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J. Biol. Chem., Vol. 275, Issue 25, 19132-19138, June 23, 2000
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,,
, and
From the Leibniz Institute of Plant Biochemistry, Department of
Natural Product Biotechnology, Weinberg 3, D-06120 Halle, Germany,
§ Karolinska Institute, Department of Medical Biochemistry
and Biophysics, Division of Physiological Chemistry II, S-17177
Stockholm, Sweden, ¶ Hewlett Packard GmbH, Hewlett-Packard-Straße
8, D-76337 Waldbronn, Germany, and Institute of Plant Genetics
and Crop Plant Research, Corrensstraße 3, D-06466 Gatersleben, Germany
Received for publication, March 14, 2000, and in revised form, April 5, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Allene oxide cyclase (EC 5.3.99.6) catalyzes the
stereospecific cyclization of an unstable allene oxide to
(9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has
previously been purified, and two almost identical N-terminal peptides
were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be
generated from tomato, which was further used to isolate a full-length
cDNA clone of 1 kilobase pair coding for a protein of 245 amino
acids with a molecular mass of 26 kDa. Whereas expression of the whole
coding region failed to detect allene oxide cyclase activity, a
5'-truncated protein showed high activity, suggesting that additional
amino acids impair the enzymatic function. Steric analysis of the
12-oxophytodienoic acid formed by the recombinant enzyme revealed
exclusive (>99%) formation of the 9S,13S
enantiomer. Exclusive formation of this enantiomer was also found in
wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on
chromosome 2 of tomato. Inspection of the N terminus revealed the
presence of a chloroplastic transit peptide, and the location of allene
oxide cyclase protein in that compartment could be shown by
immunohistochemical methods. Concomitant with the jasmonate levels, the
accumulation of allene oxide cyclase mRNA was transiently induced
after wounding of tomato leaves.
Jasmonic acid (JA)1 and
its methyl ester, collectively named jasmonates, consist of a
cyclopentanoic ring where an acetic acid and a pentenyl side chain are
attached (Fig. 1). These side chains are either oriented in the
cis (3R/7S) or the trans
form (3R/7R), and a number of structurally
related compounds have been described and found to occur ubiquitously
in plants (1). The first physiological role of JA found in 1971 was
inhibition of plant growth (2). Since then, jasmonates were identified
as signals of altered gene expression in various plant responses to
biotic and abiotic stresses as well as of distinct stages of plant
development (3, 4). In tomato leaves, JA was recognized as an essential
intermediate in the wound-induced signaling cascade following herbivore
attack (5), and for numerous cell suspension cultures JA was described as a signal of elicitor-induced phytoalexin synthesis (6). Beside
expression of defense genes such as proteinase inhibitors (5),
defensins (7), or thionines (8), the syntheses of phytoalexins (9),
alkaloids (10), and volatiles such as terpenoids (11) are the most
intriguing JA responses, caused in most cases by up-regulation of
specific enzymes (9, 12). JA responses were identified by means of
altered expression of specific genes, by JA-insensitive and
JA-deficient mutants, by JA-deficient transgenes, or by corresponding
endogenous rises of jasmonates including inhibitor studies. More
recently, the JA precursor
(9S,13S)-12-oxo-(10,15Z)-phytodienoic acid (OPDA) was suggested to be the preferential signal of JA-mediated responses such as tendril coiling (13) or terpenoid biosynthesis (11).
Among developmental processes, pollen maturation (14) and seedling
growth (15) are JA-dependent.
The biosynthesis of JA proceeds via an oxylipin pathway (Fig. 1),
starting with the lipoxygenase-catalyzed insertion of molecular oxygen
into position 13 of linolenic acid followed by the dehydration of
the resulting fatty acid hydroperoxide
((13S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid) by allene oxide synthase (AOS; EC 4.2.1.92) to an allene oxide
(1). This allene oxide is then cyclized by allene oxide cyclase (AOC;
EC 5.3.99.6) to OPDA. After reduction of the ring double bond by a
reductase and three rounds of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-oxidation, (+)-7-iso-JA, i.e. (3R,7S)-JA, is formed. Vick and
Zimmerman (16) already proposed a similar biosynthetic scheme in 1983, but the formation of OPDA from
(13S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic
acid was believed to be the result of a single enzyme called
hydroperoxide cyclase. In 1988, Hamberg (17) showed that this step was
performed by two enzyme activities. One of them, a membrane-bound
activity purified later as AOS (18), catalyzed the formation of an
unstable allene oxide, which rapidly decays by chemical hydrolysis with a half-life of 25 s to 
- and 
-ketol and OPDA (Fig.
1). OPDA only amounts to 10-15% of the
total degradation products and is racemic, consisting of the
cis isomers 9S,13S and
9R,13R. However, in the presence of a second,
soluble enzyme activity (AOC) that was purified recently (19), the
amount of - and -ketols decreased, and the
9S,13S enantiomer of OPDA was formed exclusively.
This specificity of AOC determines the stereochemistry of the side chains in the naturally occurring jasmonate structure.
View larger version (20K):
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Fig. 1.
Scheme of jasmonate biosynthesis. The
reaction sequence after the lipoxygenase-catalyzed formation of the
linolenic acid hydroperoxide is shown. This scheme includes the
enzymatic conversion of the allene oxide
(12,13S)-epoxyoctadecatrienoic acid to enantiomeric OPDA and
later to jasmonic acid as well as the chemical decomposition to -
and -ketol and racemic OPDA, which has, up to now, only been shown
to occur in vitro. 13(S)-HPOT,
(13S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic
acid.
In addition, the lipoxygenase-derived products can be converted by a divinyl ether synthase, a reductase, a peroxygenase, and a hydroperoxide lyase (20). Due to these facts and the unspecific ketol formation following the AOS step, the AOC can be regarded as the first enzyme specific for JA synthesis. Interestingly, correct isomeric structure of OPDA formed by AOC is kept only by one of two reductases isolated so far (21).
Several forms of lipoxygenases, AOSs, and OPDA reductases have been
cloned from different plant species and have been characterized biochemically, leading to hints of their physiological significance (21-25). So far, for AOC only biochemical data of the purified enzyme
including substrate specificity are available (19, 26). In order to
analyze the physiological importance of that step of JA biosynthesis,
we cloned AOC. Here, we describe a full-length cDNA isolated from
tomato leaves coding for a protein of 26 kDa that was localized in
chloroplasts and whose 5'-truncated version exhibited AOC activity.
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EXPERIMENTAL PROCEDURES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Plant Material--
Corn (Zea mays L. cv. Boss),
barley (Hordeum vulgare L. cv. Salome) and tomato
(Lycopersicon esculentum Mill. cv. Moneymaker) were grown in
soil under greenhouse conditions with 16 h light (with a minimum
intensity of 130 µmol·m
2·s1)
at 25 °C. Primary barley leaves were harvested 7 days after germination, cut into 5-cm segments starting 1 cm below the leaf tip,
and floated in Petri dishes on a 1 M sorbitol solution.
Developing corn seeds were harvested 2 weeks after anthesis. Tomato
plants were grown for 8 weeks, and the second leaf was excised and
wounded using a fabric pattern wheel and subsequently floated on
distilled water under continuous white light (120 µmol·m2·s1) for the
indicated times.
Analysis of Endogenous JA and OPDA-- Quantitative determination of levels of nonesterified OPDA and linolenic acid and steric analysis of OPDA were performed in unwounded leaves of tomato (15-20 g) or leaves (3-5 g) that had been wounded in situ for 0.5 h (OPDA) and 3 h (linolenic acid), respectively, by use of a hemostat. The tissue was shock-frozen in liquid nitrogen, and the powder was extracted with ethanol. [2H5]OPDA and [2H5]linolenic acid were added to a small part of the extracts, and the levels of OPDA and linolenic acid were determined by mass spectrometry. The remaining parts of the extracts were subjected to solid phase extraction and reversed-phase high pressure liquid chromatography, and the isolated OPDA was subjected to steric analysis as described by Ziegler et al. (26). JA levels were determined as described by Kramell et al. (27).
Determination of Peptide Sequences and PCR Cloning-- AOC was purified from corn seeds as described (19). The purified protein was subjected to SDS-PAGE and electroblotted on a polyvinylidene difluoride membrane. The protein band was excised and subjected to N-terminal sequencing as well as to internal sequencing after Lys C digestion by automated Edman degradation.
For PCR cloning total RNA was extracted from developing corn seeds,
barley leaves, and tomato leaves according to standard protocols (28).
The RNA of all three tissues was used in a reverse transcriptase-PCR
kit (Titan TM, Roche Molecular Biochemicals) with the following
primers: upstream, 5'-CAA GAA CTT TAC GT(A/C/G/T) TA(T/C) GA(A/G)-3';
downstream, 5'-dT23(C/G)(A/C/G/T)-3'. The following
temperature program was used: 30 min at 42 °C for the reverse
transcription reaction followed by 30 cycles of 94 °C for 1 min,
48 °C for 1.5 min, 72 °C for 2 min, and a final extension at
72 °C for 10 min. The PCR products were blunt end-ligated into pBSK
Bluescript (Stratagene) and sequenced. The clone pTomAOC4 was used as a
probe to screen 5 × 105 plaque-forming units of a
cDNA library made of 5 µg of mRNA from a tomato cell culture
using the 
-ZAP cDNA library kit (Stratagene, Heidelberg,
Germany) according to the manufacturer's instructions. Prehybridization was performed at 65 °C in 6× SSC, 5× Denhardt's reagent, 1% SDS, 100 µg/ml salmon sperm DNA for 3 h, followed by hybridization at 65 °C overnight with the 32P-labeled
insert from clone pTomAOC4 in the prehybridization solution. Filters
were washed three times at 65 °C in 2× SSC plus 0,1% SDS for 15 min each. Positive plaques were purified and converted into phagemids
by in vivo excision. Sequence analysis of plasmid and
phagemid clones was performed with the dideoxynucleotide chain termination method using the Thermo Sequenase Cycle Sequencing Kit
(Amersham Pharmacia Biotech).
Overexpression of AOC--
Either the whole coding region or a
5'-deletion starting at nucleotide 235 as seen in Fig. 2 was amplified
by PCR with a 5' NdeI and a 3' EcoRI restriction
site, respectively, and cloned into the cloning sites of the expression
vector pJC20 and pJC40 (29). The resulting plasmids were introduced
into the host strain BL21DE3(pLysS). The bacteria were grown in LB
medium up to an A600 of 0.5 absorbance units and
induced by 1 mM isopropyl--thiogalactopyranoside for
4 h. The bacteria were collected by centrifugation, subjected to
two freeze thaw cycles, and lysed by sonication in 20 mM
Tris-Cl, pH 7.5, 0.5 M NaCl, 0.1% Tween 20, and 10%
glycerol. The extracts were centrifuged, and the supernatant was used
for the determination of AOC activity. The enzyme activity was either
determined using the radioactivity assay as described by Ziegler
et al. (19) or by estimating the enantiomeric composition of
OPDA according to Ref. 26. The recombinant protein expressed in pJC40
was purified by affinity chromatography on
Ni2+-nitrilotriacetic acid-agarose (Quiagen), and the
purity was checked by SDS-PAGE. The purified recombinant AOC was used
to raise rabbit polyclonal antibodies.
Southern Analysis and Genetic Mapping-- 5-10 µg of genomic DNA were digested with restriction enzymes and separated by agarose gel electrophoresis. Southern blotting onto nylon membranes was performed as described by the manufacturer. For genetic mapping, 47 F2 plants were used from the mapping population described (30). Onto this interspecific population (L. esculentum × Lycopersicon pennellii) more than 1000 markers have been mapped previously. Since DNA from the same plants was used, the mapping data could be integrated into this map. The hybridization of the AOC gene and the integration into the mapping framework were done according to standard procedures (30).
Immunocytochemistry--
Small pieces of young leaves were
fixed, embedded in polyethylene glycol, and cut as described (31).
Cross-sections (2-µm thickness) were immunolabeled by incubation with
rabbit anti-AOC antibodies (diluted 1:2000) followed by a goat
anti-rabbit-IgG antibody conjugated with BODIPY (Molecular Probes,
Inc., Eugene, OR). After immunolabeling, sections were mounted in
citifluor/glycerol. Control experiments were performed by using
preimmune serum. The fluorescence of immunolabeled AOC was visualized
with a Zeiss "Axioskop" epifluorescence microscope using the proper
filter combination.
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Protein Sequencing and Cloning of AOC-- From the sequencing of the purified corn AOC protein (19), one internal sequence of 6 amino acids (SPAYLR) and two N-terminal peptides (AKDARPTKVQELYVYEIN and KAKDARPTKVQELYVYEI) were obtained. The latter were almost identical and only differed by one additional Lys residue at the N-terminal end. Previously, a dimeric nature of AOC was suggested by the molecular size of about 48 kDa as estimated by gel filtration compared with the migration of the AOC protein as a 22-kDa band on SDS-PAGE (19). The finding of two almost identical N-terminal peptides supports the suggestion that AOC is a homodimeric protein. Additionally, both N-terminal sequences lack the start methionine residue. This could be caused by post-translational processing, which might be necessary for dimer formation or for the transport across intracellular membranes.
The obtained peptide sequences served as templates for the generation
of oligonucleotide primers to perform a reverse transcriptase-PCR-based cloning approach for AOC. As upstream primers, degenerate
oligonucleotides directed against different regions of the N-terminal
sequence were used, whereas for the downstream primer, an oligo(dT)
anchor was chosen. Since developing corn seeds showed a high AOC
activity, we used the RNA from that tissue as a template. Irrespective
of the primer combinations and PCR conditions, no specific PCR fragment could be amplified, suggesting that either the peptide information is
not specific enough to amplify the desired product or that the mRNA
is of very low abundance or even absent in this tissue. In order to
exclude the second possibility as far as possible, we focused on
tissues, where an accumulation of endogenous JA levels can be induced,
presumably preceded by an increase in AOC expression. Therefore,
sorbitol-stressed barley leaf segments (32) and wounded tomato leaves
(33) were used as a source for RNA. No specific PCR products were
obtained with the barley RNA, but reverse transcriptase-PCR with RNA
from wounded tomato leaves resulted in a weak band of about 850 bp.
Sequencing of this PCR fragment revealed that it also encodes the
internal sequence of six amino acids obtained from the purified corn
protein. This PCR product was used as a probe to screen a tomato
cDNA library, resulting in the isolation of a 1-kilobase pair
clone. This size approximately corresponds to the size of the signal
detected on the Northern blots, suggesting that a full-length cDNA
clone was obtained. The first start codon in frame with the peptide
sequences from the purified corn AOC is located at position 47 and is
preceded by a stop codon at position 16. The protein coding region
encompasses 732 bp encoding a protein of 244 amino acids with a
calculated molecular mass of 26 kDa (Fig.
2). The difference of about 4 kDa between
the deduced molecular mass of the tomato protein and that from the
subunit of the purified corn enzyme as determined by SDS-PAGE could, in
part, be due to the post-translational removal of amino acids at the N
terminus.
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Overexpression of AOC--
In order to identify the protein
encoded by the cDNA clone as an AOC, we performed overexpression to
measure AOC activity. At first, the whole coding region was cloned into
the expression vector pJC20 and, for purification, into the His tag
vector pJC40. After induction by isopropyl--thiogalactopyranoside,
only a low expression of the recombinant protein was observed on
SDS-PAGE, but after Ni2+-nitrilotriacetic acid-agarose
chromatography of the His-tagged protein, one band of the expected size
of 26 kDa could be detected. However, neither the crude bacterial
extracts nor the purified protein showed AOC activity. Considering that
the purified protein from corn was N-terminally processed, the lack of
enzymatic activity in the full-length tomato protein could be due to
the failure of the right post-translational modification in the
bacteria. To explore this possibility, we amplified a fragment from the tomato sequence, which produces a truncated protein starting at amino
acid residue 64. This corresponds in length to the processed N terminus
of the purified corn protein. In that case, the start codon was
introduced by a NdeI restriction site. After induction of
the bacteria, SDS-PAGE revealed a prominent band of 22 kDa that was
absent in control bacteria transformed only with the empty vector (Fig.
3). The same band was also observed in
noninduced bacteria, which might be due to an insufficient repression
of the bacterial expression system in the absence of inducer. The bacterial extracts were then examined for AOC activity. As shown in
Table I, the control bacteria showed no
activity at all, whereas in induced, transformed bacteria a high
specific activity of about 15,000 nmol of OPDA/mg of protein was
detected. As expected from the protein pattern on SDS-PAGE, the
noninduced bacteria also showed AOC activity, but in this case a
10-fold lower specific activity was obtained. The AOC activity of the
recombinant protein was sensitive to proteinase K digestion and was
inhibited by 12,13-epoxyoctadecenoic acid, a specific AOC inhibitor
(19, 26), which further supports the specificity of the recombinant
protein. One special feature of the AOC reaction is the competition
between the chemical decomposition of the unstable allene oxide
substrate, giving rise to racemic OPDA and the enzymatic conversion to
enantiomeric OPDA. Therefore, the ultimate identification for a protein
as AOC can only be achieved by the proof that the enzyme specifically
forms (9S,13S)-OPDA. The steric analysis of the
reaction products showed that the recombinant protein almost
exclusively forms the (9S,13S)-OPDA enantiomer. Proteinase K digestion and the addition of 12,13-epoxyoctadecenoic acid
decreased the amount of that OPDA enantiomer to levels seen after
chemical decomposition. Altogether, the results on the AOC activity
showed that the isolated clone from tomato codes for AOC.
Interestingly, the specific activity of the N-terminal, His-tagged protein expressed in the pJC40 vector was about 10-fold lower than that
of the untagged protein. Together with the observation that only the
truncated protein was active, it seems possible that additional amino
acids at the N terminus might somehow impair dimer formation.
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Intracellular Localization of AOC--
Most enzymes of the
oxylipin pathway have been reported to reside in the chloroplast.
N-terminal amino acid sequence analysis of cloned lipoxygenase from
barley (34), Arabidopsis (35), and tomato (36) as well as
partially performed import studies (35, 36) indicated the occurrence
and function of putative chloroplast signal peptides. Also, the AOS
from flax (23) and Arabidopsis (37) carry a putative
chloroplast signal peptide, and the barley AOS co-purified with
chloroplasts (25). Biochemical data revealed the presence of the enzyme
activities of lipoxygenase, hydroperoxide lyase, and AOS in the outer
envelope membrane (38). In case of AOS and lipoxygenase, their
chloroplastic location was also shown immunocytochemically (12, 25).
Inspection of the N-terminal region of the tomato AOC also revealed
structural features for a chloroplastic signal peptide. It is highly
enriched in Ser residues (26% in the first 50 amino acids); the start
Met is followed by an Ala residue, and the first 10 amino acids are devoid of any charged residue (39). Computer analysis of the first 100 amino acids using the ChloroP version 1.1 program (available on the
World Wide Web) predicts a chloroplastic localization with a putative
cleavage site between position 93 and 94. However, this cleavage site
is highly unlikely, since the purified mature protein from corn starts
at amino acid residue 64 in the tomato sequence. Other predicted
possible cleavage sites would be at residues 41, 52, and 60. To
establish the localization of AOC experimentally, we performed an
immunocytological approach using an antibody directed against the
recombinant AOC. Cross-sections of tomato leaf tissues probed with that
antibody showed a significant fluorescence label in the chloroplasts
(Fig. 4). The autofluorescence of
chloroplasts is shown by cross-section of tissues that were treated
with preimmune serum. This confirms the data from the sequence analysis
indicating that AOC is a chloroplastic protein. In contrast to AOS,
which was co-purified with outer envelope membranes (38), AOC is a
soluble protein (17, 19). Since its substrate is highly unstable, it
seems reasonable to expect AOC to be in close proximity to AOS in order
to convert the substrate efficiently to
(9S,13S)-OPDA. In order to study this point
further, the levels and stereoconfiguration of endogenous OPDA were
determined in tomato leaves. In a typical experiment using unwounded
leaves, the levels of OPDA and nonesterified linolenic acid were 2 and 206 ng/g, fresh weight, respectively. Levels of OPDA and linolenic acid
increased to 187 ng/g (90-fold) and 1813 ng/g (9-fold), respectively, within 30 and 180 min, respectively, upon mechanical wounding, which
was in the range observed previously (40). Steric analysis of the
wound-induced OPDA showed it to be due exclusively (>99%) to the
(9S,13S)-stereoisomer. Additionally, no ketols or
racemic OPDA have been detected in plants up to now (41), suggesting that chemical degradation of the allene oxide is improbable in vivo. One may suggest, therefore, that AOS and AOC are either localized close to each other or even loosely associated. This putative
interaction of AOS and AOC is now under study using the corresponding
clones in a yeast two-hybrid system.
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Genomic Analysis and Mapping of the AOC Gene--
Southern blot
analysis with genomic DNA from tomato revealed a hybridization pattern
that is in agreement with a single gene for AOC in the tomato genome
(Fig. 5A). Clear polymorphism
was detected with several restriction enzymes in the parents of the standard tomato mapping population (30). All segregating fragments could be mapped to a single locus on chromosome 2 of tomato (Fig. 5B).
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Physiology of AOC Expression--
The accumulation of JA has been
shown to be an important part of a signal transduction cascade in
response to wounding (33, 40, 42, 43). In tomato, a chloroplastic
lipoxygenase is up-regulated upon wounding (36), and
Arabidopsis plants co-suppressed with a specific
chloroplastic lipoxygenase failed to respond with increased JA levels
upon wounding (35). Moreover, the strict spatial and temporal
regulation of the second enzyme of the pathway, AOS, during the wound
response in Arabidopsis underscores the importance of the
activation of JA biosynthetic enzymes in the accumulation of JA (24,
44). Because AOC is the enzyme that catalyzes the first committed step
in the sequence leading to JA, establishing the stereochemistry of the
naturally occurring enantiomer, we were interested in knowing whether
the expression of AOC coincides in time with the increase in JA levels
after wounding. As seen in Fig. 6, AOC
mRNA levels start to increase 30 min after wounding of tomato
leaves. The maximum induction was observed after 2 h, and at
8 h the control level was reached again. This correlated well with
the JA levels measured after wounding, which also showed a transient
accumulation with a peak at 1 h (40, 43, 45). Moreover, this
corresponds to the exclusive formation of the
(9S,13S)-stereoisomer 30 min after wounding
mentioned above. This result suggests an important physiological
function also for AOC in the regulation of JA levels during the wound
response in tomato.
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Another, highly attractive function for AOC might be in the development of floral organs. A data base search with the cloned tomato AOC revealed its identity with the tomato clone TPP15 (46). This yet unidentified clone was isolated by differential hybridization of tomato pistil cDNA libraries and was found to be highly expressed in pistils, mature petals, red fruits, and, to a lower level, in stamens. In young, developing flower buds, no expression was detected. Arabidopsis mutants shown to be defective in JA signaling, such as the coi1 mutant, or JA-deficient, such as the fad3-2 fad7-2 fad8 mutant, are both male sterile (14, 47), indicating the importance of JA in flower development. It was also shown that the AOS gene is highly transcribed in floral organs of Arabidopsis thaliana, suggesting that JA might be produced in flowers (44). This corresponds to elevated levels of JA repeatedly found in flowers (48). It will be interesting to analyze the expression of AOC in floral organs as well as during development and its correlation to the corresponding levels of jasmonates and octadecanoids.
Conclusion--
Through the pioneering work of Vick and Zimmermann
(16) and Hamberg (17), the enzymes for JA biosynthesis were elucidated. In the last decade, the characterization and cloning of these enzymes
have been greatly advanced, and clones of lipoxygenase (22), AOS (23,
25, 37), and OPDA reductases (49, 50) are already available. With the
described isolation of a cDNA clone coding for AOC, all enzymes
leading to the first physiologically active cyclopentenone, OPDA, are
now cloned. Additionally, this enzyme may be of major importance, since
it determines the stereochemistry of the cyclopentanones and has a
pivotal role in directing one of the oxylipin metabolic pathways to the
biosynthesis of the jasmonates. Using the AOC clone and those for the
other enzymes, extensive physiological studies and biotechnological
applications are now possible to reveal the participation of each of
the biosynthetic enzymes in the stress-induced or developmentally
regulated levels of JA.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Margret Köck (Martin-Luther University Halle, Germany) for providing the tomato cDNA library. We thank Silvia Wegener for technical assistance and Christine Dietel for typing the manuscript.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 363/C5, by Swedish Medical Research Council Project 03X-5170, and by Swedish Council for Forestry and Agricultural Research Project 301.0401.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ 272026.
The first two authors contributed equally to this work.
** To whom correspondence should be addressed: Leibniz Institute of Plant Biochemistry, Dept. of Natural Product Biotechnology, Weinberg 3, D-06120 Halle, Germany. Tel.: 49-345-5582-237; Fax: 49-345-5582-162; E-mail: cwastern@ipb.uni-halle.de.
Published, JBC Papers in Press, April 6, 2000, DOI 10.1074/jbc.M002133200
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ABBREVIATIONS |
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The abbreviations used are:
JA, jasmonic acid;
AOC, allene oxide cyclase;
AOS, allene oxide synthase;
-ketol, 12-oxo-13-hydroxy-(9Z,15Z)-octadecadienoic acid;
-ketol, 12-oxo-9-hydroxy-(10E,15Z)-octadecadienoic acid;
OPDA, 12-oxo-(10,15Z)-phytodienoic acid;
PCR, polymerase
chain reaction;
PAGE, polyacrylamide gel electrophoresis.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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