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Plant Physiol. (1998) 117: 1095-1101
The Biosynthesis of Salicylic Acid in Potato Plants1
Jean-Luc Coquoz,
Antony Buchala, and
Jean-Pierre Métraux*
Département de Biologie, Route Albert-Gockel 3, Université de Fribourg, CH-1700 Fribourg, Switzerland
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ABSTRACT |
Spraying potato (Solanum
tuberosum L.) leaves with arachidonic acid (AA) at 1500 µg
mL 1 led to a rapid local synthesis of salicylic acid (SA)
and accumulation of a SA conjugate, which was shown to be
2-O- -glucopyranosylsalicylic acid. Radiolabeling
studies with untreated leaves showed that SA was synthesized from
phenylalanine and that both cinnamic and benzoic acid were
intermediates in the biosynthesis pathway. Using radiolabeled
phenylalanine as a precursor, the specific activity of SA was found to
be lower when leaves were treated with AA than in control leaves.
Similar results were obtained when leaves were fed with the labeled
putative intermediates cinnamic acid and benzoic acid. Application of
2-aminoindan-2-phosphonic acid at 40 µM, an inhibitor of
phenylalanine ammonia-lyase, prior to treatment with AA inhibited the
local accumulation of SA. When the putative intermediates were applied
to leaves in the presence of 2-aminoindan-2-phosphonic acid, about 40%
of the expected accumulation of free SA was recovered, but the amount
of the conjugate remained constant.
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INTRODUCTION |
Since the discovery that SA is produced upon infection of cucumber
(Métraux et al., 1990 ) or tobacco (Malamy et al., 1990) leaves
prior to the expression of systemic resistance, much effort has been
devoted to demonstrate the role of SA in the resistance of plants to
disease (Raskin, 1992; Delaney et al., 1994). SA was originally
proposed as a putative signaling molecule for the induction of SAR on
the basis of the results obtained with cucumber (Métraux et al.,
1990; Rasmussen et al., 1991) and tobacco (Malamy et al., 1990; Enyedi
et al., 1992; Malamy and Klessig, 1992). In tobacco the same
defense-related genes that were expressed locally and systemically upon
infection with tobacco mosaic virus were also activated by treatment
with SA (Ward et al., 1991). More recent results have shown that
transgenic tobacco plants expressing bacterial salicylate hydroxylase,
which metabolizes SA, are unable to express SAR and even show enhanced
susceptibility to pathogens (Gaffney et al., 1993). SAR was restored in
wild-type scions grafted onto a transgenic rootstock, indicating that
SA may not be the primary mobile signal but a necessary component for
SAR (Vernooij et al., 1994a). It has been suggested that SA is required
for signal transduction at the local level and that its mode of action
may include inhibition of catalase activity, leading to increased
levels of H2O2 (Vernooij et
al., 1994b).
Rapid production of H2O2
(oxidative burst) upon treatment of plants with elicitors or upon
inoculation with avirulent pathogens may act as a local trigger of
programmed cell death (Levine et al., 1994), which very often precedes
SAR, but in tobacco leaves expressing SAR no increase in
H2O2 was detected (Ryals et
al., 1995). It has also been shown in potato (Solanum
tuberosum L.) that treatment with the biotic elicitor AA, which
induces SAR (Cohen et al., 1991; Coquoz et al., 1995), only gives rise
to local accumulation of SA (Coquoz et al., 1995) and that in rice no
changes in SA occur upon inoculation with pathogens (Silverman et al.,
1995). However, both in potato (Coquoz et al., 1995) and in rice
(Silverman, 1995), there appears to be a direct correlation between the
amount of endogenous SA and resistance to pathogens. Moreover, in
potato expressing the NahG gene, induced resistance is strongly
decreased, indicating that SA might also be a necessary component in
the induction for SAR in potato (Yu et al., 1997). Clearly, information
on the pathway leading to the synthesis of SA and the fate of
endogenous or exogenous SA is important for an understanding of the way
in which SA is related to SAR.
Early studies showed that in higher plants SA derives from the
shikimate-phenylpropanoid pathway (Zenk and Müller, 1964). Two
routes from Phe to SA have been found that differ at the step involving
hydroxylation of the aromatic ring: Phe is converted into CA by PAL and
the latter is either hydroxylated to form ortho-coumaric acid followed by oxidation of the side chain (El-Basyouni et al., 1964;
Chadha and Brown, 1974), or the side chain of CA is initially oxidized
to give BA, which is then hydroxylated in the ortho position (Zenk and Müller, 1964; Ellis and Amrhein, 1971). When BA was fed
to various plants, the synthesis of SA, and particularly of a glucosyl
conjugate of SA, was observed (Klämbt, 1962). More recently,
Yalpani et al. (1993) have shown that in tobacco SA is synthesized via
BA and that healthy tobacco leaves contain a large pool of conjugated
BA. BA 2-hydroxylase was detected in the leaves of healthy plants and
the activity increased significantly upon inoculation with tobacco
mosaic virus (Léon et al., 1993). In cucumber leaves
radiolabeling studies showed that SA is synthesized locally and
systemically from Phe upon inoculation with tobacco necrosis virus or
Pseudomonas lachrymans (Meuwly et al., 1995).
The fate of SA in plants has also been studied (for review, see Lee et
al., 1995); glycosides, esters, and amide conjugates have been
identified in different plants, but the major conjugate is usually
2-O--glucopyranosyl SA. In some cases, dihydroxybenzoic acids were also formed from SA. In tobacco decarboxylation of SA was
detected, and it was suggested that glucoside formation precedes
decarboxylation (Edwards, 1994). Hydroxy-BAs were rapidly decarboxylated by peroxidase in cell-suspension cultures of some leguminous plants, but both SAs and BAs were preferentially
glycosylated to form ester conjugates and were thereby protected
against oxidative carboxylation (Barz et al., 1978).
With the exception of rice (Silverman et al., 1995) and potato (Coquoz
et al., 1995), most of the plants in which the relationship between SA
and SAR has been studied do not contain much endogenous SA. In this
paper we report results on the biosynthesis of SA in healthy potato
leaves and in leaves treated with AA in which SA production is strongly
induced.
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MATERIALS AND METHODS |
Culture of the Plants and the Pathogen
Potato (Solanum tuberosum L. cv Bintje) plants were
propagated in vitro from a clone obtained from the Swiss Federal
Agronomy Station (Changins, Nyon, Switzerland) as previously described (Coquoz et al., 1995). Phytophthora infestans (isolate 191, race 1, 2, 3; mating-type A1) was cultured and
plants were inoculated as described previously (Coquoz et al.,
1995).
Treatment of the Plants with AA
The third leaf of 4-week-old plants was excised with a razor blade
and the petiole was immediately immersed in deionized water. The leaves
were sprayed with a sonicated suspension of AA (1500 µg
mL1) as described by Cohen et al. (1991) and
the plants were maintained at room temperature. Control plants were
sprayed with deionized water.
Radiolabeling Experiments
Two hours after the treatment with AA, the leaves were transferred
to aqueous solutions (20 µL) containing one of the following labeled
substances: [U-14C]BA (20 nCi, 13 mCi
mmol1; Sigma),
[3-14C]CA (20 nCi, 53.88 mCi
mmol1; Isotopchim, Ganagobie-Peyruis, France),
or [ring U-14C]Phe (20 nCi, 116 mCi
mmol1; Amersham). After 30 min the plants were
returned to deionized water. When appropriate, plants were treated with
an aqueous solution of AIP (40 µM) 1 h before the
treatment with AA. Labeling experiments were carried out at least twice
with similar results.
Leaf Disc Experiments
Two hours after treatment of leaf 3 with AA or water, leaf discs
(13 mm in diameter) were punched out and immediately vacuum infiltrated
with buffer solution (100 mM sodium acetate, pH 5.5) containing radiolabeled precursors: [U-14C]BA
(20 nCi, 13 mCi mmol1; Sigma) or [ring
U-14C]Phe (20 nCi, 116 mCi
mmol1; Amersham) as described by Meuwly et al.
(1995).
Extraction and Analysis of Phenolic Compounds by HPLC
Phenolic compounds were identified and quantified as described by
Meuwly and Métraux (1993) with the following modifications. Three-hundred nanograms of the internal standard (o-anisic
acid) was added per sample. Samples were resuspended in 400 or 800 µL of the starting mobile phase (15% [v/v] acetonitrile in 10 mM KH2PO4, pH
3.5) for free and conjugated phenolic compounds, respectively. Samples
(80 µL for free or 60 µL for conjugated phenolic compounds) were
separated on a deactivated reverse-phase column (25 cm × 0.4 mm,
LC-SAL, Supelco, Bellefonte, PA).
Identification of SA and Conjugate by GC-MS
Plant material was extracted with 70% ethanol as described above,
and the organic phase evaporated in vacuo to dryness. The material was
dissolved in anhydrous pyridine (100 µL) and
N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane (100 µL) was added. The mixture was heated (30 min, 80°C), evaporated to dryness, and redissolved in
n-hexane (20 µL). Samples (5 µL, splitless) were taken
for analysis by GC-MS (HP 5890 coupled to a 5970 mass specific
detector, Hewlett-Packard). Separations were carried out on a capillary column (25 m × 0.22 mm × 0.25 µm) coated with methyl
silicone (BP5X, SGE, Melbourne) using He carrier gas at 1 mL
min1. The injection port and the ion source
were maintained at 200°C and 290°C, respectively; the initial
temperature was held at 140°C for 2 min and then programmed at 4°C
min1 to 290°C. MS analysis was carried
in the electron-impact mode at an ionizing potential of 70 eV. The
per-trimethylsilyl derivative of authentic
2-O--glucopyranosyl SA, a gift from D.F. Klessig (Waksman
Institute, Rutgers, NJ) (Hennig et al., 1993), eluted at 36 min. Other
phenolic substances were identified by comparison of their retention
time and mass spectrum with those of authentic standards.
An aliquot of the extract was incubated with -glucosidase from
almond emulsin (Fluka) in sodium acetate buffer (50 mM, pH 5.5, 12 h) and analyzed as above.
Determination of PAL Activity
Plant material was homogenized in liquid N2
and suspended (5 g mL1 fresh weight) in
extraction buffer (100 mM sodium tetraborate, pH 8.8)
containing 10 mM DTT and 50 mg mL1
PVP. After centrifugation (20,000g; 15 min), the supernatant was freed of low-Mr plant material on a column
of Sephadex G-25 equilibrated with extraction buffer containing 20%
(v/v) glycerol, but without PVPP. Extract (200 µL) was added to 100 mM borate buffer (pH 8.8) containing 10 mM DTT,
60 mM Phe, and [ring U-14C]Phe (116 mCi mmol1). The mixture was incubated (1 h,
30°C) and the reaction stopped by the addition of 6 M HCl
(100 µL). The reaction products were partitioned with ethylacetate
(1.3 mL) and the radioactivity (CA) in the organic phase was counted by
liquid scintillation.
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RESULTS |
Identification of SA, 2-O--Glucopyranosyl SA, and
Other Phenolic Compounds in Potato Leaves
HPLC of acidic compounds from potato leaves soluble in aqueous
methanol showed the presence of SA, BA, ferulic acid, caffeic acid, or
CA. The presence of these compounds inter alia was confirmed by GC-MS
analysis of their pertrimethylsilyl derivatives (not shown). The
presence of SA conjugates was established by measuring an increase in
the amount of free SA after acid hydrolysis of the material. The
identity of the SA conjugate as 2-O--glucopyranosyl SA
was established by comparison of the retention time and electron-impact mass spectrum (Fig. 1) of the
pertrimethylsilyl derivative with those of an authentic sample, which
was chemically synthesized and characterized (Grynkiewicz et al.,
1993). It should be noted, however, that the mass spectrum is not very
informative, since the major ion fragments derive from the sugar
moiety. Treatment with a -glucosidase from almond emulsion
followed by analysis by GC-MS showed that complete hydrolysis of the
putative glucoside had taken place and that SA was released. The
presence of significant amounts of other SA conjugates was negligible,
since treatment of extracts with 1 M NaOH at room
temperature (saponification of ester conjugates) did not release SA and
treatment with 7 M NaOH at 100°C (hydrolysis of amide
conjugates) did not give more SA than acid hydrolysis (hydrolysis of
glycoside conjugates).
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| Figure 1.
A, Electron-impact mass spectrum of the
pertrimethylsilyl derivative of putative salicylglucopyranoside
(retention time, 35.95 min on gas-liquid chromatography) in extracts of
potato leaves treated with 1500 µg mL1 AA. B,
Electron-impact mass spectrum of the pertrimethylsilyl derivative of
authentic salicylglucopyranoside (retention time, 36 min).
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Accumulation of SA in Detached Potato Leaves after Treatment with
AA
When detached potato leaves from 4-week-old plants were sprayed
with a suspension of AA (1500 µg mL1), free
SA began to accumulate after 4 h to reach a maximum (11 µg
g1 fresh weight) after 6 h (Fig.
2). In the same time period no significant increase in the amount of conjugated SA was observed.
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| Figure 2.
Level of free (A) and conjugated (B) SA in
control (white bars) and AA-treated (shaded bars) potato leaves.
Detached leaves were treated with water (control) or 1500 µg
mL1 of AA. Results are means ± SE of
three replicates. FW, Fresh weight.
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Phe as a Precursor for the Synthesis of SA
When detached leaves were fed [14C]Phe,
radioactivity was found to accumulate in components that were
chromatographically (HPLC) identical to CA, BA, and SA (Fig.
3), suggesting that CA and BA were
intermediates in the biosynthetic pathway to SA.
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| Figure 3.
HPLC chromatograms showing incorporation of
radioactivity from [14C]Phe into CA, BA, and SA in
control (A) and treated (B) leaves, as well the amounts of SA
synthesized in control (C) and treated (D) leaves 6 h after
treatment.
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Treatment with AA of leaves fed with the same amounts of
[14C]Phe had no marked effect on the amount of
total radioactivity taken up in leaves and led to the accumulation of
similar amounts of radioactivity in the free SA of control leaves
(12.4 ± 4.9 nCi per leaf) when compared with leaves treated with
AA (10.1 ± 4.13 nCi per leaf). However, the specific activity of
the free SA in the untreated leaves was markedly higher (20.8 ± 5.7 nCi µg1) than in AA-treated leaves
(4.21 ± 1.2 nCi µg1) (Fig.
4), suggesting that not all of the free
SA that accumulated after treatment with AA derived directly from the
radioactive pool of Phe. The amount of conjugated SA did not decrease
after treatment with AA, showing that the decrease in the specific
activity of the free SA was not due to hydrolysis of conjugated SA
(Fig. 2).
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| Figure 4.
Levels of free SA (A), radioactivity in SA (B),
and specific activity of free SA (C) in detached leaves treated with
deionized water (control) or AA and fed with [14C]Phe.
Results are means ± SE of three replicates. FW, Fresh
weight.
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CAs and BAs as Intermediates for the Synthesis of SA
To confirm the role of CA as an intermediate in the path from Phe
to SA, labeled CA was fed to potato leaves under the conditions described above. After 6 h, less radioactivity was found in SA of
the treated leaves (17.6 ± 5.1 nCi per leaf) than in the control leaves (39.2 ± 8.5 nCi per leaf) and the specific activity of the
free SA decreased from 145.9 ± 19.5 nCi
µg1 in the control leaves to 7.86 ± 0.2 nCi µg1 in the treated leaves (Fig.
5). However, radioactivity was also found
in free BA both in the control (22.95 ± 2.3 nCi/leaf) and in
treated (23.2 ± 1.3 nCi/leaf) leaves. It is clear that some of
the radioactivity also followed the lignin pathway. Analogous experiments were carried out using labeled BA. There was no marked difference between control (31.7 ± 4.4 nCi per leaf) and treated (27.2 ± 7.2 nCi per leaf) leaves in the radioactivity
incorporated into free SA, but the specific activity of the free SA was
higher in the control leaves (62.9 ± 7.4 nCi
µg1) than in the treated leaves (6.9 ± 1.9 nCi µg1). When carboxyl-labeled BA was
used instead of ring-labeled BA, similar results were obtained,
indicating that no decarboxylation process occurred in the
transformation of BA to SA (data not shown).
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| Figure 5.
Levels of radioactivity (A) and specific activity
(B) of free SA in detached leaves treated with deionized water
(control) or AA and fed within control (white bars) and AA-treated
(gray bars) potato leaves fed with [14C]CA or
[14C]BA. Results are means ± SE of
three replicates.
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The Role of PAL in the Synthesis of SA
Since incorporation of radioactivity into SA occurs when both
labeled Phe and CA are fed to leaves, the role of PAL merited closer
attention. The activity of PAL in leaf homogenates was determined and
the effect of prior application of the specific inhibitor AIP (Zon and
Amrhein, 1992) was measured. PAL activity was almost completely
inhibited at concentrations greater than 20 µM. Petiole
feeding of AIP was carried out 2 h before treatment of the leaves
with AA, corresponding to the optimal condition for inhibition in
planta. No difference in the amount of SA, either in the free or
conjugated form, was found in the presence of AIP in control leaves
after 6 h (Fig. 6A). However, the
amounts of both free and conjugated SA in leaves treated with AA in the
presence of AIP were lower than in leaves treated with AA in the
absence of AIP (Fig. 6B). Feeding CA or BA to leaves 2 h after
treatment with AA in the presence of AIP gave rise to the accumulation
of free SA, but only about 40% of the amount measured in leaves
treated with AA in the absence of AIP (Fig. 6C). This low value may be due of the fact that when fed CA or especially BA, plants rapidly produce substantial amounts of the corresponding conjugate (data not
shown), which is then unavailable for the synthesis of SA.
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| Figure 6.
Levels of free (white bars) and conjugated (gray
bars) SA in detached potato leaves 6 h after treatment with
deionized water (A) or AA (B). Leaves (B and C) were fed with AIP (20 µM) 1 h before treatment. CA (3.4 mM) or
BA (8.2 mM) was added 2 h after treatment (C). Results
are means ± SE of three replicates. FW, Fresh
weight.
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Experiments with Leaf Discs
To verify that the results obtained above were not dependent on
the labeling procedure by petiole uptake, we decided to use vacuum-infiltrated leaf discs as an alternative. Figure
7 shows that when leaf discs were treated
with AA, they produced more SA than controls (Fig. 7A). Again it was
observed that the specific activity of the SA in treated leaves was
significantly lower than in control leaves. Other preliminary
experiments showed that the leaf discs behaved similarly to the
detached leaves (data not shown) and it was therefore concluded that
the results obtained with the latter gave valid information concerning
the biosynthesis of SA and were not artifacts.
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| Figure 7.
Leaf disc experiment. Levels of free SA (A),
radiolabeled SA (B), and SA specific activity (C) were measured 6 h after AA treatment. Radiolabeled CA was vacuum infiltrated 2 h
after AA treatment. Results are means ± SE of three
replicates. FW, Fresh weight.
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DISCUSSION |
Treatment of potato leaves with AA has been shown to induce SAR
against subsequent infections with P. infestans or
Alternaria solani (Cohen et al., 1991; Coquoz et al., 1995).
Application of AA also leads to a substantial accumulation of SA that
remains limited to the treated areas of the plant (Coquoz et al.,
1995). We have now examined the biochemical pathway leading to the
production of SA after treatment with AA and compared it with the
pathway in untreated, healthy plants.
Radiolabeled SA was detected after feeding plants, both untreated and
treated with AA, with [U-14C]Phe,
[U-14C]CA, or
[U-14C]BA, indicating that the pathway from Phe
via CA and BA functions in potato. This was confirmed by the finding
that both CA and BA were labeled upon feeding Phe as a precursor. No
radiolabeled o-coumaric acid was found when using labeled
Phe or CA as precursors (data not shown), showing that the pathway from
Phe to SA proceeds mainly via BA, as is the case for tobacco
(Yalpani et al., 1993) and cucumber (Meuwly et al., 1995). However,
irrespective of the labeled precursor used, the specific activity of SA
was found to be considerably lower in the plants treated with AA than
in the untreated, control plants. The reduction was not due to an inhibition of uptake of the precursors, since in all cases the pretreatment with AA had no marked effect on the amount of
radioactivity found in the metabolites. This suggests that at least
some of the SA produced in leaves from plants treated with AA might
derive from an alternative pathway or may result in an inefficient
tissue distribution of the labeled precursors that were fed via the
petiole. To rule out the latter possibility we also carried out
experiments using vacuum-infiltrated leaf discs (compare with Meuwly et
al., 1995). Using [U-14C]CA as a precursor,
these experiments showed essentially the same dilution of specific
activity as that observed after petiole feeding (Fig. 7). Another
possibility is that in leaves treated with AA in which the pathway for
SA is activated, SA is synthesized from pools of conjugated BA or CA in
the vacuole or in the chloroplasts. The examination of samples after
acid hydrolysis of the putative conjugates did not indicate the
presence of large pools of conjugated CA or BA, but it should be
noted that the estimation of both BA and CA by HPLC using UV or
fluorescence detection is not very sensitive. This is in contrast to
the data on tobacco leaves, in which pools of conjugated BA of about
100 µg g1 fresh weight were observed (Yalpani
et al., 1993).
The inhibition of PAL by AIP with a concomitant reduction in the amount
of SA that accumulated in leaves after treatment with AA (Fig. 6B)
confirmed that this step is limiting in the synthesis of SA. There was
no evidence for a contribution to SA synthesis by a PAL-independent
pathway, e.g. directly from chorismic acid or via anthranilic acid
(data not shown). However, when CA or BA was added back to leaves
treated with AIP and AA, only a partial recovery of SA levels was
obtained (Fig. 6C) perhaps due to directing of CA to lignin
biosynthesis or to the formation of conjugates sequestered in the
vacuole. It may be argued that AIP is toxic and has an influence on the
latter stages of SA synthesis, but this is thought unlikely, since
similar concentrations of AIP had almost no effect on the synthesis of
SA when BA was added back to cucumber leaves (Meuwly et al., 1995).
Incomplete reversal of the AIP inhibition of PAL by CA or BA might also
indicate that exogenous CA or BA do not efficiently reach the cells
where the synthesis of SA occurs or that these metabolites are
preferentially conjugated. This could also explain the decrease in the
specific activity of SA observed after labeling with CA or BA. A
possible alternative way to carry out the labeling experiments could
involve using suspension-cultured cells. However, preliminary
experiments showed that although the addition of AA to the culture
medium did lead to changes, e.g. coloration of the cell walls, no
increase in the amount of SA, either free or conjugated, was observed. In addition, a number of unidentified, fluorescent compounds were detected upon HPLC (not shown). These could represent putrescine and
tyramine conjugates of hydroxycinnamic acids or 4-hydroxybenzaldehyde, since it was shown that such compounds are induced in
suspension-cultured potato cells treated with an elicitor preparation
from P. infestans (Keller et al., 1996).
In conclusion, the results presented here indicate that the pathway
from Phe via CA and BA functions both in untreated leaves and leaves
treated with AA, but that the size of the intermediate pools has a much
bigger influence than originally thought. Analogous pools in cucumber
were found to be much smaller and the turnover of the intermediates
much faster such that they were not detected upon feeding labeled Phe
(Meuwly et al., 1995). It should also be noted that healthy potato
plants accumulate a considerable amount of conjugated SA (Coquoz et
al., 1995) not found in the other healthy plants where SA biosynthesis
occurs. The synthesis of SA induced by AA clearly involves PAL and is
unlikely to take place via a PAL-independent pathway. Future studies
will be directed at elucidating the rate-limiting steps of the
biosynthesis of SA and examining more closely the pools of
intermediates.
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FOOTNOTES |
1
This work was partially supported by the Swiss
National Science Foundation (grant no. 31-34098.92).
*
Corresponding author; e-mail jean-pierre.metraux{at}unifr.ch; fax
41-26-300-97-40.
Received January 5, 1998;
accepted April 6, 1998.
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ABBREVIATIONS |
Abbreviations:
AA, arachidonic acid.
AIP, 2-aminoindan-2-phosphonic acid.
BA, benzoic acid.
CA, cinnamic acid.
PAL, Phe ammonia-lyase.
SA, salicylic acid.
SAR, systemic
acquired resistance.
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ACKNOWLEDGMENTS |
The authors are grateful to P. Meuwly and W. Mölders for
helpful discussions and are indebted to H.-R. Hohl and G. Collet for
providing the potato clone and the pathogen, respectively; to D.F.
Klessig for supplying a sample of the SA conjugate; to N. Amrhein for
supplying the AIP, and to L. Grainger and G. Rigoli for their excellent
technical help.
| |
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Abstract
Introduction