Wounding Stimulates the Accumulation of Glycerolipids Containing Oxophytodienoic Acid and Dinor-Oxophytodienoic Acid in Arabidopsis Leaves

Article (PDF Available)inPlant physiology 142(1):28-39 · October 2006with36 Reads
DOI: 10.1104/pp.106.082115 · Source: PubMed
Abstract
Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.
Breakthrough Technologies
Wounding Stimulates the Accumulation of Glycerolipids
Containing Oxophytodienoic Acid and
Dinor-Oxophytodienoic Acid in Arabidopsis Leaves
1[W]
Christen M. Buseman
2
, Pamela Tamura, Alexis A. Sparks, Ethan J. Baughman
2
, Sara Maatta, Jian Zh ao
3
,
Mary R. Roth, Steven Wynn Esch, Jyoti Shah, Todd D. Williams, and Ruth Welti*
Division of Biology (C.M.B., P.T., A.A.S., E.J.B., S.M., M.R.R., S.W.E., J.S., R.W.) and Department of
Biochemistry (J.Z.), Kansas State University, M anhattan, Kansas 66506; and University of Kansas Mass
Spectrometry Laboratory, University of Kansas, Lawrence, Kansas 66045 (S.W.E., T.D.W.)
Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-
localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI)
collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS
in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-
flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidyl-
glycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of
these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and
16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-
containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded
leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023
nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA
MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of
OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each
containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing
OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic
oxylipin biosynthesis.
Oxylipins are cyclic or acyclic oxidation products of
fatty acids and are effectors in many biological path-
ways, including plant development and defense re-
sponses (Creelman and Mulpuri, 2002; Turner et al.,
2002). Jasmonic acid (JA), oxophytodienoic acid
(OPDA), and other cyclic oxylipins make up the jas-
monate family (Creelman and Mulpuri, 2002; Turner
et al., 2002; Weber, 2002). The role of JA in defense
response was suggested in 1992 (Farmer and Ryan,
1992), when jasmonate synthesis was discovered to
occur in response to wounding. More recently it has
been demonstrated that OPDA is also capable of sig-
naling and modulating gene expression (Kutchan,
1993; Blechert et al., 1995; Stintzi et al., 2001; Taki et al.,
2005). Current understanding is that each plant or plant
tissue produces its own oxylipin signature, or specific
mixture of various oxylipins that elicit appropriate
responses to particular environmental cues (Weber
et al., 1997).
JA synthesis begins in the plastid and is completed in
the peroxisome. The conversion of precursor linolenic
acid (18:3) to OPDA (Fig. 1) occurs in the plastid. The
pathway involves formation of a hydroperoxide by a
lipoxygenase, formation of an epoxide by allene oxide
synthase, and formation of a cyclopentenone ring by
allene oxide cyclase to produce OPDA. Alternatively,
12,13-epoxyoctadecatrienoic acid can be converted
nonenzymatically to a ketol (Hamberg, 1988). 9S,13S-
OPDA is transported from the plastid to the peroxi-
some, where it is reduced by 12-oxo-phytodienoic
1
This work was supported by grants from the National Science
Foundation (grant nos. MCB 0455318 and DBI 0520140). Support of
the Kansas Lipidomics Research Center was from the National
Science Foundation’s EPSCoR program (grant no. EPS–0236913)
with matching support from the State of Kansas through Kansas
Technology Enterprise Corporation and Kansas State University, as
well as from Core Facility Support from the National Institutes of
Health (grant no. P20 RR016475) from the INBRE program of the
National Center for Research Resources. Accurate mass analysis was
performed at the University of Kansas Mass Spectrometry Labora-
tory, using a Micromass Q-TOF2, which was purchased with funds
from the University of Kansas, the University of Kansas Mass
Spectrometry Laboratory, and Kansas National Science Foundation
EPSCoR. This is contribution 06–180–J from the Kansas Agricultural
Experiment Station.
2
Present address: The University of Texas Southwestern Medical
Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390.
3
Present address: Children’s Nutrition Research Center, Depart-
ment of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
* Corresponding author; e-mail welti@ksu.edu; fax 785–532–6653.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Ruth Welti (welti@ksu.edu).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.106.082115
28 Plant Physiology, September 2006, Vol. 142, pp. 28–39, www.plantphysiol.org Ó 2006 American Society of Plant Biologists
acid 10,11 reductase. Subsequently, three cycles of
b-oxidation result in shortening of the 18-carbon com-
pound to 12-carbon JA. In 1997, Weber et al. identified
dinor-OPDA (dnOPDA; [7S,11S]-10-oxy) as a 16-carbon
analog of OPDA, formed from hexadecatrienoic acid
(16:3) through the same pathway.
In 2001, Stelmach and coworkers determined that
80% to 90% of the OPDA in unstimulated Arabidopsis
(Arabidopsis thaliana) shoots is esterified in complex
lipid species. They purified and identified a novel
OPDA-containing galactolipid, 1-OPDA, 2-16:3 mo-
nogalactosyldiacylgl ycerol (MGDG). More recently,
Hisamatsu and colleagues (2003, 2005) have chemi-
cally characterized four additional OPDA-containing
galactolipids isolated from Arabidopsis leaves. These
compounds are 1-OPDA, 2-dnOPDA MGDG (annotated
by these authors as Arabidopside A), OPDA-OPDA
MGDG (Arabidopside B), 1-OPDA, 2-dnOPDA diga-
lactosyldiacylglycerol (DGDG; Arabidopside C), and
OPDA-OPDA DGDG (Arabidopside D). The relation-
ship between esterified OPDA or dnOPDA and the
free fatty acid forms of these compounds has not yet
been elucidated. Figure 1 shows two possibilities for
this relationship. One potential metabolic route in-
cludes the release of trienoic fatty acids (18:3 and 16:3)
from galactolipids, followed by the free fatty acids
being converted in three steps to OPDA and dnOPDA,
which could then be incorporated into galactolipids.
The second potential pathway is the conversion of 18:3
and 16:3 to OPDA and dnOPDA, while these acyl
groups remain esterified to the galactolipids or phos-
phatidylglycerol (PG), followed by the release of
OPDA and dnOPDA as free fatty acids.
In this work, we take advantage of recent advances
in electrospray ionization (ESI) tandem mass spec-
trometry (MS/MS) methods to identify 13 additional
OPDA-, dnOPDA-, and/or related ketol-containing
MGDG, DGDG, and PG species, in addition to the one
identified by Stelmach et al. (2001) and the four iden-
tified by Hisamatsu and colleagues (2003, 2005). In
Figure 1. Biosynthetic and proposed biosynthetic pathway for 18-carbon ketols, OPDA, and OPDA-containing MGDG.
Analogous pathways are thought to convert 16:3 to dnOPDA and 16-carbon ketols, and dnOPDA to JA via two cycles of
b-oxidation. The relationship of OPDA- and dnOPDA-containing MGDG, DGDG, and PG to this process is not clear, as
indicated by the question marks, but two possibilities are that (1) trienoic fatty acids are released from galactolipids (or PG),
converted to OPDA or dnOPDA, then reincorporated into complex lipids, or (2) conversion of trienoic fatty acids to OPDA or
dnOPDA takes place on the complex lipids, and these species are later released.
OPDA- and Dinor-OPDA-Containing Chloroplast Polar Lipids
Plant Physiol. Vol. 142, 2006 29
particular, we utilize the collisionally induced disso-
ciation (CID) and time-of-flight (TOF) capabilities of
a hybrid quadrupole (Q)-TOF mass spectrometer
to rapidly identify acyl chains present in a complex
mixture of lipids. This method facilitates the choice of
acyl groups for acyl precursor scanning, which is used
to identify the parent polar complex lipids (precursors)
containing each acyl chain. The presence of each of these
polar complex lipid species is confirmed by accurate
mass analysis of the acyl fragments of the original and
hydrogenated polar complex lipids. Furthermore, a
quantitative method for oxylipin-containing galactoli-
pids was developed, based on scanning for intact mo-
lecular precursorions ofcharacteristic fattyacyl moieties
produced by CID. In the quantitative method, the levels
of oxylipin-containing galactolipids are measured di-
rectly from unfractionated lipid extracts.
The levels of seven OPDA- and dnOPDA-containing
species are examined in Arabidopsis leaves, unstimu-
lated, and duri ng their response to wounding. We
show that various oxylipin-containing species are syn-
thesized and degraded at different rates. Identification
of the additional oxylipin-containing species, devel-
opment of a quantitative method for their analysis,
and our kinetic observations lay the groundwork for
further studies aimed at clarifying the biosynthetic
pathway and role of complex lipids containing oxy-
lipins in plants.
RESULTS
Detection of Oxylipins by CID -TOF Analysis
Fatty acyl species present in wounded and un-
wounded Arabidopsis leaves were identified with an
ESI Q-TOF hybrid mass spectrometer. Subjecting a
plant-derived lipid extract, ionized by ESI in negative
ion mode, to CID in the collision cell of a Q-TOF mass
spectrometer without selecting precursor lipid species
(i.e. with radio frequency/direct current mass selec-
tion in the Q off) produces a spectrum that includes
acyl anio ns of fatty acids derived from both free fatty
acids and complex lipids (Figs. 2 and 3). This exper-
iment is accomplished by increasing the volta ge offset
on the collision to 35 V while acquiring data in MS1
mode (no precursor selection), thus activating all ions
produced at once. We call this analysis CID-TOF here
to distinguish it from traditional product ion analysis
also performed with the Q-TOF mass spectrometer.
Accurate mass data produced by the TOF analyzer can
be correlated with chemical formulas to identify the
Figure 2. CID-TOF spectra of anions from lipids of unwounded (A) and wounded (B; after 5 h) Arabidopsis leaves. Unselected
molecular ions in an extract were subjected to CID and analyzed with a TOF analyzer. Table I provides additional information
about each peak. Note that the spectra are expanded 10 3 from approximately m/z 262 to 273 and 5 3 from 290 to 310. Insets
show ranges in which there are two peaks at similar m/z.
Buseman et al.
30 Plant Physiol. Vol. 142, 2006
acyl anions present (Table I). CID-TOF analysis pro-
vides a reproducible and excellent semiquantitative
view of acyl species present. It can only be considered
semiquantitative, mainly because the propensity of an
acyl species in a polar diacyl glycerolipid to form an
anion depends on its position (Murphy, 1993). For
example, phospholipids typically produce acyl anion
peaks from the 2 position approximately twice as
readily as from the 1 position (Murphy, 1993). Thus,
because biological lipids typically contain diacyl lipids
with specific fatty acyl chains nonrandomly distrib-
uted between the 1 and 2 positions, a CID-TOF spec-
trum will provide a somewhat biased representation
of the total acyl species in the mixture, with overrep-
resentation of the more easily produced acyl anions.
CID-TOF analysis does, however, provide an excellent
starting point for sample to sample comparisons.
The spectra shown in Figure 2 show the total acyl
species present from such an analysis before and 5 h
after wounding of Arabidopsis leaves. Species that
increase in amount after wounding include prominent
anions with mass-to-charge ratio (m/z) 263.1656 and
291.1968 and smaller peaks at m/z 281.1733 and
309.2065. The best matches between these masses
and chemical formulas for the two largest peaks are
C
16
H
23
O
3
and C
18
H
27
O
3
(Table I). Whereas typical fatty
acid anions contain two oxygen atoms, these anions
contain three oxygen atoms, suggesting that these
anions are derived from oxylipin species.
Table II shows oxylipin species described in the
literature with anion formulas corresponding to those
detected. There are three described oxylipin anions
with the formula C
18
H
27
O
3
. Catalytic hydrogenation,
which removes only carbon-carbon double bonds, was
used to identify the oxylipins present from among
these oxylipins. After hydrogenation of the mixture,
prominent peaks at nominal m/z 263 and 291 disap-
peared and new peaks appeared at m/z 267.1970 and
295.2275 (Fig. 3; Table I). As shown in Table II, of the
described oxylipins with anion formulas of C
18
H
27
O
3
,
only one, OPDA, gains only 4 atomic mass units (amu)
upon hydrogenation. Similarly, hydrogenation of
the peak at m/z 263 to 267 is consistent with iden tifi-
cation of that peak as dnOPDA and not a 16-carbon
analog of either ketotrienoic acid or colnelenic acid.
The peak at m/z 309.2065 is consistent with a chemical
formula of C
18
H
29
O
4
. This formula is consistent with
the presence of either or both characterized 18-carbo n
ketols, 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic
acid (12,13-a-ketol) and 9-hydroxy-12-oxo-10(E),15(Z)-
octadecadienoic acid (g-ketol), which can be formed
nonenzymatically from allene oxide (Hamberg, 1988;
Ziegler et al., 2000; Fig. 1), as well as with HPOTE
and di-HOTE. Hydrogenation resulted in a peak at
Figure 3. CID-TOF spectra of anions from lipids of unwounded (A) and wounded (B; after 5 h) Arabidopsis leaves after catalytic
hydrogenation. Unselected molecular ions in an extract were subjected to CID and analyzed with a TOF analyzer. Table I
provides additional information about each peak. Note that the spectra are expanded 20 3 from approximately m/z 262 to 282
and 5 3 from 290 to 310. Insets show areas in which there are two peaks at similar m/z.
OPDA- and Dinor-OPDA-Containing Chloroplast Polar Lipids
Plant Physiol. Vol. 142, 2006 31
313.2359; this mass would be expected when two
carbon-carbon double bonds are lost during catalytic
hydrogenation, consistent with the identification of
the peak at m/z 309.2065 as a-org-ketol, but not
HPOTE or di-HOTE, although the data do not rule
out the presence of complex lipids containing small
amounts of these other oxylipin components. The low
magnitude of the m/z 281.1733 (Fig. 2) did not allow
detection of a corresponding hydrogenated acyl spe-
cies via CID-TOF analysis .
Complex Lipids Containing dnOPDA, OPDA, and Ketols
Precursor scanning with an ESI triple-Q mass spec-
trometer was used to discover the complex lipid
species that contain the oxylipin speci es found in lipid
extracts of wounded Arabidopsis. Figure 4 shows
peaks identified in the range m/z 740 to 830; peaks in
the range m/z 740 to 1,050 are shown in Supplemental
Figure 1. In Figure 4, sections A and B depict precursor
ions of head group-derived fragments, while sections
C to G depict precursor ions of specific acyl anions.
Section A shows a scan specific for PG species. Section
B depicts lipid specie s containing a fragment of m/z
235, which corresponds to the mass of a negative ion of
glycero-Gal minus a water molecule. Thus, this section
represents MGDG and DGDG species. Due to the
presence of ammonium acetate (NH
4
OAc) in the in-
fusion solvent, both [M 2 H]
2
and [M 1 OAc]
2
ions
are formed, and each MGDG molecular species is
represented by two peaks, although some of the MGDG
[M 1 OAc]
2
peaks are at higher m/z than shown in
Figure 4. Sections C and D depict the lipid species
containing the normal acyl species 16:3 and 18:3,
respectively, while sections E to G depict the lipid
species containing dnOPDA, OPDA, and the 18-carbon
ketol, respectively. It was not possible to use pre-
cursor scanning to elucidate the species containing
a 16-carbon ketol because the 16-carbon ketol has the
same nominal mass (m/z 281) as the normal chain acyl
species oleic acid (18:1). Thus, a scan for precursors of
m/z 281 would depict those lipid species containing
either a 16-carbon ketol or 18:1. The difference in the
scales of sections C and D (in the 10
8
range) as
compared to sections E and F (10
6
range) and section
G (10
5
range) indicates the greater prominence of the
normal chain species (i.e. 16:3 and 18:3; sections C and
D) relative to the oxylipin species (dnOPDA, OPDA,
and ketols; sections E–G).
Sections E to G, in combination with the previous
sections, provide tentative identification for each pu-
tative oxylipin molecular species by identifying the
nominal masses of its acyl chains. The indicated spe-
cies account for most, but not all, of the complex lipid
species in this m/z range containing the investigated
oxylipins. The peak identifications shown in Figure 4,
as well as additional identifications indicated in Sup-
plemental Figure 1, were confirmed by Q-TOF (accu-
rate mass) analysis of product (acyl) ions in extract
fractions. Before performing the Q-TOF analysis, the
extract was fractionated by silicic acid chromatography
to produce fraction 2, enriched in MGDG, and frac tion
Table I. Identification by TOF analysis of major anionic fragments, produced by CID in the negative
mode, in the m/z range 230 to 310 in an extract from wounded Arabidopsis, and identification of the
fragments in the m/z range 250 to 320 in a hydrogenated extract from wounded Arabidopsis
In each case, the mass calibration was locked by setting m/z of the glycerol-Gal fragment produced by
MGDG and DGDG to its theoretical value of 253.0923.
Experimental m/z from
(Wounded)
Theoretical m/z Difference (ppm) Formula Interpretation
Figure 2
235.0820 235.0818 10.9 C
9
H
15
O
7
Glycero-Gal 2 water
241.0104 241.0113 23.7 C
6
H
10
O
8
P Inositol-P 2 water
249.1846 249.1855 23.6 C
16
H
25
O
2
16:3
(253.0923) 253.0923 10.0 C
9
H
17
O
8
Glycero-Gal
253.2164 253.2168 21.6 C
16
H
29
O
2
16:1
255.2320 255.2324 21.6 C
16
H
31
O
2
16:0
263.1656 263.1647 13.4 C
16
H
23
O
3
dnOPDA
277.2155 277.2168 24.7 C
18
H
29
O
2
18:3
279.2317 279.2324 22.5 C
18
H
31
O
2
18:2
281.1733 281.1753 27.1 C
16
H
25
O
4
16-carbon ketol
281.2471 281.2481 23.6 C
18
H
33
O
2
18:1
291.1968 291.1960 12.8 C
18
H
27
O
3
OPDA
309.2065 309.2066 20.3 C
18
H
29
O
4
18-carbon ketol
Figure 3
(253. 0923) 253.0923 10.0 C
9
H
17
O
8
Glycero-Gal
255.2311 255.2324 25.1 C
16
H
31
O
2
16:0
267.1970 267.1960 13.7 C
16
H
27
O
3
Hydrogenated dnOPDA
283.2614 283.2637 28.1 C
18
H
35
O
2
Stearic acid (18:0)
295.2275 295.2273 10.7 C
18
H
31
O
3
Hydrogenated OPDA
313.2359 313.2379 26.3 C
18
H
33
O
4
Hydrogenated 18-carbon ketol
Buseman et al.
32 Plant Physiol. Vol. 142, 2006
3, enriched in PG. DGDG was enriched in both frac-
tions 2 and 3. Using fractionated material for the
product ion analysis reduced the number of lipid
species present, allowing more certain identification of
acyl pairs. The discovery scans implied that the PG
peaks at m/z 755 and 757 (A) contained OPDA (F), and
their m/z values indicated that the acyl species paired
with OPDA were palmitoleic (16:1) and palmitic (16:0)
acids, respectively. Product ion analysis of these spe-
cies in PG-containing fraction 3 indicated that both
OPDA and 16:0 were indeed present in the species at
m/z 757 and OPDA and 16:1 were present in the
species at m/z 755 (Supplemental Table I). The latter
species is depicted in Figure 5. Similarly, each of the
MGDG and DGDG species listed in Table III were
identified by product ion mass analysis of the [M 2 H]
2
ions in fractions 2 and 3 using Q-TOF MS, and detailed
results of these analyses are shown in Supplemental
Table I.
Identifications were further substantiated by analy-
sis of unfractionated and fractionated wounded Arabi-
dopsis leaf extracts that were subjected to catalytic
hydrogenation. Precursor scanning of a catalytically
hydrogenated, unfractionated extract of wounded
Arabidopsis (Supplemental Fig. 2) and Q-TOF product
ion analysis of the catalytically hydrogenated and
fractionated extract (Supplemental Table II) reveal
the expected, hydrogenated product of each of the
species identified in Table III and Supplemental Table
I. These data are consistent with the notion that the
Table II. Previously described oxylipin anions with chemical formulas identified in Figure 2, and their predicted formulas and masses after
catalytic hydrogenation
The shorthand used in this table indicates total carbons: number of double bond equivalents-oxygen atoms. Double bond equivalents include
carbon-carbon double bonds, carbon-oxygen double bonds, and rings. The double bond equivalent does not include the carbonyl of a fatty acid; i.e. a
saturated fatty acid would be listed as having no double bonds. The left-hand side of the table shows oxylipin species described in the literature with
the chemical formulas detected in the spectra shown in Figure 2. The right-hand side of the table shows the acyl species that would be formed from the
species on the left after catalytic hydrogenation. The bold type indicates the species that were actually detected (Fig. 3).
Naturally Occurring Oxylipins with
Chemical Formulas Identified in Figure 2
Oxylipins Potentially Formed
after Catalytic Hydrogenation
Mass of Anion
Detected
Formula Shorthand Potential Compound References Showing Structure Mass of Anion Formula Shorthand
291.1960 C
18
H
27
O
3
18:4-O OPDA
a
Creelman and
Mulpuri (2002)
295.2273 C
18
H
31
O
3
18:2-O
291.1960 C
18
H
27
O
3
18:4-O Keto-octadecatrienoic acid
(13-KOTE shown; other
isomers possible)
b
Ble
´
e and Joyard (1996);
Vollenweider et al.
(2000)
297.2430 C
18
H
33
O
3
18:1-O
291.1960 C
18
H
27
O
3
18:4-O Colnelenic acid
c
Weber et al. (1999) 299.2586 C
18
H
35
O
3
18:0-O
309.2066 C
18
H
29
O
4
18:3-2O a-org-Ketol acid (a-ketol
shown; see Fig. 1 for
g-ketol)
d
Ble
´
e (1998) 313.2379 C
18
H
33
O
4
18:1-2O
309.2066 C
18
H
29
O
4
18:3-2O Hydroperoxy-octadecatrienoic
acid (13-HPOTE shown;
other isomers possible)
e
Ble
´
e and Joyard (1996);
Hamberg et al. (2003)
315.2535 C
18
H
35
O
4
18:0-2O
309.2066 C
18
H
29
O
4
18:3-2O Dihydroxy-octadecatrienoic
acid (2,13-diHOTE shown;
other isomers possible)
f
Hamberg et al. (2003) 315.2535 C
18
H
35
O
4
18:0-2O
263.1647 C
16
H
23
O
3
16:4-O dnOPDA
g
Creelman and
Mulpuri (2002)
267.1960 C
16
H
27
O
3
16:2-O
263.1647 C
16
H
23
O
3
16:4-O Keto-16:3 269.2117 C
16
H
29
O
3
16:1-O
263.1647 C
16
H
23
O
3
16:4-O 16-Carbon analog of
colnelenic acid
271.2273 C
16
H
31
O
3
16:0-O
281.1753 C
16
H
25
O
4
16:3-2O 16-Carbon ketol 285.2066 C
16
H
29
O
4
16:1-2O
281.1753 C
16
H
25
O
4
16:3-2O Hydroperoxy-16:3 287.2222 C
16
H
31
O
4
16:0-2O
281.1753 C
16
H
25
O
4
16:3-2O Dihydroxy-16:3 287.2222 C
16
H
31
O
4
16:0-2O
a b
c d
e f g
OPDA- and Dinor-OPDA-Containing Chloroplast Polar Lipids
Plant Physiol. Vol. 142, 2006 33
oxylipin acyl species in the plastid-localiz ed complex
lipids include the same acyl specie s (OPDA, dnOPDA,
and a ketol or ketols) that were found to be increased
during wounding by CID-TOF MS scanning of
wounded Arabidopsis leaf extracts.
Interestingly, two of the most prominent OPDA-
containing MGDG species were the dioxylipin-
containing species, OPDA-dnOPDA (Fig. 4, E and F)
and OPDA-OPDA (F), while the largest DGDG species
was OPDA-OPDA (Supplemental Fig. 1F, peaks o and
o’). These are the species denoted by Hisamatsu et al.
(2003, 2005) as Arabidopsides A, B, and D, respec-
tively, and two of these lipid species are depicted in
Figure 5. The species denoted by Hisamatsu et al.
(2005) as Arabidopside C, OPDA-dnOPDA DGDG
(Supplemental Fig. 1, ArC and ArC’ in sections E and
F), was also detected in the discovery scans.
Levels of OPDA and dnOPDA-Containing Galactolipids
after Wounding
Precursor scanning was used to quantify the levels
of the major OPDA- and dnOPDA-containing galacto-
lipid molecular species in leaves without wounding
and after mechanical wounding of the leaves. Stearoyl
(18:0)-18:0 MGDG and 18:0-16:0 MGDG, prepared
by catalytic hydrogenation of purified MGDG, served
as internal standards for MGDG quantification,
and 18:0-18:0 DGDG and 1 8:0-16:0 DGD G, similarly
prepared, served as internal standards for DGDG
quantification.
In unwounded leaves, the level of each of the major
OPDA- and dnOPDA-containing galactolipid molec-
ular species was low, at less than 25 pmol per mg of
leaf dry weight (Fig. 6). Upon wounding, the levels of
these species, in particul ar the amounts of the species
containing two oxylipins, rose sharply in the first
15 min (Fig. 7). OPDA-dnOPDA MGDG rose about
200-fold, OPDA-OPDA MGDG rose over 400-fold, and
OPDA-OPDA DGDG rose over 1,000-fold in the first
15 min, while OPDA-16:3 MGDG, 18:3-dnOPDA
MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG
rose 9-, 3-, 2-, and 7-fold, respectively. Between 15 and
45 min after wounding, the levels of OPDA-dnOPDA
MGDG and OPDA-OPDA MGDG increased another
1.2- to 1 .4-fold each while the levels of OPDA-16:3
MGDG, 18:3-dnOPDA MGDG, OPDA-18:3 MGDG,
and OPDA-18:3 DGDG each rose 2- to 5-fold. Between
45 min and 20 h after wounding, levels of all the
OPDA- and dnOPDA-containing galactolipid species
Figure 4. Discovery scans: precursor scanning of an unfractionated extract from wounded Arabidopsis. All scans were performed
in the negative mode. The peak designations were verified by determination of the exact masses of the acyl product ions as
indicated in Supplemental Table I. Most peaks indicate the [M 2 H]
2
ions, while peaks indicated by labels in parentheses indicate
the [M 1 OAc]
2
adducts of MGDG. A, Scan for precursors of m/z 227 indicates PG species (Welti et al., 2003). B, Scan for
precursors of m/z 235, a glycero-galactose minus water fragment, indicates MGDG. C, Scan for precursors of m/z 249 or the acyl
anion of 16:3. D, Scan for precursors of m/z 277 or the acyl anion of 18:3. E, Scan for precursors of m/z 263 or the acyl anion of
dnOPDA. F, Scan for precursors of 291 or the acyl anion of OPDA. G, Scan for precursors of m/z 309 or the acyl anion of the ketols
derived from 18:3. The marked species are MGDG unless explicitly designated as PG. Note the relative scales of the sections. In
addition, please note that sections A to F have breaks in the vertical scales. The same scales are used in Supplemental Figure 1.
Buseman et al.
34 Plant Physiol. Vol. 142, 2006
dropped 0% to 60% with the decrease tending to be
more pronounced for the species with two oxylipin
chains. Still, at 20 h after wounding, OPDA-dnOPDA
MGDG was increased about 100-fold, OPDA-OPDA
MGDG was increased 300-fold, and OPDA-OPDA DGDG
was increased 600-fold over the level in nonwounded
tissues, while OPDA-16:3 MGDG, 18:3-dnOPDA MGDG,
OPDA-18:3 MGDG, and OPDA-18:3 DGDG were in-
creased 21-, 5-, 4-, and 18-fold. Figure 8 shows the
ratios of measured species containing two oxylipin
chains to measured species containing one oxylipin
chain at each time point. This view of the data high-
lights the rapid increase in dioxylipin species, as com-
pared to monooxylipin species, immediately after
mechanical wounding.
DISCUSSION
This work utilizes CID-TOF and traditional product
ion spectra from a Q-TOF mass spectrometer, plus
discovery scans on a triple-Q mass spectrometer, to
identify and quantify numerous plastid complex lipid
species containing oxylipins. In all, 18 (17, plus par-
tially characterized Arabidopside C) mo lecular species
of MGDG-, DGDG-, and PG-containing oxylipins were
identified, including the five described previously
(Stelmach et al., 2001; Hisamatsu et al., 2003, 2005).
The major species detected were galactolipids con-
taining OPDA and dnOPDA. In addition, ketol acids
were found to be increased during wounding and were
found to be present in MGDG and DGDG molecular
Figure 5. Three of the OPDA-containing
lipid species from leaves of wounded
Arabidopsis. Please note that the acyl
position assignments and the assign-
ment of the double bond position in the
16:1 acyl chain are not based on mass
spectral data in this work. The assign-
ments utilized in producing this figure
reflect the work of others (Hisamatsu
et al., 2003, 2005), knowledge of the
16- and 18-carbon acyl positional
specificity in plastidically produced
lipids, and knowledge of the structure
of 16:1 typically found in plastidically
produced PG (Miquel et al., 1998;
Somerville et al., 2000).
OPDA- and Dinor-OPDA-Containing Chloroplast Polar Lipids
Plant Physiol. Vol. 142, 2006 35
species. This is a powerful combination of MS methods
for identification and quantification of species of com-
plex polar lipids.
Dramatic Increases of OPDA and dnOPDA Galactolipids
after Wounding
Each of seven major OPDA- and dnOPDA-containing
species was found to increase rapidly upon wounding.
Species containing two OPD A or dnOPDA chains rose
200- to 1,400-fold, while species with a single OPDA or
dnOPDA chain rose 6- to 30-fold. Compared to previ-
ous data on the levels of a single species, OPDA-16:3
MGDG, after woundi ng (Stelmach et al., 2001), these
data are very similar, but the time course in this study
tends to indicate faster production of the OPDA-
containing species after wounding. Stelmach et al.
(2001) determined that the level of OPDA-16:3 MGDG
rose to about 20 mg per g fresh weight. If dry weight
(minus lipids) were 7% of fresh weight, then 20 mg
OPDA-16:3 MGDG/g fresh weight would correspond
to about 0.4 nmol/mg dry weight, which is the level
measured in this study. In the study by Stelmach et al.
(2001) the highest level of OPDA-16:3 MGDG was
found to occur at about 10 h, while in this study,
OPDA-16:3 MGDG rose insignificantly between 45 min
and 4 h after wounding and decreased somewhat by
20 h after wounding.
The presence of the ketol acids in the galactolipids
(Table I) suggests that a minor portion of the epox-
yoctadecatrienoic acid formed during wounding is
hydrolyzed nonenzymatically and that the nonenzymatic
pathway contributes to the formation of esterified
oxylipin species. Nonenzymatic reactions of epoxyoc-
tadecatrienoic acid result in 85% to 90% a-andg-ketols,
and 10% to 15% racemic (9S,13S and 9R,13R) OPDA
(Hamberg, 1988; Ziegler et al., 2000). Thus, the rela-
tively low level of the 18-carbon ketol acid in compar-
ison to OPDA in wounded leaves (Fig. 2B; m/z 309
versus 291) implies that the major pathway is the
enzymatic synthesis of OPDA, presumably by allene
oxide synthase. On the other hand, the formation of
free a -ketol has been shown to increase in response to
exposure to the jasmonates, JA and dnOPDA (Weber
et al., 1997).
Potential Metabolic Pathways for Oxylipin-Containing
Polar Lipids
The discovery that MGDG and DGDG species con-
taining two OPDA chains or an OPDA and a dnOPDA
chain (as depicted in Fig. 5) are the major species
formed in the initial response to wounding could imply
that lipo xygenase acts directly on plastid-localized
lipid species (Fig. 1, right-hand side), rather than on
free fatty acids released from these lipids. Although
the most abundant lipoxygenase in plastids, LIPOXY-
GENASE2, appears to be required for JA synth esis
(Bell et al., 1995) and seems to act on free fatty acids,
especially 18:3, at least in barley ( Hordeum vulgare;
Bachmann et al., 2002), it has been demonstrated that a
lipoxygenase from soybean (Glycine max) can act di-
rectly on intact phospholipids (Brash et al., 1987;
Tokumura et al., 2000). 18:3-16:3 MGDG is the most
abundant polar complex lipid species in wild-type
Arabidopsis leaves, and 18:3-18:3 MGDG and 18:3-18:3
DGDG are the next most abundant galactolipid spe-
cies. Thus, the high level s of OPDA-dnOPDA MGDG
production and next highest levels of OPDA-OPDA
DGDG and OPDA-OPDA MGDG production after
wounding are consistent with the notion of direct
conversion of esterified 18:3 and 16:3 to OPDA and
Table III. Lipid molecular species identified by accurate mass
product ion analysis of fractionated extracts from wounded
Arabidopsis (Supplemental Table I)
The assignments of position are based on previous work showing that
in species containing 16- and 18-carbon acyl chains, the 16-carbon
acyl species are at position 2 (Miquel et al., 1998). Position assignments
are not based on the mass spectral data. These identifications were
confirmed by analysis of catalytically hydrogenated molecular species
(Supplemental Table II).
[M 2 H]
2
Ion of m/z Interpretation
PG 755 OPDA/16:1
757 OPDA/16:0
MGDG 759 OPDA/16:3
759 18:3/dnOPDA
773 OPDA/dnOPDA
777 18-Carbon ketol/16:3
777 18:3/16-carbon ketol
787 OPDA/18:3
791 18-Carbon ketol/dnOPDA
791 OPDA/16-carbon ketol
801 OPDA/OPDA
805 18:3/18C ketol
819 OPDA/18C ketol
DGDG 949 OPDA/18:3
963 OPDA/OPDA
967 18:3/18C ketol
981 OPDA/18C ketol
Figure 6. Levels of OPDA and dnOPDA-containing galactolipids in
unwounded Arabidopsis. Error bars are
SD, n 5 5.
Buseman et al.
36 Plant Physiol. Vol. 142, 2006
dnOPDA, respectively. Lipoxygenase might be more
likely to act on two acyl chains on the same lipid than
on two acyl chains on distinct molecules, due to the
proximity of the second acyl substrate in an intact polar
complex lipid. If the dioxylipin species are produced on
the intact plastid polar complex lipids, then decreases
with time of dioxylipin species and concomitant in-
creases in species with a single oxylipin chain may result
from a deacylation of the dioxylipin species, followed by
reacylation with a normal-chain fatty acid.
The very rapid and very large increase in levels of
the OPDA-dnOPDA and OPDA-OPDA species lend
credence to the assertion that these species are created
on the intact galactolipids rather than via the free fatty
acid pathway (Fig. 1). OPDA-dnOPDA and OPDA-
OPDA galactolipids rose 200- to 1,000-fold in the first
15 min after wounding, while free OPDA has been
shown to rise from 3- to 15-fold in the first several
hours after wounding (Weber et al., 1997; Stelmach
et al., 2001; Stintzi et al., 2001). Other free fatty acids,
such as 18:3, also increase upon wounding, though to a
lesser extent, about 2-fold (Zien et al., 2001). While the
production of dioxylipin galactolipid species alterna-
tively might be driven by high levels of free OPDA and
dnOPDA in relation to other fatty acyl species in the
time frame soon after wounding, this is difficult to
envision, given the unimpressive increase in OPDA
relative to other free fatty acids. In addition, free
OPDA is present at relatively low absolute levels
after wounding as compared to OPDA-dnOPDA and
OPDA-OPDA galactolipids. Using the estimate of dry
weight minus lipid being equal to 7% of fresh weight,
OPDA levels after wounding range from 0.035 to 0.085
nmol/mg dry weight (minus lipid; Weber et al., 1997;
Stelmach et al., 2001; Stintzi et al., 2001), as compared
to peak levels of more than 2.5 nmol/mg dry weight
(minus lipid) for the most prevalent oxylipin-containing
galactolipid species, OPDA-dnOPDA MGDG. Thus,
while these data do not exclude the possibility that
OPDA and dnOPDA are synth esized in their free
forms and incorporated into the plastid membrane
lipids, the rapid and large increase favors the hypoth-
esis that intact galactolipid molecules are the sub-
strates for OPDA and dnOPDA formation, rather than
the oxygenated species being formed as free fatty
acids, followed by incorporation into galactolipids.
The enzymes involv ed in production of complex plas-
tid lipid species containing oxylipins remain to be
elucidated. Analyses of lipid species level s in mutants
Figure 7. Levels of the major oxylipin-containing
MGDG and DGDG species during response to
wounding in Arabidopsis leaves. Wounded leaves
were collected at the time points indicated, lipids
were extracted, and galactolipids containing es-
terified oxylipins were analyzed using multiple
acyl precursor scanning. A, MGDG species. B,
DGDG species. The zero time point represents
unwounded plants. Error bars are
SD, n 5 5.
Figure 8. Ratios of measured galactolipid species containing two
oxylipins and galactolipid species containing one oxylipin. For
MGDG, ([OPDA-dnOPDA MGDG] 1 [OPDA-OPDA MGDG])/
([OPDA-16:3 MGDG] 1 [18:3-dnOPDA MGDG] 1 [OPDA-18:3
MGDG]) and for DGDG, (OPDA-OPDA DGDG)/(OPDA-18:3
DGDG) are plotted as a function of the time after wounding. The
zero time point represents unwounded plants. Error bars are
SD, n 5 5.
OPDA- and Dinor-OPDA-Containing Chloroplast Polar Lipids
Plant Physiol. Vol. 142, 2006 37
with alterations in enzymes catalyzing acylation and
deacylation reactions are in progress.
In a wounded leaf, galactolipid species containing
OPDA represent several percent of the total galactolip-
ids (typically totaling approximately 100 nmol/mg
dry weight). If OPDA- and dnOPDA-containing polar
complex lipid species are precursors of free OPDA and
dnOPDA, then these species may serve as a reservoir
for the production of free OPDA and dnOPDA at later
times. It is also plausible that these lipids, with their
folded-back acyl chains, might create a drastic altera-
tion of plastid membrane structure that could affect
the function of other membrane components.
CONCLUSION
A combination of MS methods has been used to
discover acyl chains present in complex lipid mix-
tures, to discover the polar complex lipid species
containing each of these chains, to verify the presence
of particular complex lipid species, and to quantify the
polar complex lipid species in unfractionat ed lipid
extracts. The ability to quantify specific, oxylipin-
containing, polar complex lipid species will facilitate
our understanding of the physiological roles of these
prominent plant lipids.
MATE RIALS AND METHODS
Plant Material
Leaves from approximately 7-week-old Arabidopsis ( Arabidopsis thaliana)
ecotype Columbia plants were used for identification and kinetic analysis of
the lipid species. Seeds were sown in Metro-Mix 360 soil (Scotts Company)
and allowed to equilibrate in a 4°C chamber, covered with clear plastic for 72 h.
The plants were transferred to a growth chamber at 22°C and 60% humidity
with a 12/12 photoperiod at 210 mmol m
22
s
21
. The plants were covered with
clear plastic for 1 week, were left partially uncovered for 72 h, and then were
uncovered for the remainder of the growth period. Two weeks after the seeds
were sown, seedlings were thinned so that four or five plants remained,
equidistant, in each 3-inch square pot. Miracle-Gro water soluble all purpose
plant food (Scotts Company) was applied every 3 weeks, beginning at the time
the seeds were sown, according to manufacturer’s instructions.
