer elicitor
activity. In fact, only when the natural L form of Gln was
coupled to the hydroxylated linolenic acid was volicitin
active as an elicitor of VOC emissions. This stereoisomer
specificity reduces the likelihood that nonspecific pH or
polarity effects are predominantly responsible for the induction
of VOC emissions and indeed points to a ligand–receptor
interaction.
In this study, we have identified a plasma membrane binding
protein for volicitin in Z. mays. The herbivore elicitor was synthesized in a radiolabeled form with tritiated Gln and tested for
biological activity using an in vitro Z. mays bioassay system
(Alborn et al., 1997). Binding saturability, reversibility, and affinity
were determined through saturation experiments and competition assays with structural analogs. The induction of volicitin
binding sites was stimulated by plant exposure to methyl
jasmonate (MeJA) or herbivore damage.
Release of volatiles collected for 2 h from six Z. mays seedlings that
had been treated with 300 pmol per plant of L-volicitin, D-volicitin,
17-hydroxylinolenic acid, L-Gln, and linolenoyl-L-Gln, with concentrations ranging from 50 mg/mL to 100 mg/mL plus buffer for a final volume
of 500 mL. The combined amount in nanograms of caryophyllene,
a-trans-bergamontene, (E)-b-farnesene, (E)-nerolidol, and (3E,7E)-4,8,
12-trimethyl-1,3,7,11-tridecatetraene was used to calculate the relative
release to that of seedlings treated with 15 mL of BAW oral secretions
plus 485 mL buffer, which is equivalent to 300 pmol natural volicitin.
Data labeled with the same letter do not differ significantly (ANOVA,
P \ 0.05).
biosynthetic pathways. The most consistently released set of
VOCs are the terpenes (Paré and Tumlinson, 1997), a group of
compounds derived from the mevalonate or nonmevalonate
isoprenoid pathway. Many of these terpenes are emitted
transiently and systemically with insect damage, suggesting
that this VOC release may be under transcriptional regulation
(Paré and Tumlinson, 1997). C6 alcohols and C6 aldehydes are
produced from a-linolenic acid and linoleic acid via their
respective hydroperoxides (Blee, 2002); the electrophilic nature of the a,b-unsaturated carbonyl motif, common to many
members of this class, has been suggested to be responsible for
their biological activity (Farmer, 2001). The shikimic acid pathway
can also generate VOCs (e.g., methyl salicylate and indole). In
fact, indole-3-glycerol phosphate lyase, a branch point enzyme
in the catalysis of free indole, was the first identified enzymatic
step selectively activated at the transcription level by the
herbivore elicitor N-(17-hydroxylinolenoyl)-L-Gln (L-volicitin)
(Frey et al., 2000).
The linolenic acid derivative volicitin induces Zea mays
seedlings to release a blend of volatile terpenoids and indole
that are qualitatively and quantitatively similar to those released
from plants damaged by caterpillar feeding (Alborn et al., 1997).
Interestingly, when a series of analogs were tested for
biological activity in triggering VOC emissions, the substitution
Figure 2. Chemical Structure of Volicitin and Analogs Assayed.
(A) L-volicitin.
(B) D-volicitin.
(C) Linolenoyl-L-Gln.
(D) 17-hydroxylinolenic acid.
(E) L-Gln.
Volicitin Binding to Z. mays Enriched Plasma Membrane
Table 1. Analysis of Marker Enzymes in Membrane Fractions Isolated
by Aqueous Two-Phase Partioning Procedure with the Total Protein and
Marker Enzyme Activities in the Microsomal, Plasma Membrane, and
Intracellular Membrane Fractions
Fraction
Protein
mg (%)
Plasma
membrane
ATPase
nmol s1 (%)
Mitochondria
membrane
Cyt c oxid.
nmol s1 (%)
ER membrane
Cyt c red.
nmol s1 (%)
MF
U3
L1
60
5.7 (9.5)
36.6 (61)
380
357 (94)
15.2 (4)
21
0.42 (2)
13.4 (62)
200
62 (31)
108 (54)
ATPase activity for plasma membrane was determined in the presence
of 0.025% (v/v) Triton X-100 and 50 mM vanadate. The enzymes
assayed for the mitochondria and ER were NADPH–cytochrome c
oxidase and NADPH–cytochrome c reductase, respectively. Cyt c oxid.,
NADPH–cytochrome c oxidase; cyt c red., NADPH–cytochrome c reductase; L1, intracellular membrane fractions; MF, microsomal fractions; U3,
plasma membrane fractions.
RESULTS
Bioactivity and Characterization of 17-Hydroxylinolenoyl
[3,4-3H(N)]–L-Gln
A biologically active tritiated form of volicitin ([3H]-L-volicitin) and
a series of nonradioactive analogs were synthesized. Synthetic
components were purified by HPLC and assayed for their ability
to induce VOC emission in Z. mays bioassays (Figure 1). After the
procedure reported by Alborn et al. (1997), the sum of emissions
from caryophyllene, a-trans-bergamotene, (E)-b-farnesene, (E)nerolidol, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene
were used to measure plant induction responses by different
elicitors. The results obtained using radiolabeled volicitin were
not significantly different from those results obtained in experiments using nonradiolabeled synthetic volicitin or Spodoptera
exigua (beet armyworm caterpillars, or BAW) oral secretions
containing natural volicitin. The synthetic analog of volicitin,
linolenoyl-L-Gln, was significantly less active in triggering VOC
emissions, with an approximate 40% relative release rate
compared with BAW regurgitant or synthetic volicitin. The other
volicitin analogs tested, including 17-hydroxylinolenoyl-D-Gln
(D-volicitin), 17-hydroxylinolenic acid, and L-Gln (Figure 2), did
not trigger Z. mays volatile emissions at a level greater than
emissions observed for the buffer control treatment.
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61% of the protein and 4% of the plasma membrane marker
(Table 1). This signified a 17-fold enrichment of the plasma
membrane marker in U3 versus L1. Conversely, the marker
enzymes for mitochondrial membranes and ER membranes
were present predominately in L1.
Binding Kinetics
The kinetics of radiolabeled volicitin binding in an enriched
plasma membrane preparation are illustrated in Figure 3. Binding
was observed as early as 1 min, and the extent of binding
increased for 7 min followed by a slight decrease through 13 min
(Figure 3). Nonspecific binding remained constant throughout
the experiment and represented only 7 to 10% of the total
binding between the 5- to 13-min period. All subsequent binding
studies were performed at 7 min after the addition of volicitin and
volicitin analogs.
The competition by volicitin and volicitin analogs was assessed in experiments in which nonradioactive volicitin and
volicitin analogs were added to the enriched plasma membrane
binding assay mixture. The competition assays were performed
by the addition of a 100-fold molar excess of analog to the
reaction mixture before the addition of 10 nM [3H]-L-volicitin
(Figure 4A). The binding of [3H]-L-volicitin to the enriched plasma
membrane preparation decreased to background levels in the
presence of unlabeled L-volicitin. D-volicitin containing the same
charge and hydrophobicity produced only a 15% decrease for
[3H]-L-volicitin bound by the enriched plasma membrane
preparation. Unlabeled L-volicitin and linolenoyl-L-Gln competed
for the binding sites with half-maximal inhibitory concentrations
Plasma Membrane Isolation
To identify different membrane fractions and the degree of
enrichment, marker enzymes ATPase (plasma membrane), cytochrome c oxidase (mitochondrial membrane), and NADPH–
cytochrome c reductase (endoplasmic reticulum [ER]) were
assayed. The upper plasma membrane enriched fraction (U3)
contained 9.5% of the total protein and 94% of the plasma
membrane marker, whereas the lower fraction (L1) contained
Figure 3. Time Course of [3H]-L-Volicitin Binding to Z. mays Enriched
Plasma Membranes.
Binding of [3H]-L-volicitin (10 nM) in the absence (closed circle, total
binding) or presence (open circle, nonspecific binding) of a 100-fold
excess of unlabeled L-volicitin over time. At the indicated times, the
membranes were collected, washed, and analyzed for radioactivity. Error
bars indicate standard error (n ¼ 6).
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The Plant Cell
of 9 and 22 nM, respectively (Figure 4B). D-volicitin and the fatty
acid and amino acids of volicitin did not produce greater than
a 15% decrease in [3H]-L-volicitin binding at concentrations up
to 1 mM. To relate our binding data with the volatile assays,
half-maximal effective concentration (EC50) of L-volicitin and
linolenoyl-L-Gln was determined by measuring volatile release
from Z. mays seedlings with increasing concentrations of elicitor
(Figure 5). EC50 values for L-volicitin and linolenoyl-L-Gln were
measured to be 58 and 165 nM, respectively.
To further characterize the binding protein–ligand interaction,
enriched plasma membrane fractions were incubated with
increasing concentrations of ligand. The experiments were
performed by incubating [3H]-L-volicitin in the presence or
absence of unlabeled volicitin. Nonspecific binding was subtracted from the total binding to determine specific binding of
[3H]-L-volicitin. A plot of specific binding as a function of
increasing [3H]-L-volicitin shows that the binding was saturated
at a level of 10 nM volicitin (Figure 6A). The saturation data was
used to calculate the apparent Kd and maximal binding (Bmax) by
Scatchard analysis (Figure 6B). The Kd for binding of [3H]-Lvolicitin to the enriched plasma membrane was determined by
taking the negative inverse of the slope in Figure 6B and
calculating a Kd of 1.3 nM. From the same plot, the Bmax was
determined by the x-intercept and indicated a Bmax of 1.26
fmolmg protein1. To determine the cooperativity of binding, the
slope generated from a Hill plot was used (Figure 6C). The Hill
coefficient was calculated to be 1.07. Assuming one ligand per
receptor, the number of volicitin binding sites per cell was
estimated at 3000.
Figure 4. Competition of Unlabeled Volicitin and Volicitin Analogs with
[3H]-L-Volicitin for Binding Sites on the Z. mays Enriched Plasma
Membranes.
(A) Competition of [3H]-L-volicitin binding to enriched plasma membranes with unlabeled L-volicitin, D-volicitin, linolenoyl-L-Gln, 17-hydroxylinolenic acid (17-hydroxy), and L-Gln. The competing analogs were
added at a 100-fold molar excess (1 mM) over [3H]-L-volicitin (10 nM).
Data labeled with the same letter do not differ significantly (ANOVA, P \
0.05).
(B) Competition analysis of [3H]-L-volicitin binding by L-volicitin, Dvolicitin, and linolenoyl-L-Gln were determined by treating the enriched
plasma membrane preparations with the indicated concentrations of
unlabeled analog and by calculating the percentage of specific binding
as a ratio of specific binding at the indicated concentration to maximal
specific binding found in the presence of 100-fold excess of unlabeled
volicitin. Closed circles, L-volicitin; open circles, D-volicitin; triangles,
linolenoyl-L-Gln. Error bars indicate standard error (n ¼ 6).
Figure 5. Saturation Analysis of L-Volicitin and Linolenoyl-L-Gln Induction of VOCs in Z. mays Seedlings.
Maximum release of volatiles collected for 2 h from six Z. mays seedlings
that had been treated with the indicated concentration of either L-volicitin
(closed circles) or linolenoyl-L-Gln (open circles). The combined amount
in nanograms of caryophyllene, a-trans-bergamontene, (E)-b-farnesene,
(E)-nerolidol, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene was
used to calculate the release of seedlings treated with 15 mL of test
solution. Error bars indicate standard error (n ¼ 6).
Volicitin Binding to Z. mays Enriched Plasma Membrane
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The reversibility of volicitin binding was studied by the addition
of unlabeled L-volicitin to enriched plasma membrane fractions 4
min after the addition of [3H]-L-volicitin (Figure 7). The addition of
unlabeled L-volicitin caused a time-dependent decrease of the
radioactive retention on the filters that equilibrated within 3 min
to 60% of the total bound. The pH protein–ligand binding
optimum measured by filter and slot-blot binding assays was
between 7 and 9, with ligand binding rapidly decreasing above
and below this pH range (Figure 8). Based on these results,
subsequent binding assays were maintained at pH 7.2.
Induction of Binding Sites
Jasmonic acid (JA) and mechanical damage are both inducers of
VOC emissions in Z. mays seedlings (Schmelz et al., 2001, 2003);
their effect on [3H]-L-volicitin binding to enriched plasma
membrane was assayed over time (Figure 9). The application of
50 mL of 1 mM MeJA produced a 23 increase in [3H]-L-volicitin
binding 2 h after treatment and a 43 increase in [3H]-L-volicitin
binding by 12 h after treatment. Beyond the 12-h period, [3H]-Lvolicitin binding decreased. The initial response to BAW wounding showed an approximate twofold increase within the first
2 h, a steady increase to an approximate fourfold peak at 16 h,
followed by a leveling of induction over the remaining 24-h time
course. By contrast, mechanical damage had only a modest 23
effect on the binding of [3H]-L-volicitin to the enriched plasma
membrane preparation.
To examine whether the increase in [3H]-L-volicitin binding in
response to mechanical damage, MeJA, and BAW required de
novo protein synthesis, plants were cotreated with 2 mM
cycloheximide and compared with plants not treated with the
protein translation inhibitor. Binding studies with radiolabeled
volicitin were performed with enriched plasma membrane
isolated 16 h after treatment with mechanical damage, MeJA,
and BAW, with or without cycloheximide. Volicitin binding for
plants treated with cycloheximide versus those without cycloheximide treatment showed a decreased binding in all cases
tested (Table 2): water (fourfold), mechanical damage (eightfold),
BAW (16-fold), and MeJA (17-fold).
To validate whether the specific binding of [3H]-L-volicitin
involved a proteinacous receptor, enriched Z.mays plasma membrane fractions were treated with the proteases trypsin and
pronase. Protease treatment reduced [3H]-L-volicitin–specific
binding by 90% in filter binding assays and to background
levels in slot-blot binding assays (Figure 10). Enriched plasma
membrane heat treatment also significantly reduced protein–
ligand binding ($80%), whereas heat-inactivated proteases had
no effect on ligand binding (data not shown). Plant exposure for
Figure 6. Saturation Analysis of [3H]-L-Volicitin Binding to Z. mays
Enriched Plasma Membranes.
