|
Plant Physiol. (1998) 118: 1345-1351
12-Oxophytodienoate-10,11-Reductase: Occurrence of Two Isoenzymes
of Different Specificity against Stereoisomers of 12-Oxophytodienoic
Acid1
Florian Schaller,
Peter Hennig, and
Elmar W. Weiler*
Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität,
D-44780 Bochum, Germany
 |
ABSTRACT |
The reduction of 12-oxophytodienoic
acid (OPDA) to
3-oxo-2(2 [Z]-pentenyl)-cyclopentane-1-octanoic acid
is catalyzed by 12-oxophytodienoate-10,11-reductase (OPR). Analysis of
the isomer preference of OPR has indicated that the activity is
composed of two isoenzymes exhibiting different stereoselectivities.
The two isoforms of OPR have been separated, using protein extracts of
Rock Harlequin (Corydalis sempervirens) as the starting material. OPRI, the enzyme reported
earlier from the same species and corresponding to the cloned OPR from
Arabidopsis, utilized
9R,13R-OPDA >>
9S,13R-OPDA but not the
13S-configured isomers, whereas the new activity, OPRII,
effectively reduced all four OPDA isomers, including the natural
9S,13S-OPDA
(cis-[+]-OPDA). OPRII activity is characterized in
detail. The enzyme's enzymatic, biochemical, and immunological
properties prove that it is a close relative of OPRI. The roles of OPRI
and OPRII in octadecanoid biology are discussed.
| |
INTRODUCTION |
The biosynthesis of octadecanoids, cyclic metabolites derived from
the C18 fatty acid  -linolenic acid (Vick and
Zimmerman, 1984 ), proceeds in three phases that occur in separate
cellular compartments. In phase I, -linolenic acid is converted to
its 13(S)-hydroperoxide, the substrate for the AOS/AOC
reaction yielding OPDA. In green tissues these reactions occur in the
chloroplast (Bell et al., 1995; Blée and Joyard, 1996; Laudert et
al., 1996; for review, see Weiler, 1997). In phase II, OPDA is reduced
to OPC-8:0 by a flavoprotein reductase, OPR (EC 1.3.1.42), a soluble, cytosolic protein that was characterized for the first time from corn
(Vick and Zimmerman, 1986), was later purified to homogeneity from Rock
Harlequin (Corydalis sempervirens; Schaller and
Weiler, 1997a), and was finally cloned from Arabidopsis
(Schaller and Weiler, 1997b). In phase III, OPC-8:0 is
subjected to  -oxidation to yield JA (Vick and Zimmerman, 1984).
Since -oxidation in plants occurs only in peroxisomes and
glyoxysomes, it is believed that phase III of the octadecanoid
biosynthetic pathway is associated with these organelles, although this
has not yet been proven.
OPR plays an important role in the biosynthesis of octadecanoids in
that the enzyme is one of the factors that determines the pool size of
OPDA and provides the substrate OPC-8:0 for the synthesis of JA.
Meanwhile, it has been shown several times that plants are able to
regulate the levels of OPDA and JA independently of each other
(Parchmann et al., 1997; Stelmach et al., 1998). Furthermore, evidence
is accumulating that OPDA is a signaling compound in its own right,
such as in White Bryony (Bryonia dioica) mechanotransduction (Weiler et al., 1994; Stelmach et al., 1998), and
that structural requirements for signaling through a JA pathway are
quite different from those required for signaling through an OPDA
pathway (S. Blechert and E.W. Weiler, unpublished data). Thus,
OPR could be a decisive factor determining the relative abundances of
OPDA versus JA in octadecanoid signaling.
OPDA extracted from tissues is the cis-(+) isomer (Laudert
et al., 1997), for which the 9S,13S configuration
has been determined at the two stereocenters (Crombie and Mistry, 1988;
Grieco and Abood, 1989; Laudert et al., 1997; Fig.
1). Some enzyme preparations, such as
flax, may yield a substantial amount of the cis-( ) isomer (9R,13R-OPDA) in addition to
cis-(+)-OPDA, and recombinant AOS, in the absence of AOC,
gives rise to racemic-cis-OPDA (Laudert et al.,
1997). Enolization leads to a slow conversion of
cis-OPDA isomers to the trans-isomers, a process
also reported for JA (Mueller and Brodschelm, 1994). It is very
difficult to analyze the in vivo situation, because thermal
isomerization occurs during GC, and a workup of the samples under
acidic conditions may lead to enolization as well. Nevertheless,
OPDA extracted from tissue is predominantly the cis isomer.
This is in contrast to the situation for JA.
From control tissue predominantly the trans isomer ()-JA
is extracted (which is at or near thermodynamic equilibrium with the
cis isomer [+]-7-iso-JA; Sembdner and Klose, 1985; Mueller and Brodschelm, 1994). When tissue is induced to produce additional JA,
such as after wounding or elicitation, the percentage of (+)-7-iso-JA relative to ()-JA increases (Mueller and Brodschelm, 1994),
suggesting that biosynthesis initially yields (+)-7-iso-JA, which then
enolizes to some extent in situ and/or during workup and analysis to
()-JA. However, it cannot be excluded at present that, whereas
(+)-7-iso-JA is being synthesized from cis-(+)-OPDA,
()-JA, at least in control tissue, may originate directly from
9S,13R-OPDA. It is obvious that this process
depends on the isomer preference of OPR. The availability of a chiral
GC-MS technique for the analysis of enantiomeric composition of OPDA
(Laudert et al., 1997) has now allowed the determination of the
stereoselectivity of OPR. It quickly became clear that the OPR activity
in a plant extract is a composite of two activities representing
enzymes of different isomer preference. These isoenzymes were separated
and their characteristics determined.
| |
MATERIALS AND METHODS |
Materials
Soybean (Glycine max L.) lipoxygenase type I-S,
-linolenic acid, and NADPH were from Sigma, and DEAE-cellulose
(DE-52) was obtained from Whatman. Immobiline DryStrip gels,
chromatofocusing gel PBE-94, and Polybuffer-74 were from Pharmacia.
Nitrocellulose was obtained from Schleicher & Schuell.
Plant Material
A cell-suspension culture of Corydalis sempervirens was
grown in Linsmaier and Skoog medium (Linsmaier and Skoog, 1965) at 25°C and 3.8 µmol photons m 2
s1 illumination on a rotary incubator (100 rpm)
for 6 to 7 d. Arabidopsis (race Columbia) plants were
grown in a greenhouse in standard soil under short days (8-h
photoperiods).
Assay of OPR Activity
Racemic cis-OPDA was obtained from
13(S)-hydroperoxylinolenic acid, as described by Laudert
et al. (1997), and purified according to the method of Graff et al.
(1990). The (+) enantiomer of cis-OPDA, 9S,13S-OPDA, was prepared using a coupled assay
of recombinant AOS and partially purified potato tuber AOC (Laudert et
al., 1997). Enantiomeric excess of 9S,13S-OPDA
over 9R,13R-OPDA in the final products was 99%.
The trans isomers were obtained from the corresponding cis forms through alkaline enolization and HPLC purification
(Hamberg and Hughes, 1988). The chemical purity, determined by GC-MS,
exceeded 98% for all octadecanoids used as the substrates or standards in this work.
For the determination of OPR enzyme activity, an appropriate amount of
enzyme preparation (300 µg of protein from crude plant extracts, 5 µg of protein containing enriched OPRI, or 13 µg of protein
containing enriched OPRII, see ``Results'') was incubated in 50 mM potassium phosphate buffer containing 0.1 mM
OPDA substrate and 1.0 mM NADPH, pH 7.5, in a total volume
of 0.5 mL. The reaction proceeded at a constant rate for at least 60 min at 25°C. Substrate consumption and product formation were
determined by capillary GC-MS, as described by Schaller and Weiler
(1997a, 1997b). To achieve this the reaction mixtures at the end of the
incubation period were acidified (pH 3.0), extracted with peroxide-free
diethyl ether, and, after removal of the ether, treated with base (1 N NaOH) for 1 h to convert the cis isomers
into their trans forms (this step was omitted when
trans-OPDA was used as the substrate). After reextraction
with ether, the octadecanoids were converted to the methyl esters by
treatment with ethereal diazomethane. The methylated fractions were
finally redissolved in CHCl3, and aliquots were
subjected to chiral capillary GC-MS (Laudert et al., 1997).
Chiral Capillary GC-MS
Separations were made on a -Dex120 column (30 m × 0.25 mm × 0.15 µm stationary-phase thickness) coated with
20% permethyl--cyclodextrin in SPB-35 (Supelco, Deisenhofen,
Germany) using a gas chromatograph (model HP 5890, Hewlett-Packard).
Mass spectra were recorded on a TSQ-7000 quadrupole mass spectrometer
(Finnigan MAT, Bremen, Germany) operated in electron-impact ionization
mode (70 eV). The carrier gas used was helium, and the column was
operated isothermically at 190°C. Details of the technique have been
reported (Laudert et al., 1997). Quantitation of substrate conversions
were based on substrate-to-product ratios derived from integrations of
reconstructed total ion current traces, as detailed by Schaller and
Weiler (1997a). These ratios are independent of absolute recoveries.
The overall absolute recovery of the procedure (from the extraction of
OPDA or OPC-8:0 from enzymatic reactions until quantitation by GC-MS) was estimated to be 85% ± 5%.
Separation of OPDA-Reductase Isoforms
Workups of tissue, ammonium sulfate fractionation, and
anion-exchange chromatography on Whatman DE-52 were performed as
described previously (Schaller and Weiler, 1997a). The protein
fractions with OPR activity obtained after anion-exchange
chromatography were pooled and the buffer exchanged to 25 mM His, pH 5.5. This material (approximately 35 mg of
protein) was loaded onto an HR 10/10 column (Pharmacia) filled with the
chromatofocusing resin PBE-94. The column was then rinsed for 10 min
with the His buffer, and proteins were eluted with 8-fold diluted
Polybuffer-74 (pH 5.5) at a flow rate of 1 mL
min1. Fractions of 2 mL (or in some cases 4 mL)
were collected for biochemical and enzymatic analyses.
Biochemical Methods
Blue native-PAGE was performed on 4% to 15% gradient gels
(Schägger et al., 1994; Oecking et al., 1997) at 4°C to 7°C
at 100 V until the sample had reached the separating gel. Separations were then carried out at 400 V. For the second dimension, the blue
native-gel strips were soaked in SDS sample buffer (>1 h) and the
denatured proteins in the gel strips were then subjected to SDS-PAGE,
according to the method of Laemmli (1970), or they were soaked in 1%
SDS and 1% -mercaptoethanol for 1 h followed by
electrophoresis, according to the method of Schägger and von Jagow (1987). For the determination of enzyme activity, proteins were
eluted from the blue native gels by incubation two times for 2 h
in 50 mM potassium phosphate buffer, pH 7.5.
The transfer of proteins from SDS-polyacrylamide gels to nitrocellulose
was achieved electrophoretically either at 4°C and 64 mA for 16 h or at 200 mA for 2 h (Towbin et al., 1979). Immunolabeling of
the OPDA reductases was performed as described by Schaller and Weiler
(1997b).
IEF of proteins was performed on precast gels (Immobiline DryStrips) in
a horizontal flat-bed chamber (Pharmacia) using the protocol of
Görg et al. (1995). For second-dimension separations, the gel
strips were attached to the sampling gel and a 12.5% separating polyacrylamide gel (Laemmli, 1970).
Protein was determined according to the method of Bradford (1976) using
BSA as a standard.
| |
RESULTS |
Chiral GC-MS of OPDA and OPC-8:0 Isomers
Both the cis and the trans enantiomers of
OPDA can be separated on a permethyl- -cyclodextrin chiral stationary
phase (Laudert et al., 1997); however, retention times for the
cis enantiomers are exceedingly long. Under the same
conditions, the cis enantiomers of OPC-8:0 were incompletely
separated (data not shown). On the other hand, the trans
enantiomers of OPDA and of OPC-8:0 could be separated on a single
column with acceptable retention times when using
permethyl--cyclodextrin as the stationary phase (Fig. 2). The identity of all compounds was
verified by full-scan electron impact mass-spectral analysis
(data not shown), and the assignments of the peaks to the enantiomeric
forms were made using either racemic or optically pure
trans-(+)-OPDA (9S,13R-OPDA) prepared from racemic cis-OPDA or cis-(+)-OPDA
(9S,13S-OPDA) by base treatment. This treatment
did not lead to any detectable racemization at C9 (data not shown). The
assignment of the OPC-8:0 enantiomers was made using standards produced
enzymatically from either racemic cis-OPDA or
9S,13S-OPDA. Again, reduction of OPDA using OPR
did not lead to racemization at C9. Thus, if cis enantiomers
of OPDA were used as the OPR substrates, conversion of the
cis to the trans isomers was carried out on
ether-extracted reaction mixtures prior to methylation and GC-MS
analysis. The rates of substrate conversion were calculated from
reconstructed ion current traces of full-scan electron impact
mass spectra using the peak areas of individual, corresponding
OPDA-OPC-8:0 pairs (total area of both peaks: 100%). Reaction rates
were calculated using several discrete data points during the linear
phase of the reaction collected during a reaction period of 1 h.
View larger version (20K):
[in this window]
[in a new window]
| Figure 2.
Separation and analysis of the enantiomers of
trans-OPDA (9R,13S-OPDA
and 9S,13R-OPDA) and of the enantiomers
of trans-OPC-8:0
(9R,13S-OPC-8:0 and
9S,13R-OPC-8:0) on a
permethyl--cyclodextrin stationary phase by capillary GC-MS. RIC,
Reconstructed total ion current. Retention times were as follows
(minutes:seconds): 9S,13R-OPC-8:0, 42:42;
9R,13S-OPC-8:0, 43:20;
9S,13R-OPDA, 46:42; and
9R,13S-OPDA, 47:54.
|
|
Evidence for the Occurrence of Isoforms of OPR Exhibiting Different
Isomer Preferences
Crude extracts from Arabidopsis leaf tissue or C. sempervirens cell cultures, when given
racemic-cis-OPDA, reduced both enantiomeric cis
forms to the corresponding OPC-8:0 enantiomers with, in each case, a
slight substrate preference for 9R,13R-OPDA (Fig.
3). OPR is a member of the OYE family of
flavoprotein reductases (Schaller and Weiler, 1997b), and these authors
showed that Saccharomyces cerevisiae OYE possessed OPR
activity. It is interesting that both S. cerevisiae crude
enzyme extracts and affinity-purified OYE prefer the
9S,13S enantiomer (i.e. the one naturally
occurring in plants) as a substrate. On the other hand, OPR purified
from the C. sempervirens cell culture, according to the
method of Schaller and Weiler (1997a), shows a clear preference for
9R,13R-OPDA over 9S,13S-OPDA as a substrate, as does OPR cloned
from Arabidopsis (Schaller and Weiler, 1997b; cf. Fig. 3). Taken
together, the data in Figure 3 suggest that in both plant species, a
second OPR isoenzyme must exist in crude extracts, which, in contrast to the purified and cloned enzymes, reduces
9S,13S-OPDA, the naturally occurring isomer.
View larger version (27K):
[in this window]
[in a new window]
| Figure 3.
Conversion of racemic-cis-OPDA by
plant and yeast enzyme preparations. Shown is the substrate consumption
of 9S,13S-OPDA (black bars) relative to
9R,13R-OPDA (white bars) after a total
reaction time of 1 h at 25°C (0.1 mM
racemic-cis-OPDA, 1 mM NADPH) during which
the reactions proceeded at a constant rate. Arabidopsis, C. sempervirens, and S. cerevisiae: 300 µg of
total soluble protein; OYE, 7.5 µg of S. cerevisiae
OYE purified by N-(4-hydroxybenzoyl)aminohexyl-Sepharose
affinity chromatography; OPDA-reductase I, 4.9 µg of purified
C. sempervirens OPR (Schaller and Weiler, 1997a);
recombinant OPDA-reductase I, 20 µg of recombinant Arabidopsis OPR
(Schaller and Weiler, 1997b) expressed in insect cells (Sf9; F. Schaller, unpublished data) and purified by
N-(4-hydroxybenzoyl)aminohexyl-Sepharose affinity
chromatography (Abramovitz and Massey, 1976). The amounts of enzyme
used for each sample were adjusted to result in comparable absolute
rates of enzymatic substrate conversion (100% = 2.8 ± 0.68 pkat).
|
|
Separation and Characterization of OPR Isoenzymes
The results obtained for the enantiomer preference of OYE (compare
Fig. 3) suggested that closely related OPR isoforms may exhibit
different substrate specificities with respect to OPDA isomers. It was
found that 9R,13R- and
9S,13S-converting activities initially copurified
when the protocol of Schaller and Weiler was used (1997a). A modified
purification scheme was therefore devised (Fig.
4A) during which separation of two OPR
activities was achieved by chromatofocusing (Fig. 4B). One activity,
eluting between 100 and 120 min, converted almost exclusively
9R,13R-OPDA and was associated with the
occurrence of a strongly immunoreactive 42-kD band using an antiserum
against C. sempervirens OPR (Schaller and Weiler, 1997a;
Fig. 4C). This activity thus corresponded to the enzyme purified
earlier (Schaller and Weiler, 1997a). A second peak of OPR activity
eluted between 60 and 75 min from the chromatofocusing gel. The
C. sempervirens OPR antiserum detected a minor
immunoreactive band at approximately 42 kD in these fractions (compare
Fig. 4C). This enzyme effectively converted both
9S,13S-OPDA and 9R,13R-OPDA to the corresponding OPC-8:0 enantiomers. The new activity is termed
OPRII activity, whereas the enzyme described earlier is now designated
OPRI.
View larger version (26K):
[in this window]
[in a new window]
| Figure 4.
Purification protocol (A), separation by
chromatofocusing (B), and immunoblot analysis (C) of C. sempervirens OPR isoforms. To determine OPR activity, 0.2-mL
aliquots of the individual 4-mL fractions were reacted under standard
conditions with either racemic-cis-OPDA ( ) or
optically active cis-(+)-OPDA
(9S,13S-OPDA) ( ). Immunoblot analysis
followed the procedure given by Schaller and Weiler (1997b). Substrate
consumption: 100% = 100 µM substrate consumed in a total
assay volume of 0.5 mL, equaling an absolute amount of 50 nmol of
substrate converted. Note that reaction rates are only linear until
about 50% of the substrate has been consumed (compare Fig. 7 for time
courses).
|
|
Protein from the OPRI and OPRII activity peaks was analyzed further by
two-dimensional gel electrophoresis using different gel systems (Fig.
5). Two-dimensional separations using IEF
as the first and SDS-PAGE as the second dimension followed by
immunoblotting (Fig. 5, top) allowed the identification of the OPRI and
OPRII polypeptides (spots in box), which were also clearly separated when the protein was analyzed prior to chromatofocusing (Fig. 5, top,
A). OPRII exhibited a slightly higher pI than did OPRI. The remaining
spots on the immunoblots represent cross-reacting polypeptides and/or
degradation products of OPR. This could be shown using blue native-gel
electrophoresis instead of IEF as the first dimension (Fig. 5, middle).
On blue native gels, OPRI and II were incompletely separated (Fig. 5A,
middle, lane a versus b versus c). The technique, however, allowed
enzymatic activity to be determined after the first-dimension
electrophoresis.
View larger version (19K):
[in this window]
[in a new window]
| Figure 5.
Gel-electrophoretic analysis of OPRI and OPRII
isoforms separated by chromatofocusing as in Figure 4B. For the
analysis, OPRI was prepared by pooling fractions corresponding to
fraction nos. 110 to 120 and OPRII corresponding to fraction nos. 62 to
70 in Figure 4B. Top, Two-dimensional analysis using IEF as the first
and SDS-PAGE as the second dimension. A, Immunoblot of the protein
fraction prior to separation of OPRI and OPRII by chromatofocusing. B,
Immunoblot analysis of the OPRI fraction. C, Immunoblot analysis of the
OPRII fraction. Middle, Two-dimensional analysis using blue native-gel
electrophoresis as the first and SDS-PAGE as the second dimension. A,
Immunoblot analysis of two-dimensional gels with SDS-PAGE as the second
dimension. Lane a, Protein fraction prior to separation of OPRI and
OPRII by chromatofocusing; lane b, OPRI fraction; lane c, OPRII
fraction. B, Blue native-PAGE analysis of the OPRII fraction. Lane a,
One-dimensional blue native-PAGE of the OPRII fraction,
Coomassie-stained gel. The asterisk marks the zone of OPR activity.
Lane b, Blue native-PAGE followed by SDS-PAGE (Laemmli, 1970)
silver-stained gel. Lane c, As in lane b, but immunoblot (only relevant
sector shown). Bottom, One-dimensional SDS-PAGE (Laemmli, 1970) using a
12.5% separating gel of the OPRI and OPRII fraction, followed by
immunoblotting. Left, Position of the nearest marker in kD. OPRI and
OPRII exhibit slightly different apparent molecular masses of 42 and
42.5 kD, respectively.
|
|
A single zone of activity was detected (Fig. 5, middle, B, lane a,
asterisk) that coincided with the major immunoreactive polypeptides in
Figure 5, middle, A. Thus, the additional immunoreactive bands
associated with the OPRI and OPRII fractions (Fig. 5, middle, A, lane a
versus b versus c) were devoid of enzymatic activity. When OPRII was
first separated from OPRI by chromatofocusing and then subjected to
blue native-gel electrophoresis, the OPRII activity zone, after
SDS-PAGE separation in the second dimension (Fig. 5, middle, B, lane
b), gave a single immunoreactive spot when the C. sempervirens antiserum raised against OPR was used (Fig. 5,
middle, B, lane c). One-dimensional SDS-PAGE of the two fractions separated by chromatofocusing followed by immunoblotting revealed OPRII
to be slightly larger (42.5 kD) than OPRI (42 kD; Fig. 5, bottom). This
difference in apparent molecular masses was also evident when the
immunoblots (Fig. 5, middle, A, lane b versus c) were compared.
Together, the data allowed us to assign OPRI and OPRII activity
unequivocally to the immunoreactive polypeptides, as identified in
Figure 5, top, A.
OPRII exhibits the same temperature optimum (35°C) but has a more
acidic pH optimum (7.5-8.0) than OPRI (8.7; Schaller and Weiler,
1997a). Like OPRI, OPRII is active as the monomer and is retained on
N-(4-hydroxybenzoyl)aminohexyl-Sepharose affinity columns (Schaller and Weiler, 1997b), a matrix originally devised to
purify yeast OYE (Abramovitz and Massey, 1976). Since
racemic-cis-OPDA had been used in earlier studies on OPRI,
it was next analyzed to determine whether the lack of conversion of the
natural 9S,13S-OPDA by OPRI was due to an
inhibitory effect of 9R,13R-OPDA. For this, racemic-cis-OPDA was incubated in the presence of increasing
amounts of OPRI. Figure 6 shows that even
when 9R,13R-OPDA is completely consumed,
9S,13S-OPDA is not utilized by the enzyme. Thus,
inhibitory effects of 9R,13R-OPDA on the
conversion of 9S,13S-OPDA can be excluded. OPRI
apparently does not utilize the natural
9S,13S-OPDA as a substrate. Likewise, no
indications for one cis enantiomer affecting the rate of
conversion of the other have been obtained for OPRII
(data not shown). The substrate concentrations giving half-maximum
rates of conversion by OPRII for the two enantiomers present in
racemic-cis-OPDA were 21 µM
(9R,13R-OPDA) and 15 µM (9S,13S-OPDA).
View larger version (15K):
[in this window]
[in a new window]
| Figure 6.
Enantiomer consumption by OPRI using
racemic-cis-OPDA as the substrate (0.1 mM)
as a function of protein concentration. ,
Racemic-cis-OPDA;  ,
9R,13R-OPDA; and  ,
9S,13S-OPDA; 100%: 25 nmol OPDA,
equivalent to quantitative conversion of cis-()-OPDA
from the racemate.
|
|
Finally, the conversions of the trans and cis
enantiomers of OPDA by OPRI or OPRII were compared using either
racemic-cis-OPDA versus 9S,13S-OPDA as
the substrates (Fig. 7, A and C) or
racemic-trans-OPDA versus 9S,13R-OPDA
(the trans isomer produced from
9S,13S-OPDA by enolization and occurring in small
amounts in plants; Fig. 7, B and D). Whereas OPRI does not convert
9S,13S-OPDA (Fig. 7C, triangles), the enzyme does
convert the corresponding trans isomer, 9S,13R-OPDA (Fig. 7D, triangles), albeit at a
lower rate compared with OPRII (Fig. 7D, squares). With respect to
these enantiomers, conversion by both enzymes was comparable
irrespective of whether optically pure substrate or the racemic
substrates were used (compare closed symbols in Fig. 7, A versus C and
B versus D). The conversion of the levorotatory cis
enantiomer 9R,13R-OPDA by OPRI and OPRII was
comparable (Fig. 7A, open symbols), whereas a sharp discrimination was
again observed for the levorotatory trans isomer,
9R,13S-OPDA, which was not readily converted by
OPRI but was effectively utilized by OPRII. Thus, OPRII is able to
utilize all four isomers of OPDA effectively, whereas OPRI is more
specific and utilizes 9R,13R-OPDA >>
9S,13R-OPDA, and the enzyme has a marginal to
zero reactivity against the two 13S-configurated OPDA
isoforms.
View larger version (24K):
[in this window]
[in a new window]
| Figure 7.
Enantiomer selectivity of OPRI and OPRII from
C. sempervirens. A, Substrate
racemic-cis-OPDA:
9S,13S-/9R,13R-OPDA.
B, Substrate racemic-trans-OPDA:
9S,13R-/9R,13S-OPDA. C,
Substrate 9S, 13S-OPDA. D, Substrate
9S, 13R-OPDA. All substrates were tested
under standard conditions at 0.1 mM. OPRI, Five micrograms
of protein corresponding to pooled fractions 110 to 120 in Figure 4B;
OPR II, 13 µg of protein corresponding to pooled fractions 62 to 70 in Figure 4B. , Conversion of
9R,13R-OPDA or
9R,13S-OPDA by OPRI;  , conversion of
9R,13R-OPDA or
9R,13S-OPDA by OPRII; , conversion of
9S,13S-OPDA and
9S,13R-OPDA by OPRI; , conversion of
9S,13S-OPDA and
9S,13R-OPDA by OPRII. A linear rate of
substrate consumption of 10% per hour corresponds to an absolute OPR
activity of 1.4 pkat. I, OPRI; II, OPRII; rac., racemic.
|
|
| |
DISCUSSION |
Our data show that two isoenzymes of OPR occur in C. sempervirens (and, by inference from the data in Fig. 3, also in
Arabidopsis). They are similar in their molecular masses, pI values,
immunological reactivities, and overall biochemical and enzymatic
properties, but they differ in their preference for OPDA isomers. The
new activity described here (OPRII) is the only one that is able to convert the natural 9S,13S-OPDA to the
corresponding 9S,13S-OPC-8:0, the precursor of
(+)-7-epi-JA. The physiological role of OPRI remains to be shown. This
enzyme does not utilize 9S,13S-OPDA as a
substrate and has a moderate activity against
9S,13R-OPDA, the trans isomer
originating from enolization of 9S,13S-OPDA. OPDA
extracted from control plant tissues is predominantly the cis-(+) enantiomer, 9S,13S-OPDA, with
a few of the corresponding trans forms (which might,
however, arise from work-up and GC analysis; Laudert et al., 1997). On
the other hand, JA isolated from control tissue is largely the
trans form, ()-JA. It seems possible that OPRI functions
in removing 9S,13R-OPDA, which might otherwise accumulate from enolization of 9S,13S-OPDA. From
this reaction product, 9S,13R-OPC-8:0, ()-JA
would be produced through -oxidation.
Another function of OPRI might be the removal of
9R,13R-OPDA. It has been shown (Baertschi et al.,
1988; Laudert et al., 1996) that AOS in vitro yields
racemic-cis-OPDA. In the presence of AOC,
9S,13S-OPDA is being formed in vitro, depending
on the activity of AOC. In some enzyme preparations, e.g. from flax, a
significant by-product of 9R,13R-OPDA is always
observed (Baertschi et al., 1988; Laudert et al., 1996). However,
cis-OPDA extracted from plant tissues is exclusively the
9S,13S enantiomer. This could mean that (a) in
vivo coupling of AOS and AOC is so effective as to avoid the formation
of 9R,13R-OPDA or (b) this enantiomer is being
removed by some other process. OPRI could be involved here.
It remains a possibility that OPRI is not an enzyme of octadecanoid
biosynthesis but in vivo has a different, yet-unidentified substrate.
This requires further work. On the other hand, the occurrence of two
OPR isoforms, only one of which would convert the endogenously
accumulating 9S,13S isomer (whereas the second isoenzyme would remove all other isomers except this one), bears some
logic in that it would allow the cell to maintain a pool of pure
9S,13S-OPDA as a precursor for (+)-7-iso-JA and
as a signal in its own right (Weiler et al., 1994; Parchmann et al.,
1997; Stelmach et al., 1998) by removing unwanted isomers originating from side reactions. Control over OPRII activity would furthermore allow the cell to generate transients of
9S,13S-OPDA accumulation without concomitant
transients in JA accumulation. Such a process has been observed
repeatedly (Parchmann et al., 1997; Stelmach et al., 1998). Full
appreciation of the roles of OPRI and OPRII will await cloning of OPRII
and the analysis of both isoenzymes in transgenic plants. This work is
in progress.
| |
FOOTNOTES |
1
This work was supported by the Deutsche
Forschungsgemeinschaft, Bonn, and Fonds der Chemischen Industrie,
Frankfurt (literature provision).
*
Corresponding author; e-mail
elmar.weiler{at}ruhr-uni-bochum.de; fax 49-234-709-4187.
Received May 11, 1998;
accepted August 25, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AOC, allene oxide cyclase.
AOS, allene oxide
synthase.
JA, jasmonic acid.
OPC-8:0, 3-oxo-2(2[Z]-pentenyl)-cyclopentane-1-octanoic acid.
OPDA, 12-oxophytodienoic acid.
OPR, 12-oxophytodienoate-10,11-reductase.
OYE, old yellow enzyme.
| |
ACKNOWLEDGMENT |
The authors wish to thank Dr. Claudia Oecking, Bochum,
for introducing them to the technique of blue native-gel
electrophoresis.
| |
LITERATURE CITED |
Abramovitz AS,
Massey V
(1976)
Purification of intact old yellow enzyme using an affinity matrix for the sole chromatographic step.
J Biol Chem
251:
5321-5326
[Abstract/Free Full Text]
Baertschi SW,
Ingram CD,
Harris TM,
Brash AR
(1988)
Absolute configuration of cis-12-oxophytodienoic acid of flaxseed: implications for the mechanism of biosynthesis from the 13(S)-hydroperoxide of linolenic acid.
Biochemistry
27:
18-24
[CrossRef][Medline]
Bell E,
Creelman RA,
Mullet JE
(1995)
A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.
Proc Natl Acad Sci USA
92:
8675-8679
[Abstract/Free Full Text]
Blée E,
Joyard J
(1996)
Envelope membranes from spinach chloroplasts are a site of metabolism of fatty acid hydroperoxides.
Plant Physiol
110:
445-454
[Abstract]
Bradford MM
(1976)
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Crombie L, Mistry KM (1988) Synthesis of 12-oxophytodienoic acid.
J Chem Soc Chem Commun 537-539
Görg A,
Boguth G,
Obermaier C,
Posch A,
Weiss W
(1995)
Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems.
Electrophoresis
16:
1079-1086
[CrossRef][ISI][Medline]
Graff G,
Anderson LA,
Jaques LW
(1990)
Preparation and purification of soybean lipoxygenase derived unsaturated hydroperoxy and hydroxy fatty acids and determination of molar absorptivities of hydroxy fatty acids.
Anal Biochem
188:
38-47
[CrossRef][Medline]
Grieco PA,
Abood N
(1989)
Cycloalkenone synthesis via Lewis acid catalyzed retro Diels-Alder reactions of norbonene derivatives: synthesis of 12-oxophytodienoic acid.
J Org Chem
54:
6008-6010
Hamberg M,
Hughes MA
(1988)
Fatty acid allene oxides. III. Albumin-induced cyclization of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid.
Lipids
23:
469-475
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Laudert D,
Hennig P,
Stelmach BA,
Müller A,
Andert L,
Weiler EW
(1997)
Analysis of 12-oxo-phytodienoic acid enantiomers in biological samples by capillary gas chromatography-mass spectrometry using cyclodextrin stationary phases.
Anal Biochem
246:
211-217
[CrossRef][ISI][Medline]
Laudert D,
Pfannschmidt U,
Lottspeich F,
Holländer-Czytko H,
Weiler EW
(1996)
Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates.
Plant Mol Biol
31:
323-335
[CrossRef][ISI][Medline]
Linsmaier EM,
Skoog F
(1965)
Organic growth factor requirements of tobacco tissue cultures.
Physiol Plant
18:
100-127
[CrossRef]
Mueller MJ,
Brodschelm W
(1994)
Quantification of jasmonic acid by capillary gas chromatography-negative chemical ionization-mass spectrometry.
Anal Biochem
218:
425-435
[CrossRef][ISI][Medline]
Oecking C,
Piotrowski M,
Hagermeier J,
Hagemann K
(1997)
Topology and target interaction of the fusicoccin-binding 14-3-3 homologs of Commelina communis.
Plant J
12:
441-453
[CrossRef]
Parchmann S,
Gundlach H,
Mueller MJ
(1997)
Induction of 12-oxo-phytodienoic acid in wounded plants and elicited plant cell cultures.
Plant Physiol
115:
1057-1064
[Abstract]
Schaller F,
Weiler EW
(1997a)
Enzymes of octadecanoid biosynthesis in plants. 12-oxo-phytodienoate 10,11-reductase.
Eur J Biochem
245:
294-299
[ISI][Medline]
Schaller F,
Weiler EW
(1997b)
Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana.
J Biol Chem
272:
28066-28072
[Abstract/Free Full Text]
Schägger H,
Cramer WA,
von Jagow G
(1994)
Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.
Anal Biochem
217:
220-230
[CrossRef][ISI][Medline]
Schägger H,
von Jagow G
(1987)
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1-100 kDa.
Anal Biochem
166:
368-379
[CrossRef][ISI][Medline]
Sembdner G,
Klose C
(1985)
()-Jasmonsäure-ein neues Phytohormon?
Biol Rundsch
23:
29-40
Stelmach BA,
Müller A,
Hennig P,
Laudert D,
Andert L,
Weiler EW
(1998)
Quantitation of the octadecanoid 12-oxo-phytodienoic acid, a signalling compound in mechanotransduction.
Phytochemistry
47:
539-546
[CrossRef][ISI][Medline]
Towbin H,
Staehelin T,
Gordon J
(1979)
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354
[Abstract/Free Full Text]
Vick BA,
Zimmerman DC
(1984)
Biosynthesis of jasmonic acid by several plant species.
Plant Physiol
75:
458-461
[Abstract/Free Full Text]
Vick BA,
Zimmerman DC
(1986)
Characterization of 12-oxo-phytodienoic acid reductase in corn.
Plant Physiol
80:
202-205
[Abstract/Free Full Text]
Weiler EW
(1997)
Octadecanoid-mediated signal transduction in higher plants.
Naturwissenschaften
84:
340-349
[CrossRef][ISI]
Weiler EW,
Kutchan TM,
Gorba T,
Brodschelm W,
Niesel U,
Bublitz F
(1994)
The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants.
FEBS Lett
345:
9-13
[CrossRef][ISI][Medline]
This article has been cited by other articles:
|
|
|
|
 
S. Cai and C. C. Lashbrook
Stamen Abscission Zone Transcriptome Profiling Reveals New Candidates for Abscission Control: Enhanced Retention of Floral Organs in Transgenic Plants Overexpressing Arabidopsis ZINC FINGER PROTEIN2
Plant Physiology,
March 1, 2008;
146(3):
1305 - 1321.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Alferez, G. Y. Zhong, and J. K. Burns
A citrus abscission agent induces anoxia- and senescence-related gene expression in Arabidopsis
J. Exp. Bot.,
July 1, 2007;
58(10):
2451 - 2462.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Hofmann, P. Zerbe, and F. Schaller
The Crystal Structure of Arabidopsis thaliana Allene Oxide Cyclase: Insights into the Oxylipin Cyclization Reaction
PLANT CELL,
November 1, 2006;
18(11):
3201 - 3217.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Breithaupt, R. Kurzbauer, H. Lilie, A. Schaller, J. Strassner, R. Huber, P. Macheroux, and T. Clausen
Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization
PNAS,
September 26, 2006;
103(39):
14337 - 14342.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Kim, E. C. Snesrud, B. Haas, F. Cheung, C. D. Town, and J. Quackenbush
Gene Expression Analyses of Arabidopsis Chromosome 2 Using a Genomic DNA Amplicon Microarray
Genome Res.,
March 1, 2003;
13(3):
327 - 340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Schaller
Enzymes of the biosynthesis of octadecanoid-derived signalling molecules
J. Exp. Bot.,
January 1, 2001;
52(354):
11 - 23.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Stintzi and J. Browse
The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis
PNAS,
September 5, 2000;
(2000)
190264497.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. M. Sanders, P. Y. Lee, C. Biesgen, J. D. Boone, T. P. Beals, E. W. Weiler, and R. B. Goldberg
The Arabidopsis DELAYED DEHISCENCE1 Gene Encodes an Enzyme in the Jasmonic Acid Synthesis Pathway
PLANT CELL,
July 1, 2000;
12(7):
1041 - 1062.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. Stra{beta}ner, A. Furholz, P. Macheroux, N. Amrhein, and A. Schaller
A Homolog of Old Yellow Enzyme in Tomato. SPECTRAL PROPERTIES AND SUBSTRATE SPECIFICITY OF THE RECOMBINANT PROTEIN
J. Biol. Chem.,
December 3, 1999;
274(49):
35067 - 35073.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Koch, T. Krumm, V. Jung, J. Engelberth, and W. Boland
Differential Induction of Plant Volatile Biosynthesis in the Lima Bean by Early and Late Intermediates of the Octadecanoid-Signaling Pathway
Plant Physiology,
September 1, 1999;
121(1):
153 - 162.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. J. Brown, J.-W. Hyun, S. Duvvuri, P. A. Karplus, and V. Massey
The Role of Glutamine 114 in Old Yellow Enzyme
J. Biol. Chem.,
January 11, 2002;
277(3):
2138 - 2145.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Stintzi and J. Browse
The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis
PNAS,
September 12, 2000;
97(19):
10625 - 10630.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction