Báo cáo khoa học: Unprecedented pathogen-inducible complex oxylipins from flax – linolipins A and B docx

Unprecedented pathogen-inducible complex oxylipins
from flax – linolipins A and B
Ivan R. Chechetkin, Fakhima K. Mukhitova, Alexander S. Blufard, Andrey Y. Yarin,
Larisa L. Antsygina and Alexander N. Grechkin
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Russia
Introduction
The lipoxygenase cascade and its product the oxylipins
[1,2], including jasmonates [3–7], play important roles
in plant signaling and defense. The primary lipoxygen-
ase products, fatty acid hydroperoxides, undergo
further metabolic conversion controlled by enzymes of
the unique CYP74 family (P450 superfamily) [8–12].
The three enzymes are hydroperoxide lyase (isomer-
ase), allene oxide synthase (dehydrase) and divinyl
ether synthase (DES; dehydrase). Enzymes from the
CYP74 family produce a large diversity of oxylipins.
For example, allene oxide synthase and allene oxide
cyclase control the biosynthesis of cyclopentenone
(9S,13S,15Z)-12-oxo-phytodienoic acid (12-oxo-PDA),
the precursor of phytohormone jasmonates [8,13,14].
Hydroperoxide lyase transforms fatty acid hydroper-
oxides into the short-lived hemiacetals, which are
spontaneously decomposed into aldehydes and aldo-
acids [15,16]. The volatile aldehydes produced by
hydroperoxide lyase are involved in cell and interplant
signaling, as well as in plant defense against pathogens
and insects [17,18].
Divinyl ethers constitute a family of oxylipins detected
in a limited number of plant species, from brown and red
algae to eurosids II (Solanaceae) [19–31]. The biosynthe-
sis of divinyl ethers is controlled by DES [10,32,33]. Divi-

nyl ethers and DESs are shown to be involved in plant
defense against pathogens [34–39]. Recently, we reported
the detection of DES activity and the divinyl ethers
Keywords
divinyl ether synthase; flax; lipoxygenase
cascade; oxylipins; pathogenesis
Correspondence
A. N. Grechkin, Kazan Institute of
Biochemistry and Biophysics, Russian
Academy of Sciences, P.O. Box 30, 420111,
Kazan, Russia
Fax: +7 843 292 7347
Tel: +7 843 231 9022
E-mail:
Website: />lab_ox_e.html
(Received 28 April 2009, revised 28 May
2009, accepted 12 June 2009)
doi:10.1111/j.1742-4658.2009.07153.x
Oxylipins constitute a large family of bioregulators, biosynthesized via
unsaturated fatty acid oxidation. This study reports the detection of an
unprecedented family of complex oxylipins from flax leaves. Two major
members of this family, compounds 1 and 2, were isolated and purified.
Their structures were evaluated using NMR and MS analyses. Both com-
pounds were identified as monogalactosyldiacylglycerol species. Compound
1 contains one a-linolenoyl residue and one residue of (9Z,11E,1¢Z,3¢Z)-12-
(1¢,3¢-hexadienyloxy)-9,11-dodecadienoic, (x5Z)-etherolenic acid at posi-
tions sn-1 and sn-2, respectively. Compound 2 possesses (x5Z)-etherolenic
acid residues at both position sn-1 and position sn-2. We suggest the trivial
names linolipin A and linolipin B for compounds 1 and 2, respectively, and
the collective name linolipins for this new family of complex oxylipins. The

linolipin content of flax leaves increased significantly in response to patho-
genesis. Thus, accumulation of esterified antimicrobial divinyl ethers may
be of relevance to plant defense.
Abbreviations
12-oxo-PDA, (9S,13S,15Z)-12-oxo-phytodienoic acid; DES, divinyl ether synthase; DGDG, digalactosyldiacylglycerol; EDE, esterified divinyl
ether; HRMS, high-resolution mass spectrometry; MGDG, monogalactosyldiacylglycerol.
FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS 4463
(9Z,11E,1¢Z)-12-(1¢-hexenyloxy)-9,11-dodecadienoic [(x
5Z)-etheroleic] acid and (9Z,11E,1¢Z,3¢Z)-12-(1¢,3 ¢-hexa-
dienyloxy)-9,11-dodecadienoic [(x5Z)-etherolenic] acid
in flax leaves [40]. Along with members of the Ranun-
culaceae [27–30], flax presents an additional example
of a plant species possessing strong DES activity in
leaves. These observations prompted us to inspect the
possible occurrence of oxylipin esters in complex lipids
from flax leaves. The resulting observations are described
in here.
A family of complex oxylipins has been detected
previously in Arabidopsis leaves [41–48]. These lipids,
named arabidopsides, are galactolipids containing
esterified oxylipin residues, namely 12-oxo-PDA and
2,3-dinor-12-oxo-PDA. Here, we report the detection
of a distinct, unprecedented family of complex oxyli-
pins in flax leaves. Their distinctive feature is the pres-
ence of esterified divinyl ether (EDE) residues. These
pathogen-inducible compounds detected in flax leaves
are named linolipins. The detection and identification
of linolipins A and B, the first members of linolipin
family, is described.
Results

Detection of linolipins
Total lipids extracted from 35-day-old flax leaves were
separated into different classes (neutral lipids, galactol-
ipids and phospholipids) using silicic acid column
chromatography. The UV spectrum of the phospho-
lipid fraction did not reveal the presence of 12-oxo-
PDA (k
max
at 221 nm) or (x5Z)-etherolenic acid (k
max
at 267 nm). The galactolipids monogalactosyldiacyl-
glycerol (MGDG) and digalactosyldiacylglycerol
(DGDG) were separated by micropreparative TLC.
Both MGDG and DGDG fractions exhibited strong
UV absorption at 267 nm and lacked a maximum at
221 nm, indicating the possible presence of divinyl
ether (x5Z)-etherolenic acid residues bound to galac-
tolipids. To examine this possibility, the molecular spe-
cies of galactolipids were separated by RP-HPLC
using an online UV spectral record with a diode array
detector.
Galactolipids extracted from unstressed flax leaves
possessed a single predominant molecular species
absorbing at 267 nm (compound 1; Fig. 1A). Inocula-
tion of plants with cells of the phytopathogenic bacte-
rium Erwinia carotovora subsp. atroseptica altered the
profile of the galactolipid molecular species. Galactoli-
pids extracted 4 h after inoculation possessed the addi-
tional molecular species 2 (Fig. 1B). By 24 h after
inoculation, more significant changes had occurred

(Fig. 1C). At this time, flax leaves had depigmented
spots ( 5% of total leaf area), which are characteris-
tic of infection. At this time point (24 h after inocula-
tion), the prominent molecular species 3 appeared
alongside molecular species 1 and 2, (Fig. 1C). All
mentioned species 1-3 exhibited k
max
at 267 nm, sug-
gesting the possible presence of EDE moieties. Neither
infected nor control plants possessed any galactolipid
molecular species with k
max
at 221 nm. This indicated
the absence of arabidopsides or any related galactoli-
pid species possessing esterified 12-oxo-PDA moieties.
GC-MS analyses of fatty acid methyl esters formed
during the transesterification of galactolipids did not
reveal the presence of 12-oxo-PDA. The galactolipid
molecular species were separated by RP-HPLC.
Compounds 1 and 2 were collected and finally purified
by cyanopropyl-phase HPLC for further structural
elucidation.
Identification of compound 1, linolipin A
Pure compound 1 possessed a UV absorbance maxi-
mum at 267 nm with a smooth shaped spectral band.
Fig. 1. RP-HPLC profiles of galactolipid molecular species from flax
leaves. Total galactolipids were extracted from flax leaves, sepa-
rated and purified as described in Materials and methods. UV chro-
matograms (267 nm) of galactolipids extracted from: (A) unstressed
plants, (B) infected plants (at 4 h after inoculation with E. carotovo-

ra atroseptica), (C) infected plants (at 24 h after inoculation with
E. carotovora atroseptica).
Unprecedented complex oxylipins from flax I. R. Chechetkin et al.
4464 FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS
The spectrum was identical to that of the divinyl ether
(x5Z)-etherolenic acid [27,40]. Compound 1 was
transesterified and the resulting fatty acid methyl esters
were subjected to GC-MS analysis. Two products were
detected, namely the methyl esters of a-linolenic acid
and (x5Z)-etherolenic acid. The electron impact mass
spectrum of (x5Z)-etherolenic acid methyl ester (M
+
at m ⁄ z 306) was identical to that described previously
[40]. Methyl esters formed through the transesterifica-
tion of compound 1 were catalytically hydrogenated.
GC-MS analysis of hydrogenation products revealed
the presence of methyl stearate and the methyl ester of
13-oxa-nonadecanoic acid. The mass spectrum of the
latter was identical to that reported previously [40].
Formation of 13-oxa-nonadecanoic acid (Me ester)
confirms the presence of (x5Z)-etherolenic acid (Me
ester) among the transesterification products.
The negative-ion mode mass spectrum of com-
pound 1 (Fig. 2 and Table S1) exhibited a quasimolec-
ular ion [M–H]
-
at m ⁄ z 787.4909 (C
45
H
71

O
11
), as well
as the adduct [M+CH
3
COO]
-
at m ⁄ z 847.5229
(C
47
H
75
O
13
). The MS⁄ MS spectrum of m ⁄ z 787.4909
showed ions at m ⁄ z 291.1954 [C
18
H
27
O
3
,(x5Z)-ethero-
lenic acid anion] and m ⁄ z 277.2120 (C
18
H
29
O
2
, a-lino-
lenic acid anion). The positive-ion mode mass

spectrum of compound 1 (Table S1) showed the ion
[M+NH
4
]
+
at m ⁄ z 806.5418 (C
45
H
76
O
11
N). The
MS ⁄ MS of ion m ⁄ z 806.5418 yielded a diagnostic frag-
ment at m ⁄ z 529.3287 (C
27
H
47
O
9
N, loss of a-linolenic
acid residue). The obtained high-resolution mass spec-
trometry (HRMS) data revealed the empirical formula
C
45
H
72
O
11
for compound 1. Both the MS and MS ⁄ MS
data are consistent with the MGDG structure contain-

ing one residue of a-linolenic acid and one of (x5Z)-
etherolenic acid.
NMR data (Fig. 3 and Table S2) provide further
evidence supporting the identification of compound 1
as a MGDG species. The chemical shift (4.18 p.p.m.)
and coupling constant (7.4 Hz) of anomeric proton
H1¢ prove the b-linkage. Other sugar proton-coupling
constants (J
2¢,3¢
= 9.7 Hz; J
2¢,3¢
= 3.3) and chemical
shifts demonstrate the presence of a single b-d-galacto-
pyranose moiety in compound 1, in full agreement
with the literature [42,44–46]. The signals of glycerol
protons H1a,b (4.32 and 4.18 p.p.m.) and H2
(5.19 p.p.m.) are shifted downfield relative to signals of
H3a,b (3.89 and 3.68 p.p.m.). This indicates the pres-
ence of ester substituents at sn-1 and sn-2, and a b-d-
galactopyranose residue at sn-3. The olefinic part of
the spectrum demonstrates the presence of one a-lino-
lenic acid residue (signals of six double-bond protons
H9¢¢ ¢¢, H10¢¢ ¢¢, H12¢¢ ¢¢, H13¢¢ ¢¢, H15¢¢ ¢¢ and H16¢¢ ¢¢
at 5.27–5.45 p.p.m.; a triplet of two interolefinic meth-
ylenes H11¢¢ ¢¢ and H14¢¢ ¢¢ at 2.81 p.p.m., four pro-
tons). The second fatty acyl moiety exhibits the signals
of eight olefinic protons: H9¢¢ (5.31 p.p.m., m), H10¢¢
Fig. 2. The high-resolution ESI-MS and
MS ⁄ MS data for compound 1. (A) The nega-
tive-ion mode MS and MS ⁄ MS fragmenta-

tion scheme of precursor ion [M–H]
-
, m ⁄ z
787.4909; (B) negative-ion mode full ESI-MS
of compound 1; (C) the MS ⁄ MS spectrum
of ion m ⁄ z 787.4909.
I. R. Chechetkin et al. Unprecedented complex oxylipins from flax
FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS 4465
(5.87 p.p.m., dd), H11¢¢ (6.07 p.p.m., ddd), H12¢¢
(6.72 p.p.m., d), H1¢¢ ¢ (6.32 p.p.m., d), H2¢¢ ¢ (5.53 p.p.m.,
ddt), H3¢¢ ¢ (6.26 p.p.m., dddt) and H4¢¢ ¢ (5,42 p.p.m., m).
These signals, their multiplicity, coupling constants
and the arrangement of the spin interactions between
them (estimated from the 2D-COSY data, Fig. S1)
enable us to identify this fatty acid moiety as (x5Z)-
etherolenic acid, (9Z,11E,1¢Z,3¢Z)-12-(1¢,3¢-hexadienyl-
oxy)-9,11-dodecadienoic acid. The spectral data fully
correspond to the literature data for (x5Z)-etherolenic
acid.
Ultimately, the MS and NMR data show that com-
pound 1 is a MGDG species possessing one a-linole-
noyl residue and one residue of the divinyl ether,
(x5Z)-etherolenic acid. However, neither the MS nor
the NMR spectral data allowed us to estimate the
exact distribution of a-linolenic and (x5Z)-etherolenic
acid moieties between the glycerol sn-1 and sn-2 posi-
tions. To examine their positions, compound 1 was
subjected to regiospecific hydrolysis by the sn-1-specific
Rhizopus arrhizus lipase. Liberated fatty acids (as Me
esters) were analyzed by GC-MS. Only the a-linolenic