(A) Enriched plasma membrane preparations were treated with increasing concentrations of [3H]-L-volicitin. The specific binding (total
binding minus nonspecific binding) is shown. The specific activity of [3H]L-volicitin was 1.2 Ci/mmol (140 dpm/fmol). Error bars indicate
standard error (n ¼ 6).
(B) Scatchard analysis of [3H]-L-volicitin binding to enriched plasma
membranes from data shown in (A). The Kd was calculated from the
negative inverse of the slope and Bmax from the x-intercept.
(C) Hill analysis of [3H]-L-volicitin binding to enriched plasma membranes
derived from the data shown in (A). The slope of the plot is the Hill
coefficient (nHill).
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The Plant Cell
Figure 7. Reversibility of [3H]-L-Volicitin Binding to Z. mays Enriched
Plasma Membranes.
Two sets of cells were treated with saturating levels of [3H]-L-volicitin
(10 nM), and the total radioactivity associated with the enriched
plasma membrane was determined at the indicated time. A 100-fold
excess (1 mM) of unlabeled volicitin was added to one set of enriched
plasma membrane preparations 4 min after the addition of [3H]-L-volicitin.
The total radioactivity associated with both sets of enriched plasma
membranes was determined at the indicated times. Error bars indicate
standard error (n ¼ 6).
16 h to BAW leaf damage or MeJA before plasma membrane
isolation resulted in induction of protein–ligand binding in both
the filter and slot-blot binding assays.
The possibility that volicitin intercalated into membranes
based on the hydrophobic nature of the ligand combined with
the Gln moiety nonselectively interacting with integral membrane proteins was ruled out by assaying membrane fractions
not enriched with plasma membrane proteins. The absence of
volicitin binding to the lower fraction from the phase partitioning procedure (Figure 10, last lane), which was enriched in
non-plasma membrane bound proteins (Table 1, fraction L1),
indicates that a protein-rich lipid bilayer is not sufficient to
cause volicitin binding. Indeed, the volicitin binding protein
appears to be located in or associated with the plasma
membrane.
per cell for systemin binding to Lycopersicon esculentum
(tomato) cell suspensions (Scheer and Ryan, 1999) and 2900
sites per cell for the peptide elicitor Pep-13 binding to
Petroselinum crispum (parsley) (Nurnberger et al., 1994). The
binding of volicitin did not facilitate additional volicitin binding
(i.e., noncooperative binding) based on the Hill plot coefficient
(n ¼ 1.07).
Correlations between ligand binding and biological activity
were used as the criterion for assessing authentic protein
binding. Several synthesized volicitin analogs were tested to
correlate binding and biological activity. Analogs tested for their
ability to competitively displace [3H]-L-volicitin bound to plasma
membrane enriched fractions (half-maximal inhibitory concentration values) closely overlapped biological activity with
D-volicitin, 17-hydroxylinolenic acid, and L-Gln inactive in triggering VOCs and ineffective in serving as an antagonist of [3H]-Lvolicitin binding to the enriched plasma membrane, whereas the
unlabeled elicitor molecules volicitin and linolenoyl-L-Gln were
competent in both triggering VOCs and competing for protein
binding sites occupied by radiolabeled volicitin.
The binding of radiolabeled volicitin was reversible, as determined by the decrease in binding within 3 min of a 100-fold
molar excess addition of unlabeled volicitin. Approximately
40% reduction of total binding was observed, suggesting that
volicitin–protein binding is quite strong. Other studies of elicitor
binding have shown similar levels of reversibility binding; for
example, with a 200-fold molar addition of cold systemin, a 50%
reduction of total binding was observed in L. esculentum cell
suspensions (Scheer and Ryan, 1999), and with a 10,000-fold
molar addition of cold Pep-13, a 60% reduction of total binding
was observed in P. crispum (Nurnberger et al., 1994).
To further characterize this volicitin binding protein, either
enriched plasma membrane preparations or whole plants were
exposed to previously identified chemical regulators. Loss of
DISCUSSION
These data provide biochemical evidence for the existence of
a plant binding protein that can selectively bind an herbivore
ligand (elicitor) that triggers the emissions of plant VOCs. We
demonstrate that radiolabeled volicitin rapidly, reversibly, and
saturably binds to enriched plasma membrane fractions. The Kd
observed by Scatchard plot analysis was 1.3 nM, indicating
a high affinity association; this binding constant reflects the
ability of low volicitin concentrations to serve as elicitors of VOC
emissions in plants. Bmax was calculated at 1.26 fmolmg
protein1 or 3000 sites per cell. Density estimates of other
defense signal receptors on the cell’s surface include 3000 sites
Figure 8. Assay pH Influences [3H]-L-Volicitin–Plasma Membrane
Binding.
[3H]-L-Volicitin (10 nM) filter binding and slot-blot assays for plasma
membrane enriched fractions adjusted to the selected pH. Slot-blot
insets display [3H]-L-volicitin binding to enriched plasma membranes
bound to nitrocellulose and analyzed by autoradiography. Data labeled
with the same letter do not differ significantly (ANOVA, P \ 0.05).
Volicitin Binding to Z. mays Enriched Plasma Membrane
Figure 9. Induction by MeJA, BAW, and Mechanical Wounding on [3H]L-Volicitin–Plasma Membrane Binding.
Saturating levels of [3H]-L-volicitin (10 nM) were used to determine the
total level of binding at the indicated times after treatment with MeJA,
BAW, or razor blade. The total binding is shown in fmolmg protein1.
Error bars indicate standard error (n ¼ 6).
ligand binding was observed when enriched plasma membrane
preparations were pretreated with proteases, indicating that
ligand binding is mediated at least in part through a proteincontaining entity. Treatment of plants with cycloheximide 16 h
before harvesting plant material caused a marked decrease in
binding of radiolabeled volicitin to the enriched plasma membrane preparation. This decrease in ligand–protein binding
suggests that the volicitin binding protein is in constant turnover
and that loss of synthesis quickly translates in an absence of the
binding protein. For example, cycloheximide treatment, which
results in an inhibition of protein synthesis, compared with water
treatment resulted in a decrease of volicitin-specific binding from
130 to 28 dpm (Table 2), suggesting that the binding protein
proceeds through two half-lives during the 16 h treatment, or
t1/2 ¼ 8 h. This rapid turnover for a volicitin binding protein
was unexpected, although precedent for such turnover rates
does exist in the case of the defense signaling receptor systemin isolated from L. esculentum, which exhibited a half-life of
7.5 h (Scheer and Ryan, 1999). Plant exposure to insect damage
or MeJA treatment triggered an induction of binding sites on
the enriched plasma membrane, suggesting that the volicitin
binding protein may be transcriptionally upregulated to enhance
the mechanism for perception of elicitor signals with herbivore
feeding. Insect wounding also induces the emission of volatile
terpenes in a wide range of plant taxa (Paré and Tumlinson,
1999), including Z. mays, and studies have implicated a role for
JA in triggering plant VOC emissions. Indeed the exogenous
application of JA to intact (Schmelz et al., 2001) or excised plants
(Dicke et al., 1999) triggers elevated VOC emissions. In some
cases, endogenous increases of JA with insect damage either
parallel or precede VOC emissions. In fact, Schmelz et al. (2003)
have observed a direct positive relationship between endoge-
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nous JA levels and VOC emissions in Z. mays seedlings with
insect damage.
Early steps in the herbivore elicitation process have yet to be
elucidated (Kessler and Baldwin, 2002). Although there are no
previous reports of herbivore-specific elicitor plant receptors,
plasma membrane binding proteins for pathogen elicitors have
been identified and characterized. Using radiolabeled b-1,3glucan and hepta-b-glucoside elicitors characterized from fungal
cell walls of Phytophthora megasperma f sp glycinea (Schmidt
and Ebel, 1987), high affinity binding proteins have been
identified in isolated legume plasma membrane fractions
(Cheong and Hahn, 1991; Cote et al., 2000). Elicitor binding
receptors for the N-acetylchitooligossaccharide elicitor have
also been detected in plasma membrane fractions from several
plant species including Oryza sativa (rice), Triticum aestivum
(wheat), Hordeum vulgare (barley), and Daucus carota (carrot)
(Okada et al., 2001, 2002). Each of these pathogen elicitor–plant
receptor interactions is characterized by nanomolar EC50
concentrations as well as Kd values in the low nanomolar range.
A comparable EC50 for VOC emissions in Z. mays with volicitin
exposure suggested that this elicitor, like plant pathogen
elicitors, is perceived by a cell-specific receptor.
The biogenetic origin of volicitin has been established by
a series of stable isotope–labeling studies that demonstrated
that BAW acquires linolenic acid (an essential fatty acid in the diet
of Lepidoptera) from plants and that the insect subsequently
hydroxylates and conjugates the fatty acid with Gln (Paré et al.,
1998). Mechanical damage causes the release of membranebound linolenic acid and initiates the octadecanoid signaling
pathway. Modification of linolenic acid by the feeding insect
appears to provide a distinct chemical cue that can selectively
bind to a specific volicitin binding protein, upregulate the binding
of volicitin to the enriched plasma membrane, and ultimately
allow the plant to differentiate between herbivore damage and
nonspecific leaf damage that triggers the lipoxygenase pathway.
In subsequent studies, how a volicitin binding protein might
function in the cell either as a membrane transporter shuttling
volicitin across the membrane and serving as the primary
messenger of VOC synthesis or as an activator of second
Table 2. Induction of [3H]-L-volicitin–Plasma Membrane Binding with
Mechanical Damage, BAW, MeJA, or Water Treatment in the Presence
or Absence of Cycloheximide
Specific Binding (dpm/mg protein)
Plant Treatment
Without
Cycloheximide
With
Cycloheximide
Mechanical Damage1
BAW1
MeJA1
Water1
Water2
260
510
455
130
128
30 6
32 6
27 6
28 6
1216
6
6
6
6
6
34
62
57
15
18
6.3
7.2
6.9
5.5
20
Binding of volicitin was performed with enriched plasma membrane
isolated 16 h after treatment, except where noted.
1 A 16 h delay between treatment and harvest.
2 No delay between treatment and harvest.
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The Plant Cell
Seedling Bioassays
Volatile analyses were performed on 10-d-old seedlings following
the protocol by Alborn et al. (1997), except that 15 mL of test solution
was added to 500 mL of 50 mM Na2HPO4 buffer titrated to pH 8.0 with
1 M NaH2PO4. The final assay concentration of volicitin and analogs
was 600 nM.
Preparation of Enriched Plasma Membranes
Figure 10. Protein Requirement for [3H]-L-Volicitin–Plasma Membrane
Binding.
[3H]-L-volicitin (10 nM) filter binding and slot-blot assays with indicated
plant and plasma membrane fraction treatments. Slot-blot insets display [3H]-L-volicitin (1.6 3 106 cpmmL1) binding to enriched plasma
membranes bound to nitrocellulose and analyzed by autoradiography.
Asterisks indicate the lower fraction (L1) containing a non-plasma
membrane enriched fraction. Data labeled with the same letter do not
differ significantly (ANOVA, P \ 0.05).
messenger(s) cascade will be examined. Because the volicitin
binding protein is detectable by slot-blot binding assays,
screening of a Z. mays cDNA phage–display library with radiolabeled volicitin probe may identify volicitin binding phage proteins(s) that can be subsequently cloned and analyzed.
METHODS
Plants, Insects, and Reagents
Z. mays plants (cv Delprim) grown from seed in Pro-Grow potting soil
supplemented with Osmocote fertilizer (Scotts-Sierra, Marysville, OH)
were maintained in an insect-free facility in which temperature and relative humidity were monitored and maintained at 298C 6 48C and 40% 6
10%, respectively. Metal halide and high-pressure sodium lamps on
a 16-h/8-h light/dark photoperiod provided a light intensity of 700 mmol
m2s1. Two-week-old plants were used for experiments described
below, except where noted, and BAW eggs obtained from USDA-ARS
(Tifton, GA) were incubated at 268C, relative humidity 90%, and
16-h/8-h light/dark cycle. Hatched larvae were reared on an artificial pinto
bean diet, following the method of King and Leppla (1984), and used at the
late third instar stage. MeJA was obtained from Bedoukian Research
(Danbury, CT) and was of $99% purity, as determined by capillary gas
chromatography–flame ionization detector analysis. MeJA (1 mM) was
prepared by adding 10 mL to 1 L of water (50 mM) and taking a 1 mL aliquot
with 50 mL of water before application. Other compounds were purchased form Sigma-Aldrich (St. Louis, MO) and were of $99% purity,
except where noted.
Ligand Synthesis
The synthesis of the L- and D-enantiomers of volicitin, linolenoyl-L-Gln,
and 17-hydroxylinolenic acid is described by Wei et al. (2003). All
synthetic compounds were analyzed by mass spectrometry using
a direct-inserted probe before use. The radiolabeling of volicitin was
prepared in the same method with the use of 1 mCi of L-[3,4-3H(N)]Gln
with a specific activity of 45 Ci/mmol (Dupont NEN, Boston, MA).
Membrane fractions enriched in plasma membrane material were
isolated from Z. mays leaves following the protocol of Robinson et al.
(1988), with all subsequent operations conducted at 48C. Described
briefly, leaves were homogenized in 15 mM Tris-HCl, pH 7.8, with 1 mM
EDTA, 3 mM DTT, 0.5 M sucrose, and 0.6% polyvinylpyrrolidone in a 1:4
ratio (g/mL) and filtered through four layers of cheesecloth. The filtrate
was centrifuged at 10,000g for 10 min, and the decanted supernatant was
spun a second time at 50,000g for 30 min. The resulting microsomal pellet
(8.4 mg protein/mL) was resuspended in phosphate buffer (5 mM
K-phosphate, pH 7.8, and 0.25 M sucrose), and the plasma membrane
fraction was isolated using the aqueous two-phase partitioning method
described by Larsson et al. (1987). Ten grams of the microsomal fractions
was added to 11.52 g dextran (Mr ¼ 500,000) and 5.76 g polyethylene
glycol (Mr ¼ 3350) in 5 mM phosphate buffer, pH 7.8, containing 0.3 M
sucrose and 3 mM KCl for a final weight of 36 g. The system was
equilibrated overnight at 48C after vigorous shaking for 30 s. The phases
were separated by centrifugation at 2000g for 10 min (HB-4 rotor; Sorvall,
Newton, CT). The upper fraction was combined with a fresh lower fraction
obtained from another phase system prepared as above, except with 10 g
of water, whereas the lower phase was combined with fresh upper phase.
The two systems were mixed, equilibrated, and centrifuged as above,
followed by repetition of same protocol. The final upper phases
containing enriched plasma membrane were combined, diluted in Tris
buffer (5 mM Tris-MES, pH 7.2, 0.1 mM DTT, and 0.25 M sucrose) and
centrifuged at 100,000g. The pellet was resuspended in the same Tris
buffer and stored at –708C. For pH assays, the pellet was resuspended in
a modified buffer (5 mM sodium citrate, 5 mM phosphate, pH 7.2, 0.1 mM
DTT, and 0.25 M sucrose) with the concentrations of citric acid and
phosphate adjusted to achieve pH values between 4 and 8. In addition, 10
mM ethanolamine buffer was used to assay binding between pH 9.0 and
10. Protein concentrations were assayed based on the method of
Bradford (1976) using BSA as a standard.
Membrane Purity Assays
Marker enzymes were used to assay plasma membrane, ER, and
mitochondria purity following the protocol of Hodges and Leonard (1972),
with all subsequent reactions done at 258C unless otherwise stated.
Plasma membrane was assayed for vanadate-inhibited, Mg21-ATPase
activity. The reaction was performed in a 1-mL reaction volume containing 3 mM ATP, 3 mM MgSO4, 30 mM Tris-MES buffer, pH 6.5, 50 mM
KCl, and 30 mg membrane protein in the presence or absence of 0.025%
(v/v) Titron X-100 and/or 50 mM Na3VO4. After 30 min incubation at 388C,
2.6 mL of a stopping reagent, 0.42% (w/v) ammonium molybdate in 1 M
H2SO4 with one part 10% (w/v) ascorbic acid, was added to each assay.
After 20 min incubation at room temperature, the absorbance at 650 nm
was measured, and the amount of inorganic phosphate was calculated
with 0.01 mmol equal to an absorbance of 0.240 (Ames, 1966). ER was
assayed for the NADPH–cytochrome c reductase, a cyanide-insensitive
enzyme. A 3-mL reaction volume with 0.1 mL of membrane suspension
(30 mg protein), 0.1 mL of 50 mM NaCN, 0.2 mL of 0.45 mM cytochrome c,
and 2.5 mL of 50 mM sodium phosphate buffer, pH 7.5, was started by the
addition of 0.1 mL of 3 mM NADPH, with the reduction of cytochrome c
change measured at 550 nm. Mitochondrial membranes were assayed
Volicitin Binding to Z. mays Enriched Plasma Membrane
for cytochrome c oxidase activity using NADPH–cytochrome c reductase. Cytochrome c was reduced by addition of excess of sodium
dithionite, followed by aeration of the solution to oxidize excess dithionite.
Reactions contained 0.1 mL of membrane suspension (30 mg protein), 0.1
mol of 0.3% Triton X-100, and 2.7 mL of 50 mM sodium phosphate, pH
7.5. Reactions proceeded by addition of 0.1 mL of reduced cytochrome c,
and change at 550 nm was measured. The rate of cytochrome c reduction and oxidation was calculated using an extinction coefficient of
18.5 mM1cm1.
Binding Experiments
Radioligand filter binding assays were based on a procedure by Scheer
and Ryan (1999). Binding was initiated by adding 50 mL of radiolabeled
volicitin (10 nM) to 100 mL of enriched plasma membrane (200 mg of
protein) in 1.5 mL Eppendorf tubes at 258C. Precisely 7 min were given for
binding, except when measuring the time course of binding. The reaction
mixtures were then filtered through GF/B binder-free glass fiber filters
(Fisher Scientific, Loughborough, Leicestershire, UK) housed in a 12-well
vacuum filtration manifold (Millipore, Bedford, MA). Filters were prewashed with 1 mL of 48C buffer (5 mM Tris-MES, pH 7.2, 0.1 mM DTT, and
0.25 M sucrose) and then rinsed with an additional 3 mL buffer once the
radioligand was applied; the amount of [3H]-L-volicitin retained by the
filter was measured by liquid scintillation (LS5000 TD; Beckman Instruments, Fullerton, CA). In competition assays, unlabeled volicitin or
a volicitin analog was added just before the addition of radiolableled
volicitin. Specific binding was calculated by subtracting nonspecific
binding (binding in the presence of excess nonradioactive volicitin) from
total binding. Binding assays were replicated (63) using saturating levels
of volicitin (10 nM) unless otherwise indicated. Volicitin analogs were
added at 100-fold molar excess (1 mM). The percentage of specific
binding used in displacement experiments was calculated by dividing the
specific binding at the indicated concentrations of competing analog by
the maximum specific binding determined in the presence of an excess of
volicitin. Enriched plasma membrane preparations incubated in filtration
buffer for 16 h at 378C showed no decrease in specific binding, indicating
the absence of endogenous protease activity.
For slot-blot binding assays, 10 mL of plasma membrane enriched
protein (1 mg/mL) was applied to nitrocellulose filters in a chilled slot-blot
manifold (Bio-Dot SF microfiltration apparatus; Bio-Rad, Hercules, CA);
temperature was maintained at 48C throughout the hybridization procedure. Membranes were presoaked for 20 min in 10 mL hybridization
buffer (20 mM potassium phosphate, 200 mM KCl, 1 mM EDTA, and 1 mM
DTT) adjusted to pH 7.9, and then incubated for 7 min in 10 mL of fresh
buffer containing the [3H]-L-volicitin probe (1.6 3 106 cpm/mL buffer).
Membranes were rinsed two times for 15 min in 10 mL hybridization buffer
and placed in x-ray film intensifying cassettes at –708C for 30 d before film
development.
The receptor number per cell estimation was based on a generic cell
with the following features: the plasma membrane contained 40% protein
by weight; the plasma membrane volume was 8.1 3 1012 mL (30 3 30 3
0.009 mm) (Larsson and Moller, 1989); and the average plasma
membrane density was 1.15 gmL1 (Larsson et al., 1987). Plasma
membrane number per microliter of extract was calculated from the
binding assay protein concentration, factoring in the enrichment of
plasma membrane protein from the marker enzyme. This value was then
divided by the receptor number per microliter calculated from Bmax,
assuming a 1:1 mole ratio of volicitin binding protein to ligand.
Protease and Heat Treatment
Enriched plasma membrane preparations (2 mg protein/mL) were
incubated in filtration buffer for 30 min in a 378C water bath with the
following protease concentrations: 50 mg/mL trypsin and 12 mg/mL
9 of 10
Pronase E (Sigma). Treatments were ended using the antiprotease
mixture of 50 mg/mL of phenylmethylsulfonyl fluoride to inhibit trypsin and
1 mg/mL of pepstatin for Pronase E. Enriched plasma membrane
preparations were then used for [3H]-L-volicitin binding assays. Control
assays contained the same antiprotease mixture with the proteases
inactivated by heating at 1008C for 15 min before binding.
Volicitin Binding Protein Induction
Binding protein induction was assayed under the following conditions: (1)
plants sprayed with 1 mM MeJA; (2) plants mechanically razor-blade
damaged covering an area of 2 cm2 on two lower leaves; and (3) plants
insect damaged with five BAW larvae covering an area of 2 cm2 on the two
lower leaves. All plants were sprayed with 50 mL of water and Tween 20
(0.025%) to account for solvent effects. Plants were maintained under
previously described growth conditions until the time of harvest. The
specific binding was determined in the water-treated, mechanically
damaged, BAW-treated, and MeJA-treated enriched plasma membrane
preparations and expressed as a fold increase over water-treated
controls. To measure the effect of protein translation on binding, plants
were sprayed with 2 mM cycloheximide concomitantly with mechanical
damage, BAW feeding, or MeJA treatment, and plants were harvested
16 h after treatment.
Statistical Analyses
Analyses of variance, using SAS statistical software version 8 (SAS
Institute, Cary, NC), were performed on all volatile collection data and
induction of binding results. Means were separated using Duncan’s
multiple range test with a P value \0.05. Significant treatment effects
were investigated when analysis of variance was significant (P \ 0.05).
ACKNOWLEDGMENTS
We thank Richard Jasoni for statistical analysis training, Mohamed A.
Farag for slot-blot binding assay instructions, and James G. Harmon for
constructive comments concerning the initial manuscript. Financial
support was provided by USDA Grant 35320-9378, the Herman Frasch
Foundation for Chemical Research, and the Robert A. Welch Foundation
Grant D-1478.
Received September 25, 2003; accepted November 28, 2003.
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