From the Department of Stress and Developmental
Biology, ¶ Department of Secondary Metabolism, Institute of Plant
Biochemistry, Weinberg 3, Halle/Saale D-06120, Germany,
§ Department of Molecular Cell Biology, Institute of Plant
Genetics and Crop Plant Research, Corrensstrasse 3, Gatersleben
D-06466, Germany, ** Centro Nacional de Biotecnologia CSIC, Universidad
Autonoma de Madrid, Campus Cantoblanco, Madrid E-28049, Spain, and the
Department of Medical Biochemistry and
Biophysics, Division of Physiological Chemistry II, Karolinska
Institutet, Stockholm S-171 77, Sweden
Received for publication, September 20, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Lipoxygenases are key enzymes in the
synthesis of oxylipins and play an important role in the response of
plants to wounding and pathogen attack. In cultured potato cells
treated with elicitor from Phytophthora infestans, the
causal agent of late blight disease, transcripts encoding a linoleate
9-lipoxygenase and a linoleate 13-lipoxygenase accumulate. However,
lipoxygenase activity assays and oxylipin profiling revealed only
increased 9-lipoxygenase activity and formation of products derived
therefrom, such as 9-hydroxy octadecadienoic acid and colneleic acid.
Furthermore, the 9-lipoxygenase products
9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoic and
9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic
acid were identified as novel, elicitor-inducible oxylipins in potato, suggesting a role of these compounds in the defense response against pathogen attack. Neither 13-lipoxygenase activity nor 13-lipoxygenase products were detected in higher amounts in potato cells after elicitation. Thus, formation of products by the 9-lipoxygenase pathway,
including the enzymes hydroperoxide reductase, divinyl ether synthase,
and epoxy alcohol synthase, is preferentially stimulated in cultured
potato cells in response to treatment with P. infestans
elicitor. Moreover, elicitor-induced accumulation of desaturase
transcripts and increased phospholipase A2 activity after
elicitor treatment suggest that substrates for the lipoxygenase pathway
might be provided by de novo synthesis and subsequent release from lipids of the endomembrane system.
The formation of hydroperoxy derivatives of polyunsaturated fatty
acids (PUFAs)1 represents the
first step in the synthesis of oxidized PUFAs, the oxylipins. Their
formation in plants may occur either by autoxidation or by the action
of enzymes. Enzymatic formation of fatty acid hydroperoxides in plants
is catalyzed by nonheme iron-containing lipoxygenases (LOXs, EC
1.13.11.12 (1, 2)) and by heme-containing Functional analyses in transgenic plants have shown the importance of
LOXs, the downstream enzymes, and the products of the LOX pathway in
the plant's response to wounding and pathogen attack. Thus, transgenic
Arabidopsis plants with decreased levels of 13-LOX do not
show the usual rise in jasmonic acid in response to wounding and are
deficient in wound-induced vsp transcript accumulation (8).
Increased susceptibility to insect attack was observed in transgenic
potato plants with reduced 13-LOX levels (9) and in
Arabidopsis plants that were deficient in the LOX substrate linolenic acid (10).
In addition to altered responses to wounding and insect attack, defense
against the fungal root pathogen Pythium mastophorum was
also impaired in plants with decreased levels of linolenic acid (11) as
well as in the Arabidopsis jar1 mutant, which is insensitive
to jasmonate (12). Similarly, the jasmonate-response mutant
coi1 exhibits a higher susceptibility to fungal pathogens such as Alternaria brassicicola and Botrytis
cinerea (13).
In contrast to the rather well studied products of the 13-LOX reaction,
such as hexenals, traumatin, and jasmonic acid (14, 15, 4), 9-LOX
products have only recently become the focus of attention. A possible
role in the establishment of resistance of potato against late blight,
caused by the oomycete Phytophthora infestans, has been
suggested for the 9-LOX-derived divinyl ethers colneleic and colnelenic
acids. This was based on the observation that they accumulate in potato
leaves after fungal infection and that they exhibit antimicrobial
activity (5). Recently, the efficient synthesis of isomeric trihydroxy
octadecenoates and octadecadienoates via epoxy alcohols
derived from the 9-LOX reaction has been shown to occur in potato
leaves (7). The accumulation of 9-hydroperoxy PUFAs in tobacco after
initiation of the hypersensitive response by cryptogein points to a
role of 9-LOXs in lipid peroxidation during the hypersensitive response
(16). Apart from these correlative data, the importance of 9-LOXs for
resistance was demonstrated in tobacco plants in which the
elicitor-induced increase in 9-LOX activity was inhibited by expression
of antisense constructs (17). In contrast to the resistant wild type
plants, transgenic plants were susceptible to infection with
Phytophthora parasitica var. nicotianae. Such a
conversion of an incompatible interaction into a compatible one
suggests a crucial role for the tobacco 9-LOX in conferring the
resistance phenotype.
In potato, three distinct classes of LOX cDNAs have been described,
which encode a tuber-specific 9-LOX (stlox1), possibly located in the cytoplasm, and two wound-inducible, probably
chloroplastic 13-LOXs (stlox2 and stlox3 (18,
19)). Although expression of stlox1 has been shown to be
induced in potato leaves in response to infection by P. infestans or after application of the elicitor arachidonic acid
(20), no detailed analysis has been performed addressing the question
whether individual LOX transcripts and other products of the LOX
pathway apart from the divinyl ethers (5) occur specifically in
response to pathogen attack in potato. We therefore used our model
system of cultured potato cells to determine the expression pattern of
the three LOX isoforms in response to treatment with an elicitor from
P. infestans. Furthermore, we performed oxylipin profiling
in elicitor-treated potato cells to obtain insight into enzymatic
properties of distinct LOX isoforms during conditions of pathogen
attack. In addition, enzymes and metabolites upstream from the LOX
pathway (Fig. 1) were studied and are
discussed with respect to possible functions in plant pathogen
interactions.
Infection Experiments--
Culturing of potato cells (cv.
Desirée), preparation of crude elicitor from P. infestans, and treatment of suspension-cultured potato cells with
elicitor was performed as described previously (21, 22).
Northern Analyses--
RNA from suspension-cultured potato cells
was isolated as described (22). Hybridizations to radioactively labeled
fragments of the different cDNA clones were carried out in 5×
SSPE, 5× Denhardt's solution, 0.1% SDS, 50% formamide, 100 µg/ml
denatured salmon sperm DNA. Filters were washed three times at 60 °C
with 3 × SSC, 0.1% SDS. As probes, the following cDNA
fragments were used: a 1.4-kb EcoRI fragment from
stlox1 (18), a 2.2-kb BamHI fragment from
stlox2 (19), a 1.8-kb PstI fragment from
stlox3 (19), a 0.9-kb EcoRI fragment from
stchiA (23), a 1.2-kb EcoRI fragment from
stpal (24), a 2.0-kb EcoRI fragment from
st4cl (25), a 0.95-kb fragment from sttht (22), a
0.3-kb EcoRI fragment from stpr1 (kindly provided
by C. Kistner), and a 1.3-kb BamHI fragment from
st25srRNA (kindly provided by J. Petters).
For the isolation of a PR10-specific probe, a PCR was carried out using
potato genomic DNA and two primers, 5'-GGGTGTCACTAGCTATACACATGAGACC-3' and 5'-CACTTAAGCGTAGACAGAAGGATTGGCG-3', covering the coding region of
the pr10 gene from Solanum tuberosum (GenBankTM accession
number M29041). The 800-bp PCR product was cloned into pCR2.1
(Invitrogen, Groningen, Netherlands) and verified by sequence analysis.
Amplification and Cloning of a Immunochemical Detection of Proteins--
Total protein was
extracted with 100 mM sodium phosphate buffer, pH 7.0, from
elicitor- and water-treated cultured potato cells. 40 µg of crude
protein extracts was subjected to electrophoresis on 10% denaturing
SDS-polyacrylamide gels and transferred onto nitrocellulose filters
(Schleicher & Schuell, Dassel, Germany) by electroblotting. Detection
of lipoxygenase proteins was performed using polyclonal antiserum
raised in rabbits against the N-terminal 539 amino acids of StLOX1
expressed in bacteria (18).
Determination of LOX Activity--
Determination of LOX activity
was performed with a Clark oxygen electrode as described (18). For HPLC
analysis of LOX activity, tissue extracts obtained from 0.5 g of
material were used. The tissue was extracted with 1 ml of 100 mM potassium phosphate buffer, pH 6.0. After continuous
shaking for 30 min at 4 °C, insoluble material was pelleted by
centrifugation at 10,000 × g for 10 min. Oxygenation
of linoleic acid was carried out by incubating different LOX
preparations with the substrate (120 µM final
concentration) for 30 min at room temperature. The reaction products
were extracted with three volumes of a chloroform/methanol mixture
(2:1, v/v) according to a previous study (27). After recovery of the
organic phase, solvents were evaporated by vacuo and the
lipids were reconstituted in 0.1 ml of the HPLC solvent. Analysis by
HPLC of the oxygenated linoleic acid derivatives was carried out as
described before (28, 29).
Determination of PLA2 Activity--
For
PLA2 activity measurements, 0.3 g of ground cells was
mixed with 500 µl of 0.1 M Tris-HCl, pH 7.5, 15 µl 0.1 M CaCl2, and 2 µl of [14C]PC
(L- Determination of Products of the LOX Pathway--
The analysis
of LOX-derived products from potato was performed as described (30).
The identity of colneleic and colnelenic acid was proven by adding 750 µl of methanol and 250 µl of 20% HCl to 10 µg of colneleic acid
and incubating it for 30 min at room temperature. After addition of 200 µl of DNPH, the derivatized aldehyde was analyzed as described
(31).
For the analysis of jasmonic acid, 0.5 g of tissue was essentially
extracted and derivatized as described previously (32). Gas
chromatography/mass spectrometry was performed with a Finnigan GCQ gas
chromatography/mass spectrometry system equipped with a capillary Rtx-5
column (5% diphenyl/95% polydimethyl siloxane, 30 m × 0.25 mm;
0.25-µm coating thickness; Restek, Germany). Helium was used as the
carrier gas (40 cm × s
For the analysis of trihydroxy oxylipins, frozen cells (1 g) were added
to 20 ml of ethanol containing 6.5 nmol of
[17,17,18,18,18-2H5]-11(R),12(S),13(S)-trihydroxy-9(Z),15(Z)-octadecadienoic
acid and homogenized at 0 °C for 3 min with an Ultraturrax operated at maximum speed. The mixture was extracted with two portions of
diethyl ether, and the material obtained was loaded onto an aminopropyl
Supelclean SPE tube (0.5 g; Supelco, Bellefonte, PA). Elution was
performed with 2-propanol-chlorofom (1:2, v/v), diethyl ether-acetic
acid (98:2, v/v), and methanol-acetic acid (98:2, v/v). The
last-mentioned eluate was taken to dryness, and the residue was treated
with diazomethane and trimethylsilylated. Analysis by gas
chromatography/mass spectrometry was carried out using a
Hewlett-Packard model 5970B mass selective detector connected to a
Hewlett-Packard model 5890 gas chromatograph fitted with a SPB-1701
capillary column (length, 15 m; film thickness, 0.25 mm). The
initial column temperature was 120 °C and raised at 10 °C/min
until 240 °C. Under these conditions, the retention times of the
methyl ester/trimethylsilyl ether derivatives of
9,10,11-trihydroxyoctadecadienoate, 9,10,11-trihydroxyoctadecenoate,
and deuterium-labeled standard were 14.3, 14.2, and 14.5 min,
respectively. The mass spectrometer was operated in the selected
ion-monitoring mode using the ions m/z 278 for
the deuterium-labeled standard and m/z 271 for
the two 9,10,11-trihydroxy derivatives. Standard curves were
constructed by plotting the intensities of m/z
271/278 versus the molar ratios of known mixtures of
9,10,11-trihydroxy acids and deuterium-labeled standard and used to
calculate the amounts of 9,10,11-trihydroxy acids present in samples analyzed.
stlox1 and stlox3 Expression Is Induced in Potato Cells in Response
to P. infestans Elicitor--
Potato cells grown in suspension respond
to elicitation by a crude preparation of P. infestans
culture filtrate with the activation of defense genes, for example,
those encoding enzymes of the phenylpropanoid pathway (33). To
determine the expression pattern of three stlox genes from
potato, RNA was isolated from cultured potato cells at different time
points after elicitor treatment and subjected to Northern analyses
(Fig. 2). Both stlox1 and
stlox3 mRNA levels increased transiently in response to
elicitor treatment, whereas the level of stlox2 transcripts
was below the detection limit both in control and elicitor-treated
cultures (data not shown). In different experiments, stlox1
transcripts were first detected 1 to 2.5 h after elicitation,
reaching maximal levels after 5 to 10 h. In contrast,
stlox3 mRNAs started to accumulate earlier, being
detectable already 30 min after initiation of treatment and declining
after 5 h. The time point of induction of stlox3 gene
expression is similar to that of the activation of genes encoding
enzymes of the phenylpropanoid pathway, such as PAL and 4-CL as well as
THT, as has been shown previously (33, 22). Hybridization to cDNAs
encoding PR1 or PR10 showed that expression of the corresponding genes
is induced later, i.e. after 5 h.
To determine if the increase in stlox transcript levels
correlates with higher protein levels, crude protein extracts were prepared from elicitor-treated as well as water-treated cultured potato
cells. Immunochemical analyses using a polyclonal antiserum against
StLOX1 showed higher levels of LOX protein 10 and 20 h after
elicitor treatment compared with water-treated controls (Fig.
3). Extracts prepared from cells treated
with elicitor for shorter periods, i.e. for 0, 1, 2.5, and
5 h, did not contain detectable amounts of LOX protein (Fig. 3 and
data not shown).
LOX Activity Increases in Elicitor-treated Potato Cells--
A LOX
enzyme activity assay performed with a Clark oxygen electrode revealed
higher linoleic acid-dependent oxygen consumption in
extracts of elicitor-treated cells (data not shown). In a second approach using HPLC analyses, both 13-HOD and 9-HOD were detected, but
only 9-HOD was measured to significantly higher amounts in extracts
from elicited cells. As shown in Fig. 4,
9-LOX activity started to increase 2.5 h after initiation of
treatment and reached five times higher levels after 5 and 10 h. A
specific increase in 9-LOX activity upon elicitor treatment is also
indicated by a corresponding shift in the ratio of 13- to 9-HOD from
33:67 in extracts of control cells to 7:93 after 5 and 10 h of
elicitor treatment (Table I). Chiral
phase HPLC revealed that more than 90% of 9-HOD was the S
enantiomer, indicating that this compound originated from enzymatic
conversion, whereas the racemic nature of the 13-HOD analyzed suggested
a nonenzymatic origin (data not shown).
Analysis of Elicitor-induced Changes in Oxylipin Pattern--
The
specific induction of a 9-LOX activity in elicitor-treated potato cells
was expected to be reflected in elevated levels of metabolites of the
oxylipin profile specific for corresponding downstream enzymes.
Therefore, this profile was recorded in elicitor-treated and nontreated cells.
Although hydroperoxy PUFAs survive the work-up procedure to a
significant extent (29), HPOD and HPOT levels were below the detection
limit. Analysis of the amounts of HOD, the representative of the
reductase branch, showed a 5-fold increase in 9-HOD levels in
elicitor-treated cells (Fig.
5A). 9-HOD started to
accumulate between 10 and 20 h after initiation of treatment.
Neither 9-HOT nor the 13-LOX-derived metabolites 13-HOD and 13-HOT
could be detected.
Among the divinyl ethers derived from the DES reaction, colneleic and
colnelenic acid, the derivatives of 9-HPOD and 9-HPOT, respectively,
have been reported to accumulate in potato leaves after infection with
P. infestans (5). In extracts of cultured potato cells,
colneleic acid was detectable as well. In addition to the conventional
HPLC analysis determining its characteristic UV spectrum, the
identification of colneleic acid was confirmed by acidic hydrolysis of
the collected substance and the subsequent detection of
(2E)-nonenal as its dinitrophenylhydrazone derivative as one
fragment (data not shown). Colneleic acid started to accumulate 5 h after initiation of treatment and reached maximal levels of about 680 pmol/g of fresh weight after 20 h (Fig. 5B). Colnelenic acid, the divinyl ether derived from linolenic acid, and the
corresponding derivatives from 13-HPOD or 13-HPOT, etheroleic and
etherolenic acid, could not be detected.
In potato leaves, a new branch within the LOX pathway, the EAS pathway,
has recently been described, which leads to the synthesis of trihydroxy
octadecenoates, the trihydroxy derivatives of linoleic acid (7). The
analysis of elicitor-treated cultured potato cells revealed the
elicitor-inducible accumulation of
9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate, together with a smaller increase in
9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoate, the corresponding derivative of linolenic acid (Fig. 5, C
and D). Only small amounts of trihydroxy octadecenoates and
trihydroxy octadecadienoic acid were measured in extracts of untreated
or water-treated cells. In elicitor-treated cells, accumulation started between 2.5 and 5 h after addition of the elicitor, and levels increased up to 30 h. For the trihydroxy derivative of linoleic acid, at least 10-fold higher levels were measured after 30 h (about 240 pmol/g of fresh weight), whereas the amounts of the trihydroxy derivative of linolenic acid (46 pmol/g of fresh weight) were significantly lower.
Changes in the level of other 9-LOX-derived products such as products
from the HPL branch were not detected, and, in accordance with the
absence of 13-LOX activity in elicited potato cells, no changes in
levels of 13-LOX-derived products were found. Neither jasmonic acid nor
13-HOD or 13-HOT were detected at increased levels of elicitor
treatment. Similarly, no C6-aldehydes such as
(3Z)-hexenal were detectable. Oxylipins derived from
autoxidation such as 12- and 16-HOT (31, 16) could also not be found in our model system. In summary, maximal formation of the oxylipins colneleic acid,
9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate, 9-HOD, and
9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoate was detected.
Origin of the Substrates for the LOX Reaction--
Because
oxylipin profiling showed that the major elicitor-inducible oxylipins
in potato cells are 9-LOX-derived metabolites of linoleic acid, studies
addressing the origin of LOX substrates were performed. Because StLOX1
appears to be a cytosolic enzyme, a microsomal origin of the substrates
was assumed. Therefore, a partial cDNA clone exhibiting high
similarity to
These findings indicate that linoleic acid might be synthesized
de novo within the PC fraction of the endomembrane system and may then serve as a source for LOX substrates. Therefore, we
analyzed whether PUFAs might be released specifically from this lipid
fraction to function as substrates for the observed formation of
oxylipins. Indeed, extracts from elicitor-treated potato cells
contained higher phospholipase A2 (PLA2)
activity than extracts from control cells as measured by the release of radioactively labeled linoleic acid from the sn2 position of PC (Fig.
7). PLA2 activity started to
increase as early as 2.5 h after elicitation and reached its
maximum after 20 h. Thus, both a desaturase, as concluded from its
transcript accumulation, and the specific action of a PLA2
appear to contribute to the provision of substrates for the LOX
pathway.
Oxylipins are important signaling and defense compounds in plants
whose synthesis may occur either enzymatically via the LOX pathway or by autoxidation. In the present study, the role of the LOX
pathway in plant-pathogen interactions was analyzed by parallel
recording of levels of mRNAs, proteins, enzyme activities, and
metabolites in potato cells in response to elicitor treatment. By using
this strategy of complex analyses it was shown that the 9-LOX reaction
is preferentially induced in elicitor-treated potato cells, leading to
accumulation of compounds derived from the reductase, DES and EAS
branches of the LOX pathway (Fig. 1, bold arrows).
Although 13-LOX expression is induced upon pathogen infection in a
number of plants (i.e. in rice (35), wheat (36, 37), or
broad bean (38)), accumulation of transcripts corresponding to
9-LOX-encoding cDNAs has so far only been reported to take place in
the solanaceous plants tobacco and potato in response to infection with
oomycetes or to treatment with oomycete-derived elicitors (39, 40, 20).
Here, we show further that, concomitant with the accumulation of
transcripts corresponding to the 9-LOX-encoding cDNA
stlox1, higher 9-LOX activity and increased levels of 9-LOX products can be measured after elicitor treatment in potato cells.
In contrast to the large amount of data available for 13-LOX products,
until recently not much was known about the products of the 9-LOX
reaction. Weber et al. (5) reported the pathogen-induced accumulation of colneleic and colnelenic acid, divinyl ethers derived
from 9-HPOD and 9-HPOT. Here, we show that colneleic acid also
accumulates in suspension-cultured potato cells upon treatment with
P. infestans elicitor, reaching maximal levels of 600 pmol/g of fresh weight and being the most prominent oxylipin in the present study. Thus, with respect to 9-LOX products, cultured potato cells react to P. infestans-derived elicitors qualitatively
similar as P. infestans-infected leaves.
The EAS from potato leaves described recently (7) has contributed
to another possible function for pathogen-induced 9-LOXs. In this step,
the synthesis of trihydroxy PUFAs is initiated. For 13-HPOD- and
13-HPOT-derived trihydroxy PUFAs, which are synthesized in
rice leaves infected with Magnaporthe grisea,
antifungal activity has been demonstrated (41). Similarly, trihydroxy
oxylipins produced from 9-HPOD or 9-HPOT via epoxy alcohols
might play a role in defense reactions as well. The specific
accumulation of 9-HPOD- and 9-HPOT-derived trihydroxy oxylipins upon
elicitor treatment of potato cells supports the idea that these
compounds might act via their antifungal activity in plant
defense reactions.
Despite these observations, the role that 9-LOXs and products of the
9-LOX pathway could play in resistance is still not elucidated. In the
interaction of potato plants with specific races of P. infestans, 9-LOX-derived divinyl ethers accumulated faster and to
higher levels in leaves of resistant potato plants than in susceptible
ones (5), whereas 9-LOX gene expression was reported not to be
significantly different in resistant and susceptible plants (20). This
suggests that accumulation of divinyl ethers in resistant plants is
regulated at the level of substrate availability or DES activity. On
the other hand, the necessity of a 9-LOX for the resistance reaction is
demonstrated by the conversion of an incompatible to a compatible
interaction in transgenic tobacco by antisense inhibition of the
elicitor-induced expression of the tobacco 9-LOX (17).
LOX products have been shown to play a role in the plant's response to
biotic and abiotic stresses both as antimicrobial compounds and as
signal molecules that lead to the activation of specific defense genes
(42, 43, 44). The role of colneleic and colnelenic acid is presumably
that of antifungal substances, because inhibition of spore germination
was demonstrated (5). Also, polyhydroxy fatty acids and aldehydes have
antimicrobial activity (41, 45). On the other hand, products of the LOX
pathway might function as signaling compounds, as has been shown for
jasmonic and 12-oxo-phytodienoic acids in the plant's response to
wounding and insect attack (15, 46) and for LOX-derived C6
volatiles, which are able to induce a specific subset of pathogen
defense genes in Arabidopsis (47). Recently, 13-HOT was
shown to be able to activate pr1 expression in barley (30).
However, addition of 9-HPOD, 9-HOD, or colneleic acid to potato cells
did not lead to accumulation of pr1, pr10, pal, 4-cl, stlox1, and
stlox3 transcripts (data not shown), suggesting that these
compounds do not act as signal molecules in potato cells.
For LOXs from potato, so far only data on the expression of genes
corresponding to the tuber-specifically expressed stlox1 sequences are available. Fidantsef and Bostock (20) demonstrated the
inducibility of stlox1 gene expression in potato leaves
after infection with P. infestans. The stlox3
mRNA accumulation, shown to occur specifically after elicitor
treatment, was not accompanied by a corresponding increase in 13-LOX
activity or 13-LOX products. This strengthens the notion that more than
the profile of mRNA accumulation is necessary to describe altered
metabolic activities.
In contrast to the potato cells described here, cultured cells of a
number of other plant species respond to treatment with pathogen-derived elicitors with the accumulation of jasmonic acid and
its precursor, 12-oxo-phytodienoic acid (48). In particular, treatment
of tobacco cells with an elicitor derived from P. parasitica var. nicotianae results in a rapid and transient increase in
jasmonic acid levels (49). Because we could not measure 13-LOX activity in extracts of two independently generated potato suspension cultures, we do not think that the failure to detect 13-LOX activity or products
is an artifact of the specific cell culture. Interestingly, no
significant changes in jasmonic acid levels were detected in P. infestans-infected potato leaves 3 days postinfection (5), raising
the possibility that infection with the oomycete P. infestans or treatment with P. infestans-derived
elicitor does not activate the 13-LOX pathway.
The availability of sufficient amounts of substrate seems to play a
crucial role in the activation of the LOX pathway as tobacco plants
overexpressing different LOX genes show no or only moderately elevated
levels of LOX-derived metabolites (50, 51). Because linoleic
acid-derived oxylipins were the major derivatives detected in
elicitor-treated potato cells, the linoleic acid pool was analyzed in
two different ways: (i) by studying mRNA accumulation of
Taken together, our results suggest that, in cultured potato cells,
elicitor treatment leads to activation of PUFA-generating enzymes,
stimulation of the 9-LOX pathway, and subsequently, to increased
formation of products of the DES, EAS, and reductase branch of the LOX
pathway. The function of these compounds for the response of potato to
pathogen attack will be analyzed by gain- and loss-of-function studies
in transgenic plants.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dioxygenases (3). In
plants, 9- or 13-LOXs have been identified based on their positional
specificity in introducing molecular oxygen into linoleic acid or
linolenic acid. Their products, the hydroperoxy PUFAs hydroperoxy
octadecadienoic acid (HPOD) and hydroperoxy octadecatrienoic acid
(HPOT), are substrates of different enzymes within the LOX pathway (see
Fig. 1). A peroxygenase and a reductase catalyze the synthesis of
hydroxy octadecadienoic acid (HOD) or hydroxy octadecatrienoic acid
(HOT) (4), whereas the activity of divinyl ether synthase (DES) leads
to formation of vinyl ether containing PUFAs such as colnele(n)ic and
etherole(n)ic acids (5). The synthesis of the signaling compound
jasmonic acid originates from 13-HPOT by the activity of an allene
oxide synthase (EC 4.2.1.92), whereas a hydroperoxide lyase (HPL) catalyzes the formation of
-oxo fatty acids and aldehydes (6). Trihydroxy octadecenoates are synthesized from linoleic acid-derived epoxy alcohols via epoxy alcohol synthase (EAS) and epoxy
alcohol hydrolase (7). Finally, LOX itself can catalyze the synthesis of keto PUFAs.
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Fig. 1.
Metabolic routes for
LOX-dependent catabolism of PUFAs in plants. The
preferential route described here for elicitor treatment of potato cell
cultures is indicated by bold arrows.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-Acyl Lipid
Desaturase cDNA Fragment from Potato--
RNA from potato cells
treated with elicitor for 5 h was used in an RT-PCR reaction using
the One-Tube-RT-PCR beads from Amersham Pharmacia Biotech (Freiburg)
with the following degenerate primers: 5'-TGG GTI AWH GCH CAY GAR TGB
GG-3' and 5'-CCA RTY CCA YTC IGW BGA RTC RTA RTG-3' ((26) MWG Biotech,
Ebersberg). The PCR product was cloned into the pCR2.1 vector using the
TA cloning kit (Invitrogen, Groningen, Netherlands) and sequenced on a
LI-COR 4200 (MWG Biotech). The partial cDNA clone had an insert of
579 bp with 97% similarity to a
12-desaturase cDNA
from Solanum commersonii (GenBankTM accession number
X92847).
-1-palmitoyl-2-linoleoyl-[linoleoyl-1-14C]-phosphatidylcholine;
PerkinElmer Life Sciences, Boston, MA) and incubated for 20 min at room
temperature. After addition of 75 µl of acetic acid, two chloroform
extractions were performed. After evaporation, the samples were
subjected to thin-layer chromatography with
chloroform:methanol:H2O (65:25:4) as solvent.
14C-Labeled linoleic acid and 14C-labeled PC
incubated with PLA2 (Sigma, Munich, Germany) were used as controls.
1). An electron energy of 70 eV, an ion source temperature of 140 °C, and a temperature of
275 °C for the transfer line were used. The samples were measured in
the NCI mode using ammonia as reactant gas, and the splitless injection
mode (opened after 1 min) with an injector temperature of 250 °C was
used. The temperature gradient was 60-180 °C at 25 °C
min
1, 180-270 °C at 5 °C min
1,
270 °C for 1 min, 270-300 °C at 10 °C min
1, and
300 °C for 25 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Gene expression in cultured potato cells
after treatment with P. infestans elicitor. RNA
was isolated from cultured potato cells treated with a crude elicitor
preparation of P. infestans (P.i.-cf)
or H2O at the time points indicated (h),
separated on formaldehyde gels, blotted onto nylon membranes, and
hybridized with radioactively labeled probes derived from the cDNA
encoding LOX1, LOX3, PR1, PR10, PAL, 4-CL, THT, and, for
standardization, with a radioactively labeled rRNA probe.
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Fig. 3.
LOX protein in elicitor-treated potato
cells. Protein was extracted from cultured potato cells after
treatment with elicitor (P.i.-cf) or H2O 0, 5, 10 and 20 h after treatment. Immunochemical analysis was performed
with antisera raised against the N-terminal 549 amino acids of
StLOX1.
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Fig. 4.
LOX activity in elicitor-treated potato
cells. 9-LOX activity was determined in protein extracts from
cultured potato cells after treatment with elicitor (black
bars) or water (white bars) at the time points
indicated.
Relative levels (%) of 9- and 13-HOD in elicitor (P.i.-cf) or
water (H2O)-treated potato cells
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Fig. 5.
Metabolic profiling of oxylipins in
elicitor-treated potato cells. Levels of 9-HOD (A),
colneleic acid (B), and
9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoate
(C), and
9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoate
(D) were determined in extracts of cultured potato cells
after treatment with elicitor (black bars) or water
(white bars) at the time points indicated. Data shown are
the means of two (A) and three independent experiments
(B, C, and D), respectively.
12-acyl lipid desaturases, which catalyze
the formation of linoleic acid from oleic acid within the sn2 position
of phosphatidylcholine (PC) within the endoplasmic reticulum (34), was
isolated from potato RNA. Expression analyses revealed that the
corresponding transcripts accumulated in response to elicitor treatment
(Fig. 6). The first increase was observed
30 min after initiation of treatment, and maximal transcript levels
were obtained after 5 h.
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Fig. 6.
Expression of
12-acyl lipid desaturase-homologous
genes in potato cells. RNA was isolated from cultured potato cells
treated with a crude elicitor preparation of P. infestans
(P.i.-cf) or H2O at the time points
indicated (h), separated on formaldehyde gels, blotted onto
nylon membranes, and hybridized with a radioactively labeled probe
derived from the partial cDNA clone encoding a protein with
similarity to
12-acyl lipid desaturases and with an rRNA
probe.
View larger version (86K):
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Fig. 7.
PLA2 activity in elicitor-treated
potato cells. PLA2 activity was detected by thin-layer
chromatography of extracts from potato cells treated with a crude
elicitor preparation of P. infestans (lanes 1-6:
1, 2.5, 5, 10, 20, 30 h after treatment) or H2O
(lanes 7-12: 1, 2.5, 5, 10, 20, 30 h after treatment).
Lanes 13 and 14 show PLA2 activity in
boiled extracts of water (lane 13) and elicitor-treated
(lane 14) potato cells, lane 15 contains
14C-labeled linoleic acid, lane 16 contains
14C-labeled PC. and lane 17 contains
14C-labeled PC incubated with PLA2.
Arrows indicate the position of linoleic acid
(LA), PC, and the site of sample application
(S).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-desaturases, which catalyze the formation of linoleic
acid from oleic acid at the sn2 position of PC within the endoplasmic
reticulum, and (ii) by measuring activity of PLA2, the
major enzyme responsible for release of these PUFAs into the cytosol.
Indeed, RNA analyses revealed an induction of
12-desaturase transcript accumulation, suggesting that
de novo synthesis of linoleic acid might occur upon
elicitation in potato as has been suggested for elicitor-induced
linoleic and linolenic acid formation in parsley (52, 53). Although we
did not detect significant changes in the levels of free PUFAs in
elicitor-treated potato cells (data not shown), the activation of a
PLA2 upon elicitation suggests that the substrate for the
synthesis of linoleic acid-derived oxylipins in potato might be
liberated from PC to serve as a substrate for StLOX1.
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ACKNOWLEDGEMENTS |
---|
We thank Angelika Weinel for excellent
technical assistance and Heiko Weichert for both his technical support
and for providing us with 13-HOT as an internal standard. Dr.
Catherine Kistner and Julia Petters are acknowledged for providing the
pr1 and 25SrRNA cDNA clones. We are grateful to Prof.
Dr. Claus Wasternack for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Molecular Biology,
Max-Planck-Institute of Chemical Ecology, Carl-Zeiss-Promenade 10, Jena
D-07745, Germany.
§§ To whom correspondence should be addressed: Tel.: 49-345-55821440; Fax: 49-345-55821409; E-mail: srosahl@ipb-halle.de.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M008606200
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ABBREVIATIONS |
---|
The abbreviations used are: PUFA, polyunsaturated fatty acid; 4-CL, 4-coumarate:CoA ligase, EC 6.2.1.12; DES, divinylether synthase; EAS, epoxy alcohol synthase; HPL, hydroperoxide lyase; LOX, lipoxygenase, linoleate:oxygen oxidoreductase, EC 1.13.11.12; HOD, hydroxy octadecadienoic acid; HOT, hydroxy octadecatrienoic acid; HPOD, hydroperoxy octadecadienoic acid; HPOT, hydroperoxy octadecatrienoic acid; PAL, phenylalanine ammonia-lyase, EC 4.3.1.5; PC, phosphatidylcholine; PLA2, phospholipase A2, phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4; PR1 and PR10, pathogenesis-related proteins 1 and 10; THT, hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase, EC 2.3.1.110; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; RT, reverse transcriptase; HPLC, high pressure liquid chromatography.
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