Recently, we reported the detection of DES activity and the divinyl ethers Keywords divinyl ether synthase; flax; lipoxygenase cascade; oxylipins; pathogenesis Correspondence A.. Both MG
Trang 1from 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: grechkin@mail.knc.ru
Website: http://www.kibb.knc.ru/eng/
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.
Trang 2(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 phospholgalactol-ipids) using silicic acid column
chromatography The UV spectrum of the
phospho-lipid fraction did not reveal the presence of
12-oxo-PDA (kmax at 221 nm) or (x5Z)-etherolenic acid (kmax
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 kmax at 267 nm, sug-gesting the possible presence of EDE moieties Neither infected nor control plants possessed any galactolipid molecular species with kmax 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).
Trang 3The 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 (C45H71O11), as well
as the adduct [M+CH3 COO]- at m⁄ z 847.5229
(C47H75O13) The MS⁄ MS spectrum of m ⁄ z 787.4909
showed ions at m⁄ z 291.1954 [C18H27O3,
(x5Z)-ethero-lenic acid anion] and m⁄ z 277.2120 (C18H29O2,
a-lino-lenic acid anion) The positive-ion mode mass
spectrum of compound 1 (Table S1) showed the ion
[M+NH4]+ at m⁄ z 806.5418 (C45H76O11N) The
MS⁄ MS of ion m ⁄ z 806.5418 yielded a diagnostic
frag-ment at m⁄ z 529.3287 (C27H47O9N, loss of a-linolenic acid residue) The obtained high-resolution mass spec-trometry (HRMS) data revealed the empirical formula
C45H72O11for 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 (J2¢,3¢= 9.7 Hz; J2¢,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.
Trang 4(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)-etheroa-lino-lenic 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 (C45H69O12) and the
adduct [M+CH3 COO]-at m⁄ z 861.4909 (C47H73O14)
The MS⁄ MS spectrum of m ⁄ z 801.4765 showed the
product ion at m⁄ z 291.1951 [C18H27O3, (x5Z)-ethero-lenic acid anion] The positive-ion mode mass spectrum
of compound 2 (Table S3) showed the ion [M+NH4]+
at m⁄ z 820.5231 (C45H76O11N) The obtained HRMS data predict the empirical formula C45H70O12 for compound 2 Both the MS and the MS⁄ MS data are consistent with a MGDG structure containing two residues of (x5Z)-etherolenic acid
1H 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)
Trang 5on 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 of1H 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.
Trang 6lipids 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.
Trang 7did 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-opsisleaves [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 carotovorasubsp 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 kmax 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
Trang 8diluted 10-fold with water, acidified with acetic acid to
pH 6.0 and passed through a Supelclean LC-C18 (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 lL of 50 mm 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 25C and thereafter acidified to pH 6.0 The
liber-ated fatty acids were separliber-ated and purified using the
Supelclean LC-NH2 (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 Rf 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)1Æcm)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 D600= 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 (BruBru-ker 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 1H NMR and 2D-COSY spectra of purified compounds were recorded with Bruker Avance 400 instru-ment (Bruker BioSpin, Rheinstetten, Germany), 400 MHz,
C2H3CN, 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 1H 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, C2H3CN, 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 1H NMR spectral data for linolipin A (1) (400 MHz, C2HCl3, 296 K)
Table S3 High-resolution electrospray MS and
MS⁄ MS data for compound 2
Table S4 1H NMR spectral data for linolipin B (2) (400 MHz, C2HCl3, 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
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