acid (Me ester), and not (x5Z)-etherolenic acid (Me
ester), was detected. At the same time, treatment with
unspecific Mucor javanicus lipase released both a-lino-
lenic and (x5Z)-etherolenic acids from compound 1.
These data demonstrate that a-linolenate and (x5Z)-
etherolenic acid moieties are esterified to the sn-1 and
sn-2 positions, respectively. Taken together, these data
enable us to identify compound 1 as 1-O-a-linolenoyl-
2-O-(x5Z)-etherolenoyl-3-O-b-d-galactopyranosyl-sn-glyc-
erol (see the structural formula in Fig. 2). This com-
pound is the first member of the unprecedented
complex oxylipins: galactolipids, featuring an EDE
moiety. We suggest the trivial name linolipin A for
compound 1 and the collective name linolipins for this
new family of complex oxylipins from flax.
Identification of compound 2, linolipin B
Compound 2 has a UV spectrum identical to that of
compound 1. In contrast to compound 1, compound 2
afforded only a single transesterification product,
namely the (x5Z)-etherolenic acid methyl ester (M
+
at
m ⁄ z 306), as shown by GC-MS data. Its identification
was also confirmed by conversion to 13-oxa-nonadeca-
noic acid (Me ester) upon the catalytic hydrogenation
of transesterification products.
The negative-ion mode mass spectrum of com-
pound 2 (Fig. 4 and Table S3) exhibited a quasimolec-
ular ion [M–H]
-

at m⁄ z 801.4765 (C
45
H
69
O
12
) and the
adduct [M+CH
3
COO]
-
at m ⁄ z 861.4909 (C
47
H
73
O
14
).
The MS ⁄ MS spectrum of m ⁄ z 801.4765 showed the
product ion at m ⁄ z 291.1951 [C
18
H
27
O
3
,(x5Z)-ethero-
lenic acid anion]. The positive-ion mode mass spectrum
of compound 2 (Table S3) showed the ion [M+NH
4
]

+
at m ⁄ z 820.5231 (C
45
H
76
O
11
N). The obtained HRMS
data predict the empirical formula C
45
H
70
O
12
for
compound 2. Both the MS and the MS ⁄ MS data are
consistent with a MGDG structure containing two
residues of (x5Z)-etherolenic acid.
1
H NMR (Fig. 3 and Table S4) and 2D-COSY data
(Fig. S1) for compound 2 showed significant similarity
to those for compound 1. First, the spectrum possessed
identical signals of glycerol and b-d-galactopyranose
moieties. Second, it possessed the same eight signals of
olefinic protons of the (x5Z)-etherolenic acid residue
between 5.25 and 5.80 p.p.m. At the same time, some
details of the spectra for compounds 1 and 2 were
clearly distinct. First, the spectrum for compound 2
lacked a strong multiplet of olefinic protons of a-lino-
lenic acid, which was present in the spectrum of com-

pound 1. This indicates the absence of an a-linolenate
moiety in compound 2, in full agreement with the MS
and MS ⁄ MS data. Second, the integral intensities of
olefinic signals in the compound 2 spectrum were twice
as large as the signal intensities for separate protons of
glycerol and b-d-galactose moieties (Fig. 3). This dem-
onstrates that compound 2 has two (x5Z)-etherolenic
acid residues esterified at the sn-1 and sn-2 positions of
glycerol.
These data enable us to identify compound 2 as
MGDG possessing two (x5Z)-etherolenic acid resi-
dues, i.e. 1,2-di- O-(x5Z)-etherolenoyl-3-O-b-d-galac-
topyranosyl-sn-glycerol (see the structural formula in
Fig. 4). We suggest the trivial name linolipin B for this
novel compound, a second member of linolipin family.
The amount of this linolipin is significantly increased
in infected flax leaves (Fig. 1).
The age dependence of linolipin content in
flax leaves
Young (14- and 23-day-old) flax leaves did not possess
EDEs (Fig. 5). However, EDEs were abundant in flax
leaves at later stages of ontogenesis, including stem
elongation (35 days old), inflorescence emergence
(63 days old) and flowering (76 days old). The EDE
content of the leaves during these stages comprised
50–71 nmolÆg
)1
of fresh weight (Fig. 5). The lack of
EDE in young flax leaves correlated with an absence
of free (x5Z)-etherolenic acid and a lack of DES activ-

ity (Fig. S2). This dependence of EDE content and
DES activity on plant age prompted us to test the
effects of (x5Z)-etherolenic acid Me ester and (2E)-
hexenal (the product of etherolenic acid decomposition)
Unprecedented complex oxylipins from flax I. R. Chechetkin et al.
4466 FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS
on flax seed germination. Both oxylipins inhibited the
germination of flax seeds (Doc. S1 and Fig. S3). Thus,
the correlation between ontogenesis and linolipin con-
tent cannot be accidental. It should be noted that
neither linolipins nor any other EDEs were detectable
in nongerminated flax seeds (not illustrated).
Influence of pathogenesis on linolipin content
Inoculation of flax plants with E. carotovora subsp.
atroseptica induced EDE accumulation in the leaves.
The levels of EDE (DGDG and MGDG) increased by
3- and 5.5-fold, respectively, 4 h after inoculation
(Fig. 6). At 24 h after inoculation, the EDE content
(both DGDG and MGDG) increased dramatically
(Fig. 6), up to 800 nmolÆg
)1
fresh weight. This accu-
mulation of linolipin was highly reliable (P £ 0.01) in
relation to two controls: (a) untreated plants and (b)
plants injected with empty medium without bacterial
cells (Fig. 6).
Discussion
The detected linolipins A and B are the first members
of the linolipin family to be characterized. They are
unprecedented complex oxylipins, namely galactolipids,

possessing EDE oxylipin residues. Linolipins are dis-
tant congeners of arabidopsides [41–48], a family of
galactolipids containing esterified (15Z)-12-oxo-10,15-
phytodienoic acid and 2,3-dinor-(15Z)-12-oxo-10,15-
phytodienoic acid moieties. A dedicated study [48] did
not reveal the presence of arabidopsides in any other
tested species except Arabidopsis thaliana and Arabid-
opsis arenosa. Thus, linolipins constitute a second fam-
ily of oxylipin-esterified galactolipids along with the
arabidopsides. Moreover, flax is the second plant
species, along with Arabidopsis, to contain oxylipin-
esterified galactolipids in their leaves.
Notably, the flax leaves exhibit high endogenous
levels of both (x5Z)-etherolenic acid and 12-oxo-PDA
[40]. However, no arabidopsides or any other complex
Fig. 3. The downfield regions of
1
H NMR
spectra of linolipins. Partial spectrum for (A)
lipolipin A and (B) linolipin B. Signals above
5.25 p.p.m. belong to olefinic protons and
those below 5.25 p.p.m. to protons of glyc-
erol and galactose moieties. The attribution
of all signals was substantiated by 2D-COSY
data.
I. R. Chechetkin et al. Unprecedented complex oxylipins from flax
FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS 4467
lipids possessing esterified 12-oxo-PDA were detected
in flax. This indicates that the biosynthesis of linolipins
in flax leaves occurs specifically, without any compet-

ing arabidopsides formation, despite of the availability
of the endogenous free 12-oxo-PDA.
The biogenetic origin of linolipins, as well as arabi-
dopsides, remains to be revealed. There are two hypo-
thetical alternative pathways for their biosynthesis.
First, the oxylipins are initially biosynthesized as free
fatty acids and then esterified to galactolipids. Second,
esterified a-linolenic acid residues are transformed into
esterified oxylipin residues (divinyl ether or 12-oxo-
PDA) in situ via the sequential action of lipoxygen-
ase ⁄ divinyl ether synthase or lipoxygenase ⁄ allene oxide
synthase ⁄ allene oxide cyclase, respectively. Notably, we
Fig. 4. High-resolution ESI-MS and MS ⁄ MS
data for compound 2. (A) Negative-ion mode
MS and MS ⁄ MS fragmentation scheme of
precursor ion [M–H]
-
, m ⁄ z 801.4765; (B)
negative-ion mode full ESI MS of com-
pound 2; (C) MS ⁄ MS spectrum of ion m ⁄ z
801.4765.
Fig. 5. Linolipin (EDE) content of flax leaves. Galactolipids were
separated and purified as described in Materials and methods. EDE
content was measured by UV absorbance of MGDG and DGDG
fractions at 267 nm. Average values and standard deviations of five
independent experiments are presented.
Fig. 6. Effect of pathogenesis on linolipin content. Flax plants were
inoculated with a cell suspension of the phytopathogenic bacterium
E. carotovora atroseptica. Dark gray columns, infected plants; white
columns, control (untreated) plants; light gray columns, second con-

trol – plants injected with empty LB growth medium. Detailed infor-
mation on the treatment procedures and on the measurement of
linolipin content in flax leaves is described in Materials and meth-
ods. Average values and standard deviations of five independent
experiments are presented. EDE, esterified divinyl ethers.
Unprecedented complex oxylipins from flax I. R. Chechetkin et al.
4468 FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS
did not observe any lipoxygenase oxidation of 1,2-dili-
nolenoyl-3-b-d-galactopyranosyl-sn-glycerol. The data,
as well as the presence of free (x5Z)-etherolenic acid
in flax leaves, enable us to propose that the divinyl
ether is first biosynthesized as a free fatty acid and
then esterified to galactolipids.
Previously Fauconnier et al. [49] reported the detec-
tion of divinyl ether colneleic acid esterified to phos-
pholipids of potato tubers. However, only the crude
phospholipid fraction was characterized. Phospholipids
were not separated, no individual species was purified
and no structural confirmation was presented [49].
Finally, the estimated esterified colneleic acid content
of potato phospholipids was extremely low (sev-
eral p.p.m.) [49]. By contrast, the linolipin content of
flax leaves reached 3% of total galactolipids. This is
comparable to the content of arabidopsides in Arabid-
opsis leaves [45].
As reported above, the linolipin content of flax
leaves is age dependent. Linolipins accumulated only
in adult plants; not in young seedlings. Consequently,
their biosynthesis and turnover depends on ontogene-
sis. Our data show that (x5Z)-etherolenic acid inhibits

the flaxseed germination and root development. This
indicates that (x5Z)-etherolenic acid possesses cyto-
static activity.
The linolipins are pathogen-inducible compounds.
The amount of linolipins in leaves is greatly increased
upon infection by the phytopatogenic enterobacterium
E. carotovora subsp. atroseptica SCRI1043. These data
indicate that the accumulation of esterified oxylipins
(linolipins) may represent a new type of plant defense
strategy. The antimicrobial activity of divinyl ethers
and their involvement in plant defense have been dem-
onstrated previously [34–39]. Recently, extensive for-
mation of arabidopside E has been observed in
response to pathogen-derived avirulence proteins in
Arabidopsis [45]. This arabidopside inhibited the
growth of bacterial pathogen in vitro [45].
DES gene expression occurs differently. There are
some plant species in which the DES gene is pathogen
induced, whereas it is silent or only weakly expressed
under normal conditions [32,34,39]. However, the DES
gene is constitutively expressed in some species of the
Ranunculaceae [27–30], garlic (Allium sativum) [23–25]
or flax [40]. Flax exhibits a specific strategy: the DES
gene is constitutively expressed, but linolipin biosyn-
thesis is strongly stimulated in response to pathogen
attack. Accumulation of separate linolipins like linoli-
pin B in response to pathogenesis is particularly great.
Apparently, the divinyl ethers can be liberated from
linolipins through the enzymatic hydrolysis of ester
bridges and act as antimicrobial agents.

Materials and methods
Materials
Lipase from Rhizopus arrhizus was purchased from
Boehringer (Mannheim, Germany). Flax plants (Linum usi-
tatissimum L., cv. Novotorzhski) were grown in gardens
near Kazan in summer 2007 and 2008. Flax leaves were
frozen in liquid nitrogen and stored at )85 °C until lipid
extraction.
Lipid extraction and fractionation
Flax leaves were cut at the petiole bases. Leaves (900 g)
were covered with 6 L of boiling isopropanol containing
butylated hydroxytoluene (0.025%). After boiling for
10 min, the hot mixture was homogenized with a blender.
The homogenate was centrifuged at 6000 g for 5 min. The
supernatant was decanted and concentrated threefold
in vacuo. The remainder was diluted twofold with hexane.
The 6000-g sediment was re-extracted with 3 vol. hexane ⁄
isopropanol 1 : 1 (v ⁄ v) and centrifuged at 6000 g for 5 min.
The supernatants were decanted and washed three times
with 6.2 m NaCl aqueous solution. The water ⁄ isopropanol
phase was re-extracted with hexane. The combined organic
phases were evaporated to dryness in vacuo. The total lipids
were separated by the silicic acid column chromatography.
Neutral lipids were eluted with chloroform ⁄ methanol 9 : 1
(v ⁄ v) and glycolipids with acetone ⁄ methanol 9 : 1 (v ⁄ v)
[46]. The glycolipids were separated by HPLC as described
below.
Separation of galactolipids by HPLC
Galactolipids were separated by RP-HPLC on two serially
connected Separon SIX columns (150 · 3 mm; 5 lm; Tes-

sek, Praha, Czech Republic) by eluting for 55 min with
methanol ⁄ water 88 : 12 (v ⁄ v) followed by elution with pure
methanol for 30 min at a flow rate of 0.6 mLÆmin
)1
with
online diode array detection (190–350 nm). The species of
galactolipids possessing a k
max
at 267 nm were collected
and purified by cyanopropyl-phase HPLC on two serially
connected Separon SIX CN columns (150 · 3 mm; 5 lm)
under elution with hexane ⁄ isopropanol, linear gradient
from 95:5 to 80:20 (v ⁄ v) within 60 min, flow rate 0.4 mLÆ
min
)1
.
Transesterification and enzymatic hydrolysis of
galactolipids
Aliquots of separate purified galactolipid molecular species
were subjected to transesterification with sodium methox-
ide. Galactolipid dissolved in 100 lL of methanol was
reacted with 100 lL of 0.5 m methanolic sodium methoxide
for 10 min at room temperature. The reaction mixture was
I. R. Chechetkin et al. Unprecedented complex oxylipins from flax
FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS 4469
diluted 10-fold with water, acidified with acetic acid to
pH 6.0 and passed through a Supelclean LC-C
18
(1 mL)
cartridge (Supelco, Bellefonte, PA, USA). The cartridge

was washed with water. The fatty acid methyl esters were
eluted from the cartridge with methanol, then redissolved
in hexane and analyzed by GC-MS (as described below).
The regiospecific analysis of fatty acid residues at the glyc-
erol sn-1 position of galactolipids was performed as follows.
Pure galactolipid molecular species (50 lg) were dispersed
in 250 lLof50mm Tris ⁄ HCl buffer, pH 7.5 by 3 min of
sonication, then the sn-1-specific lipase from Rhizopus
arrhizus (25 U) was added. The mixture was incubated for
2 h at 25 °C and thereafter acidified to pH 6.0. The liber-
ated fatty acids were separated and purified using the
Supelclean LC-NH
2
(3 mL) cartridges (Supelco), as
described previously [31]. The resulting free fatty acids were
methylated with diazomethane and analyzed by GC-MS.
Age dependence of linolipin content in flax
leaves
Galactolipids were extracted from leaves of 10-, 23-, 35-,
63- and 76-day-old flax plants (2.5 g), purified by column
chromatography as described above and purified by micro-
preparative TLC using 20 · 20 cm plates with silica gel LS
5 ⁄ 40 (Chemapol), solvent acetone ⁄ benzene ⁄ water
(91 : 30 : 8, v ⁄ v). Broad zones with R
f
0.2–0.26 and 0.63–
0.77 containing DGDG and MGDG were scraped from the
plates. DGDG and MGDG were eluted from silica with
ethanol. The UV spectra of DGDG and MGDG were
recorded with Cary 50 Bio spectrophotometer (Varian, Palo

Alto, CA, USA). The amounts of EDE [i.e. the galactoli-
pid-bound (x5Z)-etherolenic acid] were estimated by
267 nm absorbance using a molar extinction coefficient
30 000 m
)1
Æcm
)1
for methyl ester of (x5Z)-etherolenic acid
[22].
Effects of pathogenesis on oxylipin profiles
When specified, plants were infected with pathogenic
enterobacterium E. carotovora subsp. atroseptica strain
SCRI1043 [50]. The cells of E. carotovora subsp. atroseptica
were cultivated on LB medium to D
600
= 0.1 [50]. A sus-
pension of bacterial cells was injected into the flax stems (at
6 cm above the soil). Control plants were injected with LB
medium only. Leaves were collected at 4 and 24 h after
inoculation and frozen in liquid nitrogen. In all experi-
ments, lipids were extracted, separated and analyzed as
described above for unstressed leaves.
Spectral studies
UV spectra were recorded with a Cary 50 Bio spectropho-
tometer. Alternatively, the UV spectra were recorded online
during the HPLC separations with SPD-M20A diode array
detector (Shimadzu Europa, Duisburg, Germany). GC-MS
analyses were performed, as described previously [40], using
a Shimadzu QP5050A mass spectrometer connected to
Shimadzu GC-17A gas chromatograph equipped with an

MDN-5S (5% phenyl 95% methylpolysiloxane) fused capil-
lary column (Supelco; length, 30 m; ID 0.25 mm; film
thickness, 0.25 lm). High-resolution ESI-MS and MS ⁄ MS
spectra of purified galactilipids were recorded with the Bru-
ker micrOTOF-Q mass spectrometer (Bruker Daltonics,
Billerica, MA, USA) with the electrospray source. The cap-
illary voltage was )4.5 kV for the positive-ion mode and
)3.5 kV for the negative-ion mode. The collision gas was
argon and the collision energy was 15 eV. Samples were
dissolved in hexane ⁄ methanol ⁄ 40 mm ammonium acetate
300 : 660 : 40 (v ⁄ v ⁄ v) and infused at 180 lLÆh
)1
into the
ESI source.
1
H NMR and 2D-COSY spectra of purified
compounds were recorded with Bruker Avance 400 instru-
ment (Bruker BioSpin, Rheinstetten, Germany), 400 MHz,
C
2
H
3
CN, 296 K.
Effects of oxylipins on seed germination
Tests of the effects of oxylipins on seed germination were
performed as described in Doc. S2.
Statistical analysis
Statistical analyses were performed using one-way ANOVA
and Student’s t-test. Average values ± SD are presented
for the indicated number of experiments. A value of

P < 0.05 was considered to be statistically significant.
Acknowledgements
The authors are grateful to Y. V. Gogolev and N. Mu-
khametshina for providing cells of the phytopathogenic
bacterium Erwinia carotovora subsp. atroseptica
SCRI1043 and their expert assistance in experiments
on pathogenesis.
1
H NMR and 2D-COSY records by
Dr Oleg I. Gnezdilov are gratefully acknowledged. The
authors thank Dr Ildar Kh. Rizvanov for helpful
discussions of mass spectral data. This work was
supported in part by Grant 09-04-01023-a from the
Russian Foundation for Basic Research and a grant
from the Russian Academy of Sciences (program
‘Molecular and Cell Biology’).
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Supporting information
The following supplementary material is available:
Fig. S1. 2D-COSY plots for compound 1 (A) and
compound 2 (B) (400 MHz, C
2
H
3
CN, 296 K).
Fig. S2. GC-MS analyses of oxylipins extracted after
13-HPOD incubation with 15 000 g supernatant of flax
leaf homogenate.
Fig. S3. Effects of (x5Z)-etherolenic acid Me ester and
(2E)-hexenal on flax seed germination.
Table S1. High-resolution electrospray MS and
MS ⁄ MS data for compound 1.
Table S2.
1
H NMR spectral data for linolipin A (1)
(400 MHz, C
2
HCl
3
, 296 K).
Table S3. High-resolution electrospray MS and
MS ⁄ MS data for compound 2.
Table S4.
1
H NMR spectral data for linolipin B (2)
(400 MHz, C
2

HCl
3
, 296 K).
Doc. S1. The influence of (x5Z)-etherolenic acid Me
ester and (2E)-hexenal on flax seed germination.
Doc. S2. Experimental procedures on seed germination
tests.
This supplementary material can be found in the
online article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
Unprecedented complex oxylipins from flax I. R. Chechetkin et al.
4472 FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS