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: 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. 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’). References 1 Grechkin AN (1998) Recent developments in biochemis- try of the plant lipoxygenase pathway. Progr Lipid Res 37, 317–352. Unprecedented complex oxylipins from flax I. R. Chechetkin et al. 4470 FEBS Journal 276 (2009) 4463–4472 ª 2009 The Authors Journal compilation ª 2009 FEBS 2 Feussner I & Wasternack C (2002) The lipoxygenase pathway. Annu Rev Plant Biol 53 , 275–297. 3 Liechti R & Farmer EE (2002) The jasmonate pathway. Science 296, 1649–1650. 4 Gfeller A & Farmer EE (2004) Keeping the leaves green above us. Science 306, 1515–1516. 5 Kessler A, Halitschke R & Baldwin IT (2004) Silencing the jasmonate cascade: induced plant defenses and insect populations. Science 305, 665–668. 6 Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA & Browse J (2007) JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling. Nature 448, 661–665. 7 Chini A, Fonseca S, Ferna ´ ndez G, Adie B, Chico JM, Lorenzo O, Garcı ´ a-Casado G, Lo ´ pez-Vidriero I, Lozano FM, Ponce MR et al. (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448, 666–671. 8 Song WC & Brash AR (1991) Purification of an allene oxide synthase and identification of the enzyme as a cytochrome P-450. Science 253, 781–784. 9 Stumpe M & Feussner I (2006) Formation of oxylipins by CYP74 enzymes. Phytochem Rev 5, 347–357. 10 Grechkin AN (2002) Hydroperoxide lyase and divinyl ether synthase. Prostag Other Lipid Mediat 68 ⁄ 69, 457– 470. 11 Lee D-S, Nioche P, Hamberg M & Raman CS (2008) Structural insights into the evolutionary paths of oxyli- pin biosynthetic enzymes. Nature 455, 363–368. 12 Li L, Chang Z, Pan Z, Fu ZQ & Wang X (2008) Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase. Proc Natl Acad Sci USA 105, 13883– 13888. 13 Liechti R & Farmer EE (2006) Jasmonate biochemical pathway. Sci STKE 2006, cm3. 14 Liechti R, Gfeller A & Farmer EE (2006) Jasmonate signaling pathway. Sci STKE 2006, cm2. 15 Grechkin AN & Hamberg M (2004) The ‘heterolytic hydroperoxide lyase’ is an isomerase producing a short- lived fatty acid hemiacetal. Biochim Biophys Acta 1636, 47–58. 16 Grechkin AN, Bru ¨ hlmann F, Mukhtarova LS, Gogolev YV & Hamberg M (2006) Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomeri- zation of fatty acid hydroperoxides into hemiacetals. Biochim Biophys Acta 1761, 1419–1428. 17 Matsui K (2006) Green leaf volatiles: hydroperoxide lyase pathway of oxylipin metabolism. Curr Opin Plant Biol 9, 274–280. 18 Baldwin IT, Halitschke R, Paschold A, von Dahl CC & Preston CA (2006) Volatile signaling in plant–plant interactions: ‘talking trees’ in the genomics era. Science 311, 812–815. 19 Galliard T & Phillips DR (1972) The enzymatic conver- sion of linoleic acid into 9-(nona-1¢,3¢-dienoxy)-non-8- enoic acid, a novel unsaturated ether derivative isolated from homogenates of Solanum tuberosum tubers. Biochem J 129, 743–753. 20 Galliard T, Phillips DR & Frost DJ (1973) Novel divi- nyl ether fatty acids in extracts of Solanum tuberosum. Chem Phys Lipids 11, 173–180. 21 Galliard T & Mathew JA (1975) Enzymic reactions of fatty acid hydroperoxides in extracts of potato tuber I. Comparison 9d- and 13l-hydroperoxyoctadecadie- noic acids as substrates for the formation of a divinyl ether derivative. Biochim Biophys Acta 398, 1–9. 22 Proteau PJ & Gerwick WH (1993) Divinyl ethers and hydroxy fatty acids from three species of Laminaria (brown algae). Lipids 28, 783–787. 23 Grechkin AN, Fazliev FN & Mukhtarova LS (1995) The lