From the Lehrstuhl für Pflanzenphysiologie,
Ruhr-Universität, D-44780 Bochum, Germany and the
¶ Institut für Pharmazeutische Chemie, Johann Wolfgang
Goethe-Universität, D-60439 Frankfurt am Main, Germany
Received for publication, November 29, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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The cyclic derivative of
13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid,
serves as a signal transducer in higher plants, mediating
mechanotransductory processes and plant defenses against a
variety of pathogens, and also serves as a precursor for the
biosynthesis of jasmonic acid, a mediator of plant herbivore defense.
Biosynthesis of 12-oxophytodienoic acid from Oxylipins are a diverse class of acyclic or cyclic oxidation
products of fatty acids, many of which have regulatory functions in the
cell (1). Among the plant oxylipins, the cyclic octadecanoids, collectively called jasmonates, have received much recent interest because of their diverse biological functions as mediators of mechanotransduction, herbivore and pathogen defense reactions, senescence, and male fertility (2-4).
Jasmonate biosynthesis is thought to start with induced release of
It is known that plant lipoxygenases may act on glycerolipids as well
as on free fatty acids (13 and references therein). This prompted us to
survey plant membrane lipids for the presence of cyclic octadecanoids.
As a result of this study, we report here that the major fraction of
OPDA in Arabidopsis thaliana occurs esterified in the
sn1 position of plastid-specific galactolipids. The
implications of this novel class of glycerolipids for octadecanoid physiology and chloroplast membrane biology are discussed.
Plant Material--
Greenhouse-grown rosettes of 6-8-week-old
A. thaliana C24 were used for all experiments. Plants were
raised individually in standard soil at 18 °C (night) and 24 °C
(day) (average temperatures) between 50 and 70% relative humidity and
no less than 150 µmol of photons m Wounding Experiments--
Wounding experiments were carried out
as previously described (14). Briefly, all leaves of a rosette were
crushed on one half of the leaf blade with a hemostat so that ~10%
of the total leaf area of the plant was wounded. After incubation for
the times indicated, 1 g of leaf tissue was collected using the
whole leaf blades of wounded leaves and analyzed for OPDA and JA
content as described in detail in Ref. 15, using
[2H5]OPDA and
[13C2]JA (vide infra) as internal
standards. Extraction (16) and quantification of lipid-bound OPDA were
done as described above.
Preparation of Protein Extracts--
Leaves of A. thaliana were homogenized in a mortar with pestle in 12% sucrose,
1 mM EDTA, 10 mM Tricine, pH 7.5, 10 mM KCl, 1 mM MgCl2, 10 mM Na2S2O4, 0.1%
bovine serum albumin using 1 ml of buffer per g of fresh weight. The
homogenate was centrifuged at 10,000 × g, and the
supernatant was used to analyze the enzymatic degradation of OPDA. To
do this, to 1 ml of the protein extract (1.5 mg of protein), 1 µg/ml
[2H5]OPDA and
[13C2]JA were added, and the assay mixture
was then incubated at room temperature. Aliquots were taken at the
times indicated and worked up for the analysis of OPDA and JA.
Lipid Extraction and Purification--
Initial experiments were
carried out using the standard procedure of Bligh and Dyer (16). It was
later found that yields and purity of the glycolipid fraction improved
when the following preparative procedure was employed. Leaf
tissue (500 g) was immersed in 600 ml of boiling methanol containing,
as an antioxidant, 200 mg of
2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene). After boiling for 15 min, the hot mixture was homogenized with a
Polytron mixer, the homogenate was filtered on a Buechner funnel and
then over two sequential paper filters, and the methanol was removed by
vacuum distillation (25 mbar, 30 °C). The aqueous phase was
adjusted to 70% (v/v) methanol (crude lipid extract).
The crude lipid extract was passed over a column of 30 g of
LiChroprep RP-18 (Merck). The column was eluted with 150 ml
isopropyl alcohol: isohexane (80:55, v/v) and further
fractionated on Chromabond NH2 (Macherey-Nagel; aliquots of
one-eighth of the LiChroprep RP-18 eluate passed over 3 g of
column material that was eluted with 10 ml of the above solvent). The
effluents and eluates of the Chromabond NH2 column were
pooled, the lipid extract was concentrated in a rotary evaporator (25 mbar, 30 °C), and the crude lipid was finally dried in a stream of
N2 (yield, ~11.3 mg from 500 g of leaf tissue). The
residue was stored in 4-6 ml of chloroform:methanol (1:1, v/v) at
The dried crude lipid was redissolved in 4-5 ml of
i-propanol:i-hexane (4:3, v/v) and centrifuged to
remove particles. Aliquots (1 ml) of the clear solution were injected
onto a semipreparative Nucleosil 100 HPLC column (Knauer; 25 mm × 8 mm inner diameter, particle size 15-25 µm) and eluted at a
flow rate of 5 ml min Analysis of 12-Oxophytodienoic Acid--
To analyze the isomeric
composition, optical purity, and quantity of OPDA, the methods
described in detail previously, based on GC-MS, were employed (15, 19, 20,). Pentadeuterated OPDA
(17[2H2],18[2H3]-cis-OPDA),
synthesized according to Ref. 19, was used as an internal standard in
all experiments.
Analysis of Jasmonic Acid--
If desired, JA was quantitated by
GC-MS concurrently with OPDA using the same procedures and samples and
an internal standard of [13C2]jasmonic acid.
[13C2]Jasmonic acid was synthesized according
to Ref. 21 as follows. [13C3]Malonic acid (49 mg, >99 atom % 13C, Isotec Inc.) was converted to its
dimethyl ester using CH2N2 and dissolved in 50 µl of 2-(2Z pentyl)-2-cyclopenten-1-one (Firmenich SA,
Geneva, Switzerland), followed by the addition of 170 µl of 0.2 M sodium methanolate. The mixture was stirred for 1.5 h under nitrogen at room temperature. The reaction was terminated with concentrated acetic acid (4 µl), and the sample was dried with nitrogen, redissolved in doubly distilled water (150 µl), and hydrolyzed under nitrogen for 18 h at 225 °C. After cooling,
the reaction mixture was diluted into 0.1 M
NaHCO3 (2-3 ml) and extracted twice with chloroform, and
the reaction product (racemic
[13C2]methyljasmonate) recovered from the
chloroform phase was treated for about 2 h at 60 °C with 0.5 M KOH to release the free acid, which was purified by HPLC
as described in Ref. 15 (yield, ~30%; purity, >99%; isotopic
abundance, >99% as analyzed by GC-MS).
Mass Spectrometry of Lipids--
Routine analyses of fatty acid
and OPDA methyl esters were performed on a Finnigan MAT Magnum
ion trap mass spectrometer in chemical ionization (reactant gas,
methanol) mode (scan range, 50-398 amu; scan rate, 1 Hz). GC settings
were as follows: DB-35 column (J & W Scientific, 30 m × 0.25 mm × 0.25 µm coat), helium carrier gas, splitless injection
mode (250 °C), and temperature program of 1 min at 80 °C,
30 °C min
LC-MS analyses were performed on a triple-quadrupole instrument (TSQ
7000, Finnigan MAT) operated in electrospray (positive or
negative ion) mode (4.5 kV; capillary temperature, 200 °C; atmospheric pressure ionization-collision-induced dissociation voltage,
15 V). The compounds to be analyzed were introduced (injection volume,
5 µl) through a Luna C18 HPLC column (Phenomenex; 150 mm × 1 mm inner diameter, 5 µm particle size) using
methanol, 20 mM ammonium acetate (90:10, v/v) as
eluent (isocratic flow rate of 50 µl min Nuclear Magnetic Resonance Spectroscopy--
The purified
OPDA-containing lipid (~20 mg) was dissolved in 0.7 ml of
CD3OD. NMR spectra were recorded with Bruker AC 200 and AMX
500 (5-mm inverse TXI probe head) instruments. NMR conditions were as follows: H-H COSY, 450 mixing pulse; HMQC,
phase-sensitive mode using time-proportional phase increment, bilinear
rotation decoupling sequence, globally optimized
alternating-phase rectangular pulses decoupled; HMBC, phase-sensitive
using time-proportional phase increment, delay tuned to long range
couplings, 71 ms.
Enzymatic Conversion of Monogalactosyl Diglyceride
(MGDG)--
MGDG was isolated from Brassica napus according
to Bligh and Dyer (16). After drying, the crude lipid was purified as
described above using solid phase extraction and semipreparative HPLC
(Nucleosil 100, 12-14-min fraction). The identity of the isolated MGDG
was verified by electrospray ionization-MS. The presence of the
18:3/16:3 molecular species was indicated by the molecular ion [M + NH4]+; m/z = 764.61. The purified MGDG (10 mg) was treated with soybean lipoxygenase
(1 mg of protein) and recombinant allene oxide synthase (2 mg of
protein representing total soluble protein from Escherichia coli lysates from cells expressing the A. thaliana
enzyme (16, 19)) in 50 mM potassium phosphate, pH 8.0, at
room temperature for 6 h. Thereafter, the reaction mixture was
reextracted (16), and the lipids were hydrolyzed for 1 h in 0.5 N KOH (100 °C). The released acyl moieties were analyzed
by GC-MS (19). To monitor the functionality of the enzymes used, the
reaction mixture contained, in addition to plant MGDG,
[2H5]linolenic acid. This allowed us to
monitor in parallel by GC-MS the conversion of the esterified
General and Biochemical Procedures--
The presence and
quantity of lipid-bound (esterified) OPDA was determined by lipase
treatment of the corresponding fractions using an adaptation of the
procedure described in Ref. 18, followed by GC-MS analysis (see above)
of the released metabolites (OPDA, fatty acids) as their methyl esters.
OPDA was released by the sn1-selective lipase from R. arrhizus (triacylglycerol lipase, EC 3.1.1.3, Roche
Molecular Biochemicals) as follows. To each fraction to be analyzed was
added an internal standard of
cis-[2H5]OPDA (250 ng), and the
sample was dried in a stream of nitrogen. The residue was then
redissolved in 1 ml of 50 mM Tris borate (pH 7.5). To this
solution were added 25 units of lipase in 50 µl of 100 mM
Tris buffer (pH 7.5); controls received only buffer. The samples were
then incubated at 37 °C (for the times indicated or overnight, if
reactions were to go to completion), acidified (20 µl of concentrated
HCl), and extracted two times with 2 ml of ethyl acetate
followed by standard workup (20). In preparative experiments aimed at
purification of the OPDA-lipid, no internal standard
([2H5]OPDA) was used.
Additional lipases tested were those from Candida rugosa
(Sigma), Mucor javanicus (Sigma), and wheat germ (Sigma),
and phospholipases tested were the phospholipases A2
(EC 3.1.1.4) from Apis mellifica (Sigma) and
Naja mossambica (Sigma). All lipases were assayed at pH 7.5;
the two phospholipases A2 were assayed at pH 8.9. Alkaline hydrolysis of lipids was for 1 h at 100 °C in
0.5 M KOH, followed by acidification and further workup as above.
Discovery of Membrane Lipid-bound 12-Oxophytodienoic
Acid--
During the course of studies on OPDA metabolism in
A. thaliana, it was noticed that
[2H5]OPDA added to leaf crude extracts was
rapidly metabolized, whereas metabolism of endogenous (i.e.
unlabeled) free OPDA followed a completely different time course; over
at least 1 h, its level actually increased, before it started to
decline also (Fig. 1). The pool from
which this OPDA was released in crude extracts could be localized to
the membrane fraction (100,000 × g pellet), whereas the 100,000 × g supernatant harbored the
OPDA-metabolizing activity. When crude lipid from A. thaliana leaves obtained through Bligh and Dyer extraction (16)
was subjected to alkaline hydrolysis (saponification conditions), it
released OPDA. Control reactions verified that this OPDA fraction was
not just dissolved in the lipid phase but covalently bound in an
alkali-labile manner, i.e. most likely esterified (Fig.
2).
To scrutinize these initial results, the crude lipid was subjected to
treatments with various lipases including phospholipases of the
A2 type (Fig. 3). Whereas
there was no (or marginal) release of OPDA by phospholipase
A2 as well as by the wheat germ lipase, the two
lipases that exhibited a preference for the sn1 position of
glycerolipids (the fungal enzymes from R. arrhizus and
M. javanicus (18)) efficiently released OPDA from the crude
lipid fraction. It was estimated that ~80-90% of the total OPDA
content of A. thaliana shoots occurred covalently bound in
the lipid fraction in plants not subjected to any stimulus inducing the
jasmonate signaling system. The endogenous activity that released OPDA
from the lipid-bound form was associated with the 5,000 × g pellet but was not detected in the 12,000 × g supernatant of crude extracts and, thus, most likely
represents a membrane-associated system. This activity was not
characterized further in the present study.
Analysis by chiral GC-MS of the isomeric status of OPDA released from
the lipid revealed that the lipase-released metabolite was the
cis(+) enantiomer,
(9S,13S)-12-oxophytodienoic acid, the same
enantiomer that is also extracted in free form from plants (19, 20).
Alkaline hydrolysis, of course, yielded predominantly the trans isomer
(9S,13R)-OPDA), which is produced from the cis form by enolization, particularly under alkaline conditions. The method
of analysis of OPDA enantiomers has been described in detail elsewhere
(19).
Purification and Structural Characterization of the
Lipase-degradable, Membrane-associated OPDA-Lipid--
The solubility
of the OPDA-lipid in aqueous methanol proved optimum around 80% (v/v),
whereas free OPDA is most soluble around 50% (v/v) methanol. Thus,
although less polar than the free acid, the OPDA-lipid turned out to be
a remarkably polar metabolite that could be extracted from the plant
material more selectively with aqueous methanol (70% v/v) than with
conventional, less polar, lipid-solvent mixtures
(e.g. see Ref. 16). Using the procedure detailed
under "Experimental Procedures," from 600 mg of crude lipid
(corresponding to 26.5 kg of leaf tissue), 20 mg of an apparently homogenous OPDA-lipid fraction was obtained for structure elucidation. During the course of the purification, the compound, in all steps, behaved most similarly to the plastid lipid MGDG. The OPDA-lipid eluted
at Rt
Treatment of the purified lipid fraction with the
sn1-specific R. arrhizus lipase (Fig. 4,
inset) resulted in the immediate release of two acidic
metabolites, which were identified by GC-MS as
MALDI-TOF analysis of the sample (Fig. 5)
revealed two masses of m/z 769.5 and
m/z 783.4, which were Na+ adducts of
metabolites having molecular masses of 746.5 and 760.4 Da,
respectively. These masses are in agreement with calculated masses of
monogalactosyl-(18:3/16:3)-diacylglycerol (compound A) and of
monogalactosyl-(OPDA/16:3)-diacylglycerol (compound B).
From the known plastid lipids, no other class could produce these two
masses with incorporation of the acyl moieties identified after lipase
treatment. The analysis thus confirmed the enzymatic data that the
purified lipid was a mixture of two components, differing in the
substituent at the sn1 position.
LC-MS and LC-MS/MS with electrospray ionization further corroborated
the proposed structure of the OPDA-lipid (compound B) in both negative
(Fig. 6A) and positive (Fig.
6B) ion mode. The compound eluted from the LC column at
Rt = 29.5 min. The mass of the molecular ion was
identified from the [M
The structure of
sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl
diglyceride could be further supported by two-dimensional NMR
experiments such as COSY, HMQC, and HMBC (Table
I, Fig. 7). The resonances of
sn1, sn2, and sn3
were assigned by inspection of chemical
shifts and multiplicities (Table I). As seen in Fig. 7, the anomeric
proton of the sugar moiety at Dynamics of MGDG-O Levels in Leaves from Wounded Plants--
It is
well established that the level of JA, a wound signal produced via OPDA
(6), rises rapidly and transiently after wounding of leaves
(e.g. see Ref. 27). We therefore analyzed the levels
of MGDG-O in parallel with those of free OPDA and JA in wounded
A. thaliana leaves (Fig. 9).
The results revealed complex dynamics different for each of the three
compounds.
Using a combination of soybean lipoxygenase and recombinant allene
oxide synthase, it is possible to convert free During recent years, it has become obvious that plants harbor a
rich variety of oxidized lipids, collectively known as oxylipins (1),
many of them inducible and of physiological importance as signaling
compounds in herbivore defense, pathogen defense, and
mechanotransduction or involved in the regulation of developmental processes such as senescence and pollen maturation (2-4). The majority
of oxylipins occur in free form (1), which is believed to
originate from fatty acids released from membrane lipids (5, 9).
Recently, however, it has become clear that esterified or amidated
fatty acids can be oxidized by enzymes of plant oxylipin biosynthesis.
Thus, lipoxygenase attacks unsaturated fatty acids of triglycerides to
convert them to hydroperoxo derivatives, which are then released by
lipases and enter We show, for the first time, that OPDA, a signal in mechanotransduction
(36) and the precursor of JA (6), a wound and pathogen defense signal
(2-4), occurs in esterified form in the sn1 position of the
plastid galactolipid MGDG. This strongly suggests a defined route of
its formation. It is likely that this route is a plastidic one,
i.e. in green tissue localized in chloroplast. The first
indication is the
(7Z,10Z,13Z)-hexadecatrienoic acid (16:3) in the sn2 position of MGDG-O. MGDG molecular species
carrying sn2-16:3 are synthesized in plastids by a
prokaryotic pathway (22, 26). A large fraction of
sn2-16:3-MGDG carries MGDG-O conceivably is a metabolite with a biological significance of
its own, and/or it may form a pool from which OPDA could be released
immediately when the need arises. Indeed, a membrane-associated enzymatic activity that liberates OPDA from MGDG-O was identified in
A. thaliana in this study but awaits its full
characterization. The data in Fig. 9, on the other hand, do not show
decreases in MGDG-O correlated with the initial accumulation of JA (and
OPDA) induced by wounding. This may indicate resynthesis of MGDG-O or simply reflect the fact that only a minor fraction of it is being converted to release OPDA. A decisive answer about precursor-product relationships awaits pulse-chase analyses of metabolic fluxes through
these metabolites and experiments with isolated chloroplasts. It is
clear, though, that wounding induces a complex, biphasic increase in
the MGDG-O level. The transient nature of this increase shows clearly
that MGDG-O is not simply a metabolic end product but that it has a
significant turnover. MGDG-O levels fluctuate quite independently from
those of JA. Thus, distinct physiological roles of MGDG-O and JA should
be envisaged. It is interesting to note that the two
sn1-specific lipases that effectively cleaved OPDA from
MGDG-O are enzymes of fungal origin and that both species, R. arrhizus and M. javanicus, are plant pathogenic
saprophytes. A potential function of MGDG-O could thus be as a reporter
of lipolytic fungal activity indicative of invading phytopathogenic fungi. OPDA released locally upon fungal attack would (directly or
through conversion to JA) signal to the plant the presence of an
intruder and help to mount immediate local defense responses. Indeed,
octadecanoids act as local, and not as systemic, defense signals in
A. thaliana (40).
-linolenic acid occurs
in plastids, mainly in chloroplasts, and is thought to start with free
linolenic acid liberated from membrane lipids by lipase action. In
Arabidopsis thaliana, the glycerolipid fraction contains
esterified 12-oxophytodienoic acid, which can be released enzymatically
by sn1-specific, but not by sn2-specific,
lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated,
purified, and characterized. Enzymatic, mass spectrometric, and NMR
spectroscopic data allowed us to establish the structure of the novel
oxylipin as
sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the
sn1-(12-oxophytodienoyl) derivative by the combined action
of (soybean) lipoxygenase and (A. thaliana) allene oxide
synthase, an enzyme ensemble that converts free
-linolenic acid to
free 12-oxophytodienoic acid. When leaves were wounded, a significant
and transient increase in the level of
(12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In
A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-linolenic acid from membrane glycerolipids (5), and the pathway
from
-linolenic acid to jasmonic acid
(JA)1 has been worked out in
detail (6, 7), whereas the processes of
-linolenic acid release are
not understood. The oxidation of
-linolenic acid to
13(S)-hydroperoxolinolenic acid catalyzed by 13-lipoxygenase
and the conversion of this intermediate to the first cyclic metabolite
of the pathway, 12-oxophytodienoic acid (OPDA), occur in
plastids and thus, in the shoot, predominantly in the chloroplast
(8-10). OPDA then leaves the chloroplast and is reduced by the
NADPH-dependent flavoprotein oxophytodienoate reductase 3 (11, 12), and the reaction product is converted to JA by
-oxidation
(11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
s
1 (photosynthetically active radiation;
supplementary light, if required, from sodium vapor lamps). Plants were
cut above soil and immediately extracted, or they were immersed in
liquid nitrogen and stored at
70 °C for minimum amounts of time.
28 °C under nitrogen, if not used directly.
1 as follows: 1 min of
eluent A, then a linear gradient in 10 min to 50% eluent B, then 27 min of isocratic flow at 50% solvent B, followed by a linear gradient
over 10 min to 100% solvent B (solvent A: isopropyl alcohol:
isohexane (4:3, v/v); solvent B: isopropyl alcohol:
isohexane:H2O (8:6:1.5, v/v/v)) (17). UV detection was at
205 nm. The lipid composition was checked in each fraction by TLC
according to (17). The presence of esterified OPDA was checked by
enzymatic treatment of each lipid fraction with Rhizopus
arrhizus lipase (18). Esterified OPDA-containing fractions
(Rt
15 min) were purified further. To do so, the
OPDA-containing lipid from each run on the silica column was pooled,
the solvent was removed in a stream of nitrogen, and the residue was
redissolved in 1 ml of acetonitrile:aqueous 50 mM ammonium
acetate (70:30, v/v), centrifuged, and applied to a Dynamax C18
reversed phase HPLC column (Rainin; 250 mm × 21.4 mm inner
diameter, 8 µm particle size, 60 Å pore size) at a flow rate
of 20 ml min
1 (isocratic elution for 100 min,
UV detection at 221 nm). The OPDA-containing fractions
(Rt
52-57 min) were pooled and
rechromatographed on the same column to yield a homogenous peak
(Rt = 55 min), which was used for all further
characterizations. From 1 kg of plant material, ~1-2 mg of
ester-OPDA lipid was obtained.
1 to 200 °C, 5 °C
min
1 to 250 °C, and 10 min at 250 °C.
Typical retention times (methyl esters) were as follows:
cis-OPDA, 16.73 min (m/z = 307 [M + H]+);
(9Z,12Z,15Z)-octadecatrienoic acid
(
-linolenic acid), 12.07 min (m/z = 293 [M + H]+);
(7Z,10Z,13Z)-hexadecatrienoic acid,
9.57 min (m/z = 265 [M + H]+).
1
obtained with an Applied Biosystems 140 B dual-piston pump). The
retention time of the OPDA-lipid was ~30 min. MS/MS measurements were
performed using argon collision gas (2 × 10
3 torr) and a collision energy at
quadrupole 2 of 10 V. MALDI-TOF-MS was performed on a PE
Biosystems Voyager instrument (matrix, dihydroxybenzoic acid) with
positive ion detection (25 kV acceleration voltage, calibrated with
human angiotensin I (Sigma)).
-linolenic acid in the MGDG substrate as well as that of free
-linolenic acid by the two enzymes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Metabolism of OPDA in aqueous crude leaf
extracts. Exogenous, labeled [2H5]OPDA
(open circles); endogenous, unlabeled compound (closed
circles). The ion intensity of 100% represents 3.4 nmol of
substance.
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Fig. 2.
Release of OPDA from the crude lipid of
A. thaliana by alkaline treatment. OPDA from
nonhydrolyzed (left and middle bars) and
hydrolyzed (saponification for 3 h at 25 °C in 1 N
KOH, right bar) lipid was quantified by GC-MS.
fw, fresh weight.
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Fig. 3.
Enzymatic release of OPDA from crude lipid of
A. thaliana, quantified by GC-MS. Cleavage of
OPDA from the lipid was most efficient using lipases, which show a
preference for the sn1 position of glycerolipids
(black bars), but was inefficient by phospholipases with
sn2 preference (white bars). A,
control (pH 7.5); B, R. arrhizus lipase;
C, wheat germ lipase; D, M. javanicus
lipase; E, control (pH 8.9); F, A. mellifica phospholipase A2; G, N. mossambica phospholipase A2; fw,
fresh weight.
15 min from the first HPLC column
(Nucleosil 100, coeluting with MGDG) and at Rt
52-57 min from the reversed phase column separating the lipid
molecular species. The purified material, upon rechromatography, eluted
from the same column as a single peak with Rt = 55 min (Fig. 4).
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Fig. 4.
Kinetic analysis of acyl release from
purified OPDA-lipid treated with sn1-specific R. arrhizus lipase. The inset shows the
normalized kinetics, whereas the elution behavior of the substrate (3 mg injected) used on reversed phase HPLC is shown in the main frame
using relative absorbance units (100% = peak maximum).
-linolenic acid and
OPDA. After a lag phase of ~5 min, when the rates of OPDA and
-linolenic acid release began to level off and the enzyme began to
attack the sn2 position, a third acidic metabolite began to
accumulate, which was identified as
(7Z,10Z,13Z)-hexadecatrienoic acid
(16:3). These results indicated that (i) a mixture of two lipids had
been obtained by the purification procedures employed (attempts to
resolve this further were unsuccessful), and (ii) compound A of that
mixture was a diacyl lipid with
-linolenic acid in the
sn1 position and 16:3 in the sn2 position, and
compound B was a diacyl lipid with OPDA in the sn1 position
and 16:3 in the sn2 position. The occurrence of 16:3 in the
sn2 position is typical for membrane lipids synthesized in
plastids (22).
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Fig. 5.
MALDI-TOF analysis of the purified OPDA-lipid
(30 µg).
Monogalactosyl-(OPDA/hexatrienoyl)-diacylglycerol is indicated by the
mass fragment with m/z of 783.4 ([M + Na]+). The fragment with m/z of
769.4 corresponds to the accompanying (18:3/16:3)-MGDG ([M + Na]+).
H]
ion
(m/z 759.5), from the acetate adduct [M + Ac
H]
(m/z 819.7), and
from the [M + H]+ ion (m/z 761.6)
as well as from several positively charged solvent adducts (see Fig.
6B) to be 760.5 Da. MS/MS of the [M + Ac
H]
ion yielded several diagnostic fragments
(cf. Fig. 6A), among them the [OPDA
H]
fragment (m/z 291.1) and the
[16:3
H]
fragment (m/z
249.3), most likely representing the carboxylate anions of the two acyl
moieties, as has been observed with other monogalactosyl diacylglycerol
species (23). No 18:3(linolenic acid)-derived ions were observed in the
29.5-min peak. Likewise, MS/MS of the [M + H]+ ion
(m/z 761.1) yielded the
-fragmentation
derivatives showing loss of the esterified 16:3 fatty acid
(m/z 349.2) and the corresponding loss of OPDA
(m/z 307.2). Thus, the mass spectrometric
analysis was in agreement with a proposed structure of a MGDG species
in which one of the acyl groups is a 16:3 fatty acid, and the other is
OPDA.
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Fig. 6.
Electrospray ionization-LC/MS/MS
analysis of the purified OPDA-lipid (100 µg). Molecular ions of
monogalactosyl-(OPDA/hexatrienoyl)-diacylglycerol are represented by
m/z = 761.6 in positive ion mode
(A) and m/z = 759.7 in negative
ion mode (B), shown by full scan MS analysis of the eluting
lipid compound (left). MS/MS analysis of characteristic ions
gave specific fragment patterns, which confirmed the presence of the
indicated acyl moieties (right).
4.22 ppm (J = 7.4 Hz, which proves the
-linkage) couples via HMBC with the
sn3 carbon at
63.9 ppm. The galactopyranosyl nature of
this sugar moiety could be proven by analysis of the spin system, showing e.g. the typical broad doublet of the H-4' at
3.82 ppm (J = 3.2 Hz). The signals C-1" and C-1
of
the unsaturated fatty acids appear at
174.6 and 175.0 ppm, making
it impossible to assign the linkage position at the glycerol skeleton.
However, the sn1 linkage of the OPDA has been proven by the
enzymatic methods already described above. The chemical shifts of the
carbon and proton resonances, as well as the multiplicity, are in
agreement with the data reported formerly for OPDA (24) and
methyl-(7Z,10Z,13Z)-hexadecatrienoate (25). The overlapping of the signals of the olefinic protons 7", 8",
10", 11", 13", 14", 15
, and 16
at
5.30-5.50 ppm did not
allow the determination of the E/Z configuration. However, the
configuration of the sn2-hexadecatrienoic acid of MGDG from A. thaliana had earlier been shown to be exclusively the
7Z,10Z,13Z isomer (26), and our GC-MS
data (see above) proved that the same isomer was present in the
sn2 position of the OPDA lipid. The collective evidence thus
firmly established the structure of the isolated metabolite to be
sn1-O-(9S,13S-12-oxophytodienoyl)-sn2- O-(7Z,10Z,13Z-hexadecatrienoyl)-monogalactosyl
diglyceride (MGDG-O, Fig. 8).
NMR data for
sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl
diglyceride
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Fig. 7.
Expanded plots of COSY, HMQC, and HMBC
spectra of
sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl
diglyceride (MGDG-O) showing signals of the
-D-galactopyranose moiety.
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[in a new window]
Fig. 8.
Structure of the OPDA-lipid identified
as
sn1-O-(9S,13S-12-oxophytodienoyl)-sn2-O-(7Z,10Z,13Z-hexadecatrienoyl)-sn3-O-( -D-galactopyranosyl)-glycerol
(MGDG-O).
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Fig. 9.
Kinetic analysis of the endogenous levels of
JA, OPDA, and MGDG-O in leaf tissue of A. thaliana after
wounding. Shown are the means of typical experiments plotted in
absolute (A) and relative (B) amounts.
fw, fresh weight.
-linolenic acid via
13(S)hydroperoxylinolenic acid to the allene oxide
12,13-epoxylinolenic acid, which then rearranges spontaneously, with
the formation, among other products, of racemic cis-OPDA
(19). When purified MGDG (17) was subjected to the same procedures,
MGDG-O was not detected, although
-linolenic acid used as a control
was readily converted (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation (28), but lipoxygenase has also been
shown to oxidize fatty acids esterified in phospholipids (29).
Phospholipases that exhibit a preference for phosphatidylcholine with
oxygenated acyl groups have been identified in plant microsomes (30).
Moreover, N-acyl(ethanol) amines have been identified in
plants (31), and these are thought to play a role in plant pathogen
defense (32). It has recently been shown that enzymes of the
octadecanoid biosynthetic pathway, namely lipoxygenase and allene oxide
synthase, act on N-acyl(ethanol) amines to yield, among
other products, the 12-oxo-N-phytodienoylamines (33).
Homologs of isoprostanes that are formed nonenzymatically on
phospholipids (34) have meanwhile been found in plants, the phytoprostanes (35). Thus, although the biological relevance of these
compounds is still not understood, the generation, in situ,
of oxylipins in plant membranes probably complements their formation
from free fatty acids, and it will be important to elucidate the
enzymology of their formation and their physiological role(s).
-linolenic acid in the
sn1 position, the precursor of OPDA. It is thus possible that OPDA is generated in situ from the
-linolenic acid
in the sn1 position of MGDG. However, we have been unable to
show conversion of MGDG to MGDG-O in vitro using soybean
lipoxygenase and A. thaliana allene oxide synthase, an
enzyme combination that converts free
-linolenic acid to
OPDA. This means that either the enzymes involved in formation of
MGDG-O in vivo differ from those involved in synthesizing OPDA or the substrate has to be presented to them in a particular environment (e.g. a bilayer membrane). Alternatively, it is
conceivable that esterified OPDA is not formed from MGDG in
situ but that free OPDA is synthesized first and then incorporated
in the membrane lipid. These issues will be addressed in future
studies. It was shown earlier that allene oxide synthase is associated
with chloroplast membranes from which it has to be released with
detergent (9, 37). The recombinant enzyme is soluble and, as such, acts
on free fatty acid 13(S)-hydroperoxides (38, 39). The
chloroplast membrane docking site of allene oxide synthase is unknown,
but the membrane association may reflect the fact that the enzyme, in vivo, acts on a membrane substrate rather than on a free
fatty acid.
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ACKNOWLEDGEMENT |
---|
We thank Rolf Breuckmann, Bioorganic Chemistry, Faculty of Chemistry, Ruhr-Universität Bochum, for the MALDI-TOF analyses.
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FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn and by Fonds der Chemischen Industrie, Frankfurt (literature provision).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: Merck KGaA ZD-A/ZFA 1, Frankfurter Str. 250, 64271 Darmstadt, Germany.
To whom correspondence should be addressed: Lehrstuhl
für Pflanzenphysiologie, Ruhr-Universität Bochum,
Universitätsstra
e 150, D-44801 Bochum, Germany. Tel.: 49 234 3224291; Fax: 49 234 3214187; E-mail:
Elmar.Weiler@ruhr-uni-bochum.de.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010743200
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ABBREVIATIONS |
---|
The abbreviations used are: JA, jasmonic acid; OPDA, 12-oxophytodienoic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GC, gas chromatography; MS, mass spectrometry; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation; MGDG, monogalactosyl diglyceride; EI, electron impact ionization.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hamberg, M. (1993) J. Lipid Mediators 6, 375-384[Medline] [Order article via Infotrieve] |
2. | Wasternack, C., and Parthier, B. (1997) Trends Plant Sci. 2, 302-307[CrossRef] |
3. | Creelman, R. A., and Mullet, J. E. (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355-381[CrossRef][Medline] [Order article via Infotrieve] |
4. | Weiler, E. W. (1997) Naturwissenschaften 84, 340-349[CrossRef] |
5. | Farmer, E. E., and Ryan, C. A. (1992) Trends Cell Biol. 2, 236-241 |
6. | Vick, B. A., and Zimmerman, D. C. (1984) Plant Physiol. (Bethesda) 75, 458-461 |
7. | Vick, B. A., and Zimmerman, D. C. (1987) Plant Physiol. (Bethesda) 85, 1073-1078 |
8. | Vick, B. A., and Zimmerman, D. C. (1981) Plant Physiol. (Bethesda) 67, 92-97 |
9. |
Blée, E.,
and Joyard, J.
(1996)
Plant Physiol. (Bethesda)
110,
445-454 |
10. | Hamberg, M. (1988) Biochem. Biophys. Res. Commun. 156, 543-550[Medline] [Order article via Infotrieve] |
11. | Vick, B. A., and Zimmerman, D. C. (1986) Plant Physiol. (Bethesda) 80, 202-205 |
12. | Schaller, F., Biesgen, C., Müssig, C., Altmann, T., and Weiler, E. W. (2000) Planta 210, 979-984[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Brash, A. R.
(1999)
J. Biol. Chem.
274,
23679-23682 |
14. | Laudert, D., and Weiler, E. W. (1998) Plant J. 15, 675-684[CrossRef][Medline] [Order article via Infotrieve] |
15. | Stelmach, B. A., Müller, A., Hennig, P., Laudert, D., Andert, L., and Weiler, E. W. (1998) Phytochemistry 47, 539-546[CrossRef][Medline] [Order article via Infotrieve] |
16. | Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 31, 911-917 |
17. | Demandre, C., Tremolières, A., Justin, A. M., and Mazliak, P. (1985) Phytochemistry 24, 481-485[CrossRef] |
18. | Williams, J. P., Khan, M. U., and Wong, D. (1995) J. Lipid Res. 36, 1407-1412[Abstract] |
19. | Laudert, D., Hennig, P., Stelmach, B. A., Müller, A., Andert, L., and Weiler, E. W. (1997) Anal. Biochem. 246, 211-217[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Schaller, F.,
and Weiler, E. W.
(1997)
J. Biol. Chem.
272,
28066-28072 |
21. | Knöfel, H. D., and Gross, D. (1988) Z. Naturforsch. 43, 29-31 |
22. | Somerville, C., and Browse, J. (1991) Science 252, 80-87 |
23. | Kim, Y. H., Yoo, J. S., and Kim, M. S. (1997) J. Mass Spectrom. 32, 968-977[CrossRef] |
24. | Bohlmann, F., Jakupovic, J., Ahmed, M., and Schuster, A. (1983) Phytochemistry 7, 1623-1636[CrossRef] |
25. | Morimoto, T., Nagatsu, A., Murakami, N., Sakakibawa, J., Tokuda, H., Nishini, H., and Iwashima, A. (1995) Phytochemistry 5, 1433-1437[CrossRef] |
26. | Browse, J., McCourt, P. J., and Somerville, C. R. (1986) Anal. Biochem. 152, 141-145[Medline] [Order article via Infotrieve] |
27. | Albrecht, T., Kehlen, A., Stahl, K., Knöfel, H. D., Sembdner, G., and Weiler, E. W. (1993) Planta 191, 86-94 |
28. | Feussner, I., Wasternack, C., Kindl, H., and Kühn, H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11849-11853[Abstract] |
29. | Brash, A., Ingram, C. D., and Harris, T. M. (1987) Biochemistry 26, 5465-5471[Medline] [Order article via Infotrieve] |
30. | Banás, A., Johansson, I., and Stysune, S. (1992) Plant Sci. 84, 137-144[CrossRef] |
31. | Schmid, H. H. O., Schmid, P. C., and Natarjan, V. (1990) Prog. Lipid Res. 29, 1-43[Medline] [Order article via Infotrieve] |
32. |
Tripathy, S.,
Venables, B. J.,
and Chapman, K. D.
(1999)
Plant Physiol. (Bethesda)
121,
1299-1308 |
33. |
van der Stelt, M.,
Noordermeer, M. A.,
Kiss, T.,
van Zadelhoff, G.,
Merghart, B.,
Veldink, G. A.,
and Vliegenthart, J. F. G.
(2000)
Eur. J. Biochem.
267,
2000-2007 |
34. | Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. N., Bader, K. F., and Roberts, J. F., II (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9383-9387[Abstract] |
35. | Mueller, M. J. (1998) Chem. Biol. 5, R323-R333[Medline] [Order article via Infotrieve] |
36. |
Blechert, S.,
Bockelmann, C.,
Fü![]() |
37. | Song, W.-C., and Brash, A. R. (1991) Science 253, 781-784[Medline] [Order article via Infotrieve] |
38. |
Song, W.-C.,
Funk, C. D.,
and Brash, A. R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8519-8523 |
39. | Laudert, D., Pfannschmidt, U., Lottspeich, F., Holländer-Czytko, H., and Weiler, E. W. (1996) Plant Mol. Biol. 31, 323-335[Medline] [Order article via Infotrieve] |
40. | Kubigsteltig, I., Laudert, D., and Weiler, E. W. (1999) Planta 208, 463-471[CrossRef][Medline] [Order article via Infotrieve] |