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Plant Physiol. (1998) 116: 231-238
Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in
Phloem Fluids of Cucumber during Systemic Acquired Resistance Is
Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase
Activity in Petioles and Stems1
Jennifer Smith-Becker*,
Eric Marois2,
Elisabeth J. Huguet3,
Sharon L. Midland,
James J. Sims, and
Noel T. Keen
Department of Plant Pathology, University of California, Riverside,
California 92521
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ABSTRACT |
Cucumber
(Cucumis sativa) leaves infiltrated with
Pseudomonas syringae pv. syringae cells
produced a mobile signal for systemic acquired resistance between 3 and
6 h after inoculation. The production of a mobile signal by
inoculated leaves was followed by a transient increase in phenylalanine
ammonia-lyase (PAL) activity in the petioles of inoculated leaves and
in stems above inoculated leaves; with peaks in activity at 9 and
12 h, respectively, after inoculation. In contrast, PAL activity
in inoculated leaves continued to rise slowly for at least 18 h. No
increases in PAL activity were detected in healthy leaves of inoculated
plants. Two benzoic acid derivatives, salicylic acid (SA) and
4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at
about the time PAL activity began to increase, reaching maximum
concentrations 15 h after inoculation. The accumulation of SA and
4HBA in phloem fluids was unaffected by the removal of all leaves
6 h after inoculation, and seedlings excised from roots prior to
inoculation still accumulated high levels of SA and 4HBA. These results
suggest that SA and 4HBA are synthesized de novo in stems and petioles
in response to a mobile signal from the inoculated leaf.
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INTRODUCTION |
Pathogen-induced necrosis on the leaves of many plant species,
including cucumber (Cucumis sativa), tobacco
(Nicotiana tabacum), and Arabidopsis, results in the
production of a phloem mobile signal that triggers systemic resistance
to subsequent pathogen attack (Kuc, 1982 ; Ryals et al., 1994). The
development of SAR depends on the rate at which the inducing pathogen
causes necrosis on the inoculated leaf. In cucumber, for example, an
incompatible bacterial pathogen that causes a rapid HR induces systemic
resistance within 2 d. A compatible pathogen that causes a slow,
spreading necrosis requires 1 week or more to induce systemic
resistance (Smith et al., 1991). Pathogen-induced necrosis on the
inoculated leaf is accompanied by the accumulation of SA at the site of
inoculation, in phloem fluids, and in healthy, uninoculated leaves
(Malamy et al., 1990; Metraux et al., 1990; Enyedi et al., 1992;
Summermatter et al., 1995). In tobacco and Arabidopsis, SA accumulation
is in turn correlated with the expression of a set of genes called SAR
genes, a subset of which encode the PR proteins (Ward et al., 1991;
Uknes et al., 1992). In cucumber SAR is correlated with the systemic
accumulation of extracellular peroxidase,  -1,3 glucanase, and
chitinase (Hammerschmidt et al., 1982; Boller and Metraux, 1988; Ji and
Kuc, 1995). Chitinase is a particularly useful marker for SAR in
cucumber, since levels of the enzyme in control leaves are extremely
low (Metraux et al., 1988).
The accumulation of SA is a requirement for SAR, as demonstrated by the
inability of tobacco plants transformed with a bacterial gene encoding
salicylate hydroxylase (nahG) to express SAR (Gaffney et
al., 1993). However, SA does not appear to be the only mobile signal
transported from the inoculated leaf, since NahG rootstocks, which do
not accumulate SA, were still able to transmit a signal for SAR to
wild-type scions (Vernooj et al., 1994). In addition, cucumber plants
inoculated on one leaf with the HR-causing pathogen Pseudomonas
syringae pv. syringae accumulated high levels of SA in
phloem exudates even when the inoculated leaf remained on the plant for
only 6 h (Rasmussen et al., 1991). Maximal levels of SA were
measured 18 h after inoculation in this system, so the majority of
systemically accumulating SA was not synthesized by the inoculated
leaf. In contrast to cucumber inoculated with HR-causing bacteria, 60 to 70% of systemically accumulating SA in tobacco mosaic
virus-inoculated tobacco was found to originate from the inoculated
leaf (Shulaev et al., 1995). In addition, removal of the inoculated
tobacco leaf prior to SA accumulation prevented SAR. It remains to be
determined whether the difference in the pattern of SA accumulation in
the two systems results from a mechanistic difference in SAR between
the two plant species or if it is due to the different rates of
necrosis induced by incompatible bacteria (24 h) versus virus (3 d).
The nature of the mobile signal that originates in the inoculated leaf
and leads to SA accumulation and SAR is unknown. The discovery that SAR
occurs in Arabidopsis has resulted in a major effort to identify
mutants in the SAR pathway (Uknes et al., 1992; Cameron et al., 1994;
Cao et al., 1994; Dietrich et al., 1994). Although the advantages of
Arabidopsis as a genetic tool are well documented, the cucumber system
offers several advantages for the biochemical study of SAR. First and
most importantly, the mobile signal for SAR is produced by cucumber
leaves within 6 h after inoculation with incompatible bacteria,
providing a narrow time frame within which to look for an active
compound (Rasmussen et al., 1991; Smith et al., 1991). Second, the
large leaves of cucumber are easy to saturate with inoculum and produce
correspondingly large amounts of mobile signal. Third, cucumber stems
exude relatively large amounts of phloem fluid when cut, allowing
direct analysis of phloem-localized compounds. In the present work we
have taken advantage of the rapid SAR response induced by an
incompatible bacterial pathogen to study the biochemical changes that
occur during the initiation of SAR in cucumber.
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MATERIALS AND METHODS |
Plant Culture and Inoculation
Cucumber (Cucumis sativa cv Wisconsin SMR 58) seedlings
were grown in a growth chamber at 28°C with 14 h of light (270 µmol s 1 m2) and
10 h of dark. Seedlings used for SA and 4HBA accumulation and PAL
activity time-course experiments were left under continuous light
during the experiment. Young plants, approximately 3 weeks old and with
two fully expanded leaves, were inoculated on the second leaf with
Pseudomonas syringae pv. syringae as described previously (Rasmussen et al., 1991). Control plants were infiltrated with water. In some experiments, inoculated and uninoculated leaves were excised with a razor blade at various times after inoculation. Greenhouse-grown plants were used for the large-scale collection of
phloem fluids.
Chitinase Assay
A sandwich ELISA assay utilizing monoclonal and polyclonal
antisera generated against cucumber chitinase was used to estimate chitinase levels in leaf homogenates (Ciba-Geigy, Research Triangle Park, NC). Purified cucumber chitinase was used as a standard. The
timing of primary signal production by inoculated leaves was determined
by removing inoculated leaves at various times after inoculation as
described previously (Rasmussen et al., 1991). Chitinase accumulation
was measured in the third leaf after 5 d. Two 1-cm-diameter leaf
discs were removed from the third leaves of four plants and stored in
liquid N2 prior to assay. The limit of detection
of chitinase in leaf extracts was approximately 150 ng/g fresh weight.
Collection of Phloem and Xylem Exudates
Time-Course Experiments
Petioles of inoculated leaves were excised 1.0 cm from the stem at
various times after inoculation. For each sample, 12.5 µL of phloem
fluid was collected from the excised petiole of each of four plants
using a micropipettor. Collected phloem exudate was dispensed
immediately into 500 µL of ice-cold methanol in a 1.5-mL Eppendorf
tube. Two samples, representing a total of eight plants, were collected
for each treatment and analyzed separately by HPLC. Methanol extracts
were vortexed and the precipitate was removed by centrifugation in a
microfuge at 10,000 rpm for 5 min. The supernatant was transferred to a
new tube and the pellet re-extracted with an additional 200 µL of
methanol. The methanol fractions were combined and the methanol was
removed by evaporation at 40°C under a vacuum. The remaining aqueous
sample was adjusted to a volume of 50 µL with distilled water and
used for HPLC analysis. The efficiency of the extraction of SA and 4HBA
was determined by adding known amounts of the compounds to control
phloem fluid prior to extraction.
For the collection of xylem exudates, inoculated or control plants were
cut with a razor blade at the stem base. Residual phloem exudate was
blotted from the cut stump for 2 min, after which time the root
pressure exudate continued to accumulate on the cut surface.
Two-hundred microliters of root pressure exudate was collected from
each of four plants and then added individually to Eppendorf tubes
containing 1 mL of ice-cold methanol. Samples were processed as
described for phloem samples except that the aqueous sample remaining
after vacuum evaporation of methanol was lyophilized. Lyophilized
samples representing root pressure exudate from four plants were
combined in 50 µL of water prior to HPLC analysis.
Leaf and Root Excision Experiments
To determine the tissue source of systemically accumulating SA and
4HBA, leaves and cotyledons of seedlings that had been inoculated on
one leaf were excised from plants 6 h after inoculation. All
leaves, including the apical bud, were excised with a razor blade
through the petiole at the base of the leaf. Phloem exudates were
collected from stems immediately above petioles of inoculated leaves
20 h after inoculation (14 h after excision of leaves and cotyledons). In a separate experiment, seedlings were submerged in
water and excised from roots at the stem base. Excised seedlings were
floated on water for 2 d prior to inoculation. Although cucumber seedlings wilt severely upon excision from roots, approximately 70% of
the seedlings survive the procedure if floated on water for at least
24 h. After 2 d seedlings were removed from water and placed
with cut stem bases in large test tubes filled with distilled water.
Leaves of excised seedlings were inoculated as described above and
phloem exudate was collected from stems 20 h after inoculation.
HPLC Analysis
Fifty microliters of aqueous phloem fluid extract was injected
onto a 5-µm C-18 column (Econosphere, Alltech, Deerfield, IL; 4.6 mm × 25 cm) fitted with a guard column. The column was
equilibrated in water containing 0.075% trifluoroacetic acid with a
flow rate of 1 mL/min. Five minutes after injection of the sample,
a linear gradient of 0 to 70% acetonitrile was applied to the column
over 20 min. UV-absorbing compounds eluting from the column were
monitored at 230 and 254 nm with a diode array detector (model 1040A,
Hewlett-Packard).
The unidentified compound eluting from the gradient at 17.3 min was
collected, dried, resuspended in 50 µL of distilled water, and
purified isocratically on the same column using 15% acetonitrile in
0.075% trifluoroacetic acid and a flow rate of 1 mL/min. The compound
eluted at 6.8 min under these conditions. The isocratically eluted
compound was dried and used for MS and proton NMR analysis. SA, which
eluted at 21.8 min on the gradient system, was identified by its
characteristic UV absorption spectrum, its co-elution with standard SA,
and its visible fluorescence upon exposure to UV (302 nm) light.
Quantitation was determined using a linear range of calibration
standards consisting of 0 to 1.3 µg/50 µL of SA and 0 to 0.6 µg/50 µL of 4HBA. The limits of detection for SA and 4HBA were 24 ng/50 µL and 70 ng/50 µL, respectively.
NMR and MS Analysis
MS analysis was performed on a spectrometer (model VG7070E,
Micromass, Manchester, UK) with an ionization potential of 30 eV and a
resolution of 2000. Proton NMR spectra were taken in methanol-d4 at 300 MHz using a spectrometer
(model QE-300, General Electric).
PAL Activity
PAL activity was determined spectrophotometrically as described by
Edwards and Kessmann (1992) in inoculated leaves, petioles of
inoculated leaves, stem segments above inoculated leaves, and leaves
directly above inoculated leaves. For each sample, 1-cm-diameter leaf
discs or 1-cm-long stem or petiole segments were collected from each of
four plants and stored in liquid N2 prior to
assay. Petiole segments were collected from the end of the petiole
closest to the stem. Stem segments were collected 2 cm above the
junctions of the petioles of inoculated leaves and stems. In one
experiment, inoculated leaves were excised from petioles 6 h after
inoculation. The excision was made at the base of the leaf so as to
leave the entire petiole intact. Petiole segments were collected at 6 and 9 h after inoculation as described above. Tissue for assay was first ground to a fine powder in liquid N2 prior
to the addition of extraction buffer. Homogenates were centrifuged for
5 min at 10,000 rpm in a microcentrifuge and the resulting supernatant was used for PAL activity and protein determination. Protein levels were measured using a protein assay kit (Bio-Rad) with BSA as standard
(Bradford, 1976). Two samples were collected in each experiment and the
experiments were repeated at least twice. se was calculated
for measurements within each experiment.
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RESULTS |
Chitinase Assay
Signal production, as measured by systemic increases in chitinase
after removal of inoculated leaves, began between 3 to 4 h after
inoculation (Fig. 1). Plants inoculated
at 50 sites on the first leaf were fully induced by 5 h. This
correlates well with the previous observations of Smith et al. (1991)
and Rasmussen et al. (1991) for the systemic increase of peroxidase
activity.
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| Figure 1.
Signal production by inoculated leaves. Cucumber
seedlings were infiltrated on the second leaf with P. syringae cells at 50 sites per leaf. The inoculated leaves were
removed from plants at various times and systemic chitinase
accumulation was measured in the third leaf 5 d after inoculation.
se values were smaller than the size of the symbols.
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HPLC, MS, and NMR Analysis
In addition to SA, a compound eluting at 17.3 min in the gradient
system with an absorbance maximum at 260 nm increased significantly with time after inoculation (Fig. 2, A
and B). Unlike SA, however, this compound was also present at low
levels in control phloem fluids. The compound was purified from phloem
fluids collected 18 h after inoculation and subjected to NMR and
MS analyses. Observation of proton NMR doublets at 6.79 and 7.85 ppm
(J = 8.5 Hz) indicated a disubstituted benzene ring with one
electronegative (oxygen) moiety and one electron-donating (carbonyl)
moiety in the para orientation. The compound was further identified as
4HBA by MS analysis.
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| Figure 2.
HPLC profile of phloem exudates. Phloem fluids
were collected 18 h after infiltration of leaves with water (A and
C) or with P. syringae (B and D). Eluting peaks were
monitored for UV A254 (A and B) and
A230 (C and D). The peaks were identified as
SA glucoside (1), 4HBA (2), and SA (3).
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A standard sample of 4HBA and the compound isolated from phloem fluids
shared identical HPLC retention times and UV spectra. SA was first
detected in phloem fluids 6 h after inoculation (Fig. 3A). The levels of both SA and 4HBA
increased with time until 15 h after inoculation, with a maximum
rate of increase between 9 and 15 h (Fig. 3, A and B). The
levels of both compounds had declined by 36 h, although free
SA was still detected. In addition to 4HBA and SA, a new compound with
A230 eluted from the column at 17.1 min in the
gradient system (Fig. 2). This peak first appeared in phloem exudates
collected 18 h after inoculation and increased with time as levels
of free SA decreased (data not shown). The UV absorbance spectrum of
the new compound was similar to that of SA and treatment of the
isolated compound with -glucosidase (Enyedi et al., 1992) released
free SA (data not shown). Therefore, at least some of the free SA
accumulating in phloem fluids was conjugated in the vascular system.
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| Figure 3.
Time course of SA and 4HBA accumulation in
cucumber phloem exudates. The levels of SA ( ,  ) and 4HBA ( ,
 ) in petiole phloem exudates from control (open symbols) or
inoculated (closed symbols) plants were monitored for 36 h after
infiltrating one leaf with water or with P. syringae
cells. ses are indicated.
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SA and 4HBA accumulating in phloem exudates did not originate from
synthesis of the compounds in leaves or roots. Both SA and 4HBA
accumulated to high levels in phloem fluid even when all of the leaves
and both cotyledons were excised from plants 6 h after inoculation
(Fig. 4). Seedlings excised from the
roots also accumulated SA and 4HBA in phloem exudates to the same
levels as seedlings that remained intact (Fig.
5). The efficiency of extraction of SA
and 4HBA from phloem exudates was 87%, and the data are corrected for
this value. Very few UV-absorbing peaks were observed in root pressure
exudate samples and no differences were detected between control and
inoculated plants at any time (data not shown).
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| Figure 4.
Accumulation of SA and 4HBA in phloem exudates
after excision of leaves. Levels of SA and 4HBA in stem phloem exudate
were determined 20 h after infiltration of one leaf on each plant
with water (C) or P. syringae cells (I). Plants were
either left intact (+Leaves), or had all leaves excised 6 h after
inoculation ( Leaves). ses are indicated.
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| Figure 5.
Effect of excision of seedlings from roots on the
accumulation of SA and 4HBA. Seedlings were either left intact (+Roots) or were excised from roots at the stem base and placed in water 2 d prior to inoculation (Roots). Levels of SA and 4HBA in stem phloem
exudate were determined 20 h after infiltration of one leaf on
each plant with water (C) or with P. syringae (I).
ses are indicated.
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PAL Activity
PAL activity began to increase by 6 h after inoculation in
inoculated leaves and by 7 h after inoculation in the petioles of
inoculated leaves and stems (Fig. 6). The
increase in activity was transient in the petiole and stem, and
activity returned to control levels by 12 and 18 h after
inoculation, respectively. The peak in PAL activity occurred 9 h
after inoculation in petioles and 12 h after inoculation in stems.
In contrast, PAL activity in the inoculated leaf continued to increase
for at least 18 h after inoculation. The increase in petiole PAL
activity was unaffected by excision of the inoculated leaf 6 h
after inoculation (Fig. 7). No
differences in PAL activity were detected between control and
inoculated plants in the leaf directly above the inoculated leaf (data
not shown). In addition, no PAL activity was detected in phloem fluid
exudates at any time after inoculation (data not shown). The
constitutive activity of PAL in petioles and stems was at least 20-fold
higher than that in leaves.
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| Figure 6.
Time course of PAL activity in different tissues
during SAR. PAL activity was measured in leaves infiltrated with
P. syringae () or water () (Inoculated Leaf); in
1-cm-long petiole segments of inoculated leaves (Petiole of Inoculated
Leaf); or in 1-cm-long stem segments immediately above inoculated
leaves (Stem) at various times after inoculation. ses are
indicated.
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| Figure 7.
Effect of leaf excision on petiole PAL activity.
Seedlings inoculated with P. syringae or water were
either left intact (+Leaf) or the inoculated leaves were excised at the
leaf base 6 h after inoculation (Leaf). PAL activity was
measured in 1-cm segments taken from the base of the petiole adjacent
to the stem at 6 and 9 h after inoculation. ses are
indicated.
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DISCUSSION |
In the classic interpretation of SAR, a mobile signal produced by
the inoculated leaf travels through the vascular tissue to uninoculated
leaves, where it induces PR gene expression and some level of SA
synthesis. In this work we provide evidence that the vascular tissue
itself may have an integral role in the perception and relay of the
mobile signal. The accumulation of SA and 4HBA in phloem exudates of
cucumber inoculated with P. syringae was preceded by a
transient increase in PAL activity in stems and petioles of inoculated
plants. The accumulation of both SA and 4HBA in phloem exudates peaked
15 h after inoculation and was unaffected by the removal of all
leaves from plants 6 h after inoculation. Similarly, the increase
in PAL activity in the petiole of the inoculated leaf was unaffected by
the removal of the leaf 6 h after inoculation. Finally, seedlings
excised from roots prior to inoculation still accumulated high levels
of SA and 4HBA in phloem exudates.
Taken together, these data suggest that systemically accumulating SA
and 4HBA were synthesized in stems and petioles, most likely in the
vascular tissue, in response to a mobile signal from the inoculated
leaf. Vascular synthesis of these compounds would explain the previous
observation that cucumber leaves infiltrated with cells of P. syringae need to remain on the plant for only 6 h to induce
SAR and systemic accumulation of peroxidase and SA (Smith et al., 1991;
Rasmussen et al., 1991). In the present work we measured the
accumulation of chitinase in uninfected leaves of induced cucumber as
an additional marker for the production of a mobile signal for SAR. Our
results are in agreement with previous studies, and demonstrate that
export of a mobile signal from the inoculated leaf occurs rapidly
between 3 and 5 h after inoculation. We first detected SA in
phloem fluids of induced plants at 6 h after inoculation, which
was slightly earlier than that reported by Rasmussen et al. (1991).
This difference is most likely due to the increased sensitivity of HPLC
analysis used in this work compared with the TLC assay used in the
previous work.
Several reports suggest that PAL is a key regulatory enzyme in the
synthesis of SA and establishment of SAR. Mauch-Mani and Slusarenko
(1996) showed that in Arabidopsis, PAL activity was essential for
accumulation of SA and expression of the HR. Recently, it was also
reported that tobacco (Nicotiana tabacum) plants
epigenetically suppressed in PAL activity were unable to express SAR
(Pallas et al., 1996). We observed that the constitutive activity of
PAL in stems and petioles was approximately 20-fold higher than in leaves. Several studies of PAL-promoter activity in transgenic plants
demonstrated activity in vascular tissues, where PAL is thought to
provide the precursors for lignin deposition in the xylem (Bevan et
al., 1989; Liang et al., 1989; Ohl et al., 1990). In addition, detailed
analyses of the promoters of PAL2 from bean and PAL1 from Arabidopsis
have begun to identify regions of the promoters specifically responsive
to environmental and developmental signals (Liang et al., 1989; Ohl et
al., 1990; Leyva et al., 1992). Although no PAL genes have been cloned
from cucumber, their regulation is likely to be as complex as in other
species.
In addition to SA, a second phenylpropanoid compound, 4HBA, accumulated
in phloem fluids of inoculated cucumber with the same kinetics as SA.
Trace levels of 4HBA have previously been detected in phloem fluids
from cucumber inoculated with Colletotrichum lagenarium
(Metraux et al., 1990). Unlike SA, however, infiltration of 4HBA into
leaves did not induce local resistance to C. lagenarium. Similarly, we found that infiltration of up to 5 mm 4HBA
into leaves did not induce the accumulation of SA in phloem exudates or local accumulation of chitinase (data not shown). Meuwly
et al. (1995) studied the incorporation of
14C-labeled Phe in inoculated and uninfected
leaves of cucumber during SAR. They did not report enhanced synthesis
of 4HBA in induced cucumber, but this may have been because of their
addition of excess cold 4-coumaric acid, a likely precursor to 4HBA
(Schnitzler et al., 1992; Fig. 8), to the
reaction to drive incorporation into SA. Treatment of carrot cells with
elicitor resulted in a transient increase in PAL activity, followed by
an increase in 4HBA covalently bound to cell walls (Schnitzler and
Seitz, 1989; Bach et al., 1993). Although 4HBA may enhance cell wall
impermeability in response to pathogens, this has not been conclusively
demonstrated. It is possible that enhanced levels of the compound in
phloem exudate during SAR in cucumber is a nonspecific consequence of PAL stimulation (Fig. 8).
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| Figure 8.
Proposed pathways of SA and 4HBA biosynthesis.
Enzymatic steps for which the enzymes have been identified include
PAL, CA4H (cinnamic acid 4-hydroxylase),
and BA2H (benzoic acid 2-hydroxylase).
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The pattern of induced PAL activity in petioles and stems suggests that
these tissues are responding to a mobile signal originating from the
inoculated leaf. The increase in PAL activity may be due to increased
expression of the gene for the enzyme and/or an increase in the rate of
depletion of its product, trans-cinnamic acid. Cinnamic acid
is a potent inhibitor of PAL activity, acting both to accelerate the
depletion of the enzyme and to inhibit de novo enzyme production
(Shields et al., 1982). One possibility is that the initial signal from
the inoculated leaf leads to the activation of an enzyme or group of
enzymes downstream from PAL that convert cinnamic acid to SA. For
example, the enzyme benzoic acid 2-hydroxylase has been characterized
in tobacco as a pathogen-induced monooxygenase that converts benzoic
acid to SA (Leon et al., 1993; Fig. 8). Synthesis of SA in cucumber has
also been suggested to proceed via a benzoic acid intermediate (Meuwly
et al., 1995; Molders et al., 1996). The activities of benzoic acid
2-hydroxylase and the enzymes responsible for side-chain shortening of
trans-cinnamic acid to benzoic acid have not been reported
in cucumber. The results of the work presented here indicate that
petioles of P. syringae-inoculated leaves and stems above
inoculated leaves are likely tissues in which to find these enzyme
activities in cucumber.
A variation of the classic model of signal transmission is suggested by
the unexpected observation of Mauch-Mani and Slusarenko (1996) that the
PAL promoter in Arabidopsis was suppressed by specific inhibition of
PAL activity in pathogen-treated tissue. This indicates that a product
of the phenylpropanoid pathway is involved in feedback stimulation of
the PAL gene. If similar feedback regulation occurs in the
cucumber/P. syringae system, PAL expression may be amplified
in vascular tissue by a product of the phenylpropanoid pathway. For
example, SA has been reported to potentiate the expression of PAL and
other defense-related genes, allowing higher levels of expression in
response to elicitors (Mur et al., 1996; Shirasu et al., 1997).
SA has also been shown to induce binding of a tobacco nuclear protein
to a sequence conserved among stress-induced proteins, including PAL
(Goldsborough et al., 1993), and to induce transcription from the as-1
element of the cauliflower mosaic virus via binding of a tobacco
cellular factor, SARP (Jupin and Chua, 1996). Although it is unlikely
that low levels of SA present in the phloem at 6 h after
inoculation are sufficient to induce the SAR response, it is
conceivable that SA may enhance its own vascular synthesis in response
to the mobile signal by direct effects on the relevant genes of the
phenylpropanoid pathway. The fact that SA has never been demonstrated
to induce its own synthesis necessitates the involvement of an
additional signal molecule to initiate the branch in the
phenylpropanoid pathway leading to SA biosynthesis.
In summary, we have shown that the first measurable effect of the
mobile signal for SAR in cucumber inoculated with P. syringae is the transient stimulation of PAL activity in the
petiole of the inoculated leaf and in the stem above the inoculated
leaf. The transient increase in PAL activity precedes a transient
increase in SA and 4HBA in phloem fluids, and suggests that the two
compounds are produced de novo in stems and petioles, perhaps in
vascular tissues. If PAL gene expression is regulated by the mobile
signal, a detailed analysis of PAL message accumulation in different
tissues during SAR should provide insight into the movement of signal from the inoculated leaf. Furthermore, future efforts to identify the
mobile signal for SAR in this system should focus on products of the HR
that are able to modulate PAL activity and initiate the synthesis of SA
in petioles and stems.
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FOOTNOTES |
1
This work was supported by a grant from
Ciba-Geigy.
2
Present address: Department of Biology, Coker
Hall 108, University of North Carolina, Chapel Hill, NC 27599-3280.
3
Present address: Institut des Sciences
Vegetales, Centre Nationale de la Recherche Scientifique, Avenue de la
Terrassse, 91198 Gif-sur-Yvette, France.
*
Corresponding author; e-mail beck{at}ucrac1.ucr.edu; fax
1-909-787-4294.
Received June 26, 1997;
accepted September 28, 1997.
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ABBREVIATIONS |
Abbreviations:
4HBA, 4-hydroxybenzoic acid.
HR, hypersensitive
response.
PAL, Phe ammonia-lyase.
SA, salicylic acid.
SAR, systemic
acquired resistance.
| |
ACKNOWLEDGMENTS |
We thank Sandy Stewart for supplying us with the cucumber
chitinase ELISA assay and Ray Hammerschmidt for helpful discussions.
| |
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Abstract
Introduction