Wounding Treatment, Sampling, and Lipid Extraction
Wounding was performed as described by Laudert and Weiler (1998). Five
to six leaves of each rosette were crushed using a hemostat so that approx-
imately 10% of the total leaf area was wounded. Care was taken to not crush
the midvein. At the indicated time after wounding, three to five wounded
leaves were collected and immediately immersed in 75°C isopropanol
containing 0.01% butylated hydroxytoluene. Extractions were continued as
described by Welti et al. (2002). The solvent from each sample was evaporated
and the sample was dissolved in 1 mL chloroform. Lipid amounts were
normalized to dry weight, as measured after extraction of the lipid (Welti et al.,
2002). Five replicate samples were collected for each experimental time point.
Chromatographic Fractionation
Activated silicic acid (Unisil, Clarkson Chemical) was mixed with chloro-
form and packed into a column (1.5 cm diameter, 40 mL column volume).
Crude extract containing lipid from 144 mg dry weight of Arabidopsis leaves
in chloroform was allowed to bind to the colu mn and was then eluted in five
fractions: fraction 1, chloroform:acetone (1:1, v/v), 200 mL; fraction 2, acetone,
400 mL; fraction 3, chloroform:methanol (19:1, v/v), 400 mL; fraction 4,
chloroform:methanol (4:1, v/v), 400 mL; and fraction 5, chloroform:methanol
(1:1, v/v), 800 mL (Christie, 1982). This scheme resulted in the elution of
MGDG primarily in fraction 2, PG primarily in fraction 3, and DGDG in both
fractions 2 and 3. The major phospholipids (phosphatidylethanolamine,
phosphatidylcholine, phosphatidylinositol, and phosphatidylserine) were
eluted primarily in fractions 4 and 5. The solvent was evaporated from each
fraction, and fractions were dissolved in chloroform before mass spectral
analysis in chloroform:methanol:300 m
M ammonium acetate (60:133:7, v/v).
Catalytic Hydrogenation
Crude lipid extract was evaporated and dissolved in ethyl acetate/meth-
anol (1:1, v/v) with platinum (IV) oxide at 1% by lipid weight. The suspension
was subjected to .1 atm H
2
at 25°C for 6 h. Hydrogenated lipid was
centrifuged to remove platinum residue, and the supernatant was evaporated
and dissolved in chloroform.
Multiple Acyl Precursor Scans
An automated ESI-MS/MS approach was used and data acquisition and
analysis were performed as described previously (Welti et al., 2002; Wanjie
et al., 2005) with minor modifications. Briefly, unfractionated lipid extracts
were introduced by continuous infusion into the ESI source on a triple-Q
MS/MS (API 4000, Applied Biosystems). Sequential precursor scans of acyl
anions in the galactolipid mass range (740–1,050 amu) of the extracts pro-
duced a series of spectra with each spectrum revealing a set of lipid species
containing either a common head group or specific fatty acyl chain. When
scans were to be used for quantification purposes, internal standards, 16:
0-18:0 MGDG (2.01 nmol), 18:0-18:0 MGDG (0.39 nmol), 16:0-18:0 DGDG (0.49
nmol), and 18:0-18:0 DGDG (0.71 nmol), were added. The internal standards
were prepared by catalytic hydrogenation of MGDG and DGDG purified
(separately) from unwounded Arabidopsis (Christie, 1982). Each of the
components, i.e. 18:0-18:0 MGDG and 18:0-16:0 MGDG (and the same species
in DGDG), were quantified by gas chromatography of the fatty acid methyl
esters. 18:0-18:0 MGDG, 18:0-16:0 MGDG, 18:0-18:0 DGDG, and 18:0-16:0
DGDG can be used as internal standards because the biological samples do
not contain these fully saturated compounds.
Samples were introduced using an autosampler (LC Mini PAL, CTC
Analytics AG) fitted with the required injection loop for the acquisition time
and presented to the ESI needle at 30 mL/min. The collision gas pressure was
set at 4 (arbitrary units), the source temperature (heated nebulizer) was 100°C,
the interface heater was on, 24.5 kV was applied to the electrospray capillary,
the curtain gas was set at 20 (arbitrary units), and the two ion source gases at
45 (arbitrary units). The collision energy was 245 V with nitrogen in the
collision cell, declustering potential was 2100 V, entrance potential was 210 V,
and exit potential was 220 V. The mass analyzers were adjusted to a resolution
of 0.7 amu full width at half height. For each spectrum, 36 continuum scans
were averaged in multiple channel analyzer mode. The background was
subtracted, the data were smoothed, and peak areas integrated using a custom
script and Applied Biosystems Analyst software.
Data Handling and Quantification
of Galactolipid Species
The m/z and peak area data were sorted using Microsoft Excel to find peaks
within each fatty acyl precursor spectrum corresponding to each target
galactolipid molecular species. Peak areas were corrected for precursor ion
isotopic distribution (Han and Gross, 2001). Peak areas of the same precursor
from its two acyl precursor scans were added, except when the precursor had
two of the same acyl chain, in which case, the peak area was used directly. The
total peak areas for each species were quantified in comparison to a standard
line in a plot of total peak area versus m/z, created with isotopically corrected
data from two galactolipid internal standards, 16:0-18:0 MGDG and 18:0-18:0
MGDG (for quantification of MGDG species), or 16:0-18:0 DGDG and 18:0-18:0
DGDG (for quantification of DGDG species).
Q-TOF MS/MS and CID-TOF MS Analysis
ESI-Q-TOF MS/MS and ESI-CID-TOF MS spectra were acquired with
a Micromass Q-TOF-2 tandem mass spectrometer (Micromass). The TOF
analyzer was tuned for maximum resolution (10,000 resolving power) with
argon in the collision cell. Micromass MassLynx software was used as the
operating software. All TOF spectra were acquired with daily mass calibration.
Buseman et al.
38 Plant Physiol. Vol. 142, 2006
For Q-TOF analysis, samples conta ining silicic acid-fractionated lipids
were infused directly in chloroform:methanol:300 m
M ammonium acetate in
water (60:133:7, v/v) at 30 mL/min into the ESI source of the Q-TOF.
Negatively charged precursor target peaks, selected by the Q tuned to
transmit at 0.8 amu full width at half height, were subjected to product ion
scanning in the negative ion mode. The collision energy was 35 V. For CID-
TOF MS analysis, radio frequency/direct current mass selection in the Q is
turned off, while the collision cell remains at 35 V offset. Unfractionated
samples in chloroform:methanol:300 m
M ammonium acetate in water
(60:133:7, v/v) were infused at 20 mL/min into the ESI source.
The unselected precursor ions were subjected to CID at 35 V in the negative
mode.
Both Q-TOF and CID-TOF mass spectra were mass corrected by locking the
m/z to the determined theoretical m/z of the ion formed from a head group
fragment with an exact mass of m/z 253.0923 in MGDG (in both Q-TOF and
CID-TOF modes) and m/z 397.1346 (in Q-TOF mode) in DGDG. With the
locked mass value set, the exact masses of product ions were calculated to ten
thousandths of an amu. Chemical formulas for the product ions were
determined using the Micromass MassLynx chemical formula tool. Each
designated chemical formula match was the best match for a formula
containing the indicated elements. Deviations between the detected m/z and
theoretical m/z, calculated by the chemical formula tool, of the best-matched
chemical formula were determined.
ACKNOWLEDGMENTS
We would like to thank Dr. Giorgis Isaac for critical reading of the
manuscript. We would also like to thank the reviewers for their helpful
comments.
Received April 13, 2006; accepted June 30, 2006; published July 14, 2006.
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    • "While studying the canonical JA biosynthetic pathway initiated from free FAs, came as an unexpected fi nding that some JAs can occur abundantly as acyl parts of plastid galactolipids (Stelmach et al. 2001 ), exceeding largely the abundance of free JAs. A number of different variants known as " arabidopsides " have successively been characterized, and contain generally one to three OPDA or dinor-OPDA moieties esterifi ed to the sn -1 or sn -2 glycerol position, or bound to the galactose headgroup itself (Fig. 16.1; Hisamatsu et al. 2005 ; Andersson et al. 2006 ; Buseman et al. 2006 ; Böttcher and Weiler 2007 ). Several of these compounds accumulate to very high levels in vegetative tissues after wounding or during the hypersensitive response to an ectopically-expressed bacterial avirulence protein (Andersson et al. 2006 ), and this accumulation depends on an intact JA signaling pathway (Kourtchenko et al. 2007 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Jasmonates (JAs) constitute a major class of plant regulators that coordinate responses to biotic and abiotic threats and important aspects of plant development. The core biosynthetic pathway converts linolenic acid released from plastid membrane lipids to the cyclopentenone cis-oxo-phytodienoic acid (OPDA) that is further reduced and shortened to jasmonic acid (JA) in peroxisomes. Abundant pools of OPDA esterifi ed to plastid lipids also occur upon stress, mainly in the Arabidopsis genus. Long thought to be the bioactive hormone, JA only gains its pleiotropic hormonal properties upon conjugation into jasmonoyl-isoleucine (JA-Ile). The signaling pathway triggered when JA-Ile promotes the assembly of COI1-JAZ (Coronatine Insensitive 1-JAsmonate Zim domain) co-receptor complexes has been the focus of most recent research in the jasmonate field. In parallel, OPDA and several other JA derivatives are recognized for their separate activities and contribute to the diversity of jasmonate action in plant physiology. We summarize in this chapter the properties of different bioactive JAs and review elements known for their perception and signal transduction. Much progress has also been gained on the enzymatic processes governing JA-Ile removal. Two JA-Ile catabolic pathways, operating through ω-oxidation (cytochromes P450) or conjugate cleavage (amido hydrolases) shape signal dynamics to allow optimal control on defense. JA-Ile turnover not only participates in signal attenuation, but also impact the homeostasis of the entire JA metabolic pathway.
    Full-text · Article · Mar 2016 · Plant Cell and Environment
    • "Increasing evidence revealed that esterified lipid-bound fatty acids serve as substrates for LOX. The resulting esterified fatty acid hydroperoxides can be further catalysed by AOS and HPL, but not by POX (Buseman et al. 2006; Nakashima et al. 2013; Savchenko et al. 2014). Phytoprostanes form another group of oxylipins, which are mainly generated by non-enzymatic oxidation of linolenate via reactive oxygen species (Andreou et al. 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Lipids are one of the major components of biological membranes including the plasma membrane which is the interface between the cell and the environment. It has become clear that membrane lipids also serve as substrates for the generation of numerous signalling lipids such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, N-acylethanolamines, free fatty acids and others. The enzymatic production and metabolism of these signalling molecules is tightly regulated and can rapidly be activated upon abiotic stress signals. Abiotic stress like water deficit and temperature stress triggers lipid-dependent signalling cascades, which control the expression of gene clusters and activate plant adaptation processes. Signalling lipids are able to recruit protein targets transiently to the membrane and thus affect conformation and activity of intracellular proteins and metabolites. In plants, knowledge is still scarce of lipid-signalling targets and their physiological consequences. This review focuses on the generation of signalling lipids and their involvement in response to abiotic stress. We describe lipid-binding proteins in the context of changing environmental conditions and compare different approaches to determine lipid-protein interactions, crucial for deciphering the signalling cascades.
    Article · Oct 2015
    • "The resulting increase in the ratio of MGDG to DGDG could lead to loss of membrane integrity. Wounding stress causes activation of phospholipase D and phospholipase A and oxidation of fatty acids on galactolipids (Narvaez-Vasquez et al. 1999; Ryu 2004; Buseman et al. 2006; Zien et al. 2001 ). The amount of oxidized galactolipids might reflect the degree of oxidative stress a plant is experiencing. "
    [Show abstract] [Hide abstract] ABSTRACT: Identifying lipids that experience coordinated metabolism during heat stress would provide information regarding lipid dynamics under stress conditions and assist in developing heat-tolerant wheat varieties. We hypothesized that co-occurring lipids, which are up-or-down-regulated together through time during heat stress, represent groups that can be explained by coordinated metabolism. Wheat plants (Triticum aestivum L.) were subjected to 12 days of high day and/or night temperature stress, followed by a 4-day recovery period. Leaves were sampled at four time points, and 165 lipids were measured by electrospray ionization-tandem mass spectrometry. Correlation analysis of lipid levels in 160 leaf samples from each of two wheat genotypes revealed 13 groups of lipids. Lipids within each group co-occurred through the high day and night temperature stress treatments. The lipid groups can be broadly classified as groups containing: extraplastidic phospholipids, plastidic glycerolipids, oxidized glycerolipids, triacylglycerols, acylated sterol glycosides, and sterol glycosides. Current knowledge of lipid metabolism suggests that the lipids in each group co-occur because they are regulated by the same enzyme(s). The results suggest that increases in activities of desaturating, oxidizing, glycosylating, and acylating enzymes lead to simultaneous changes in levels of multiple lipid species during high day and night temperature stress in wheat. This article is protected by copyright. All rights reserved.
    Full-text · Article · Oct 2015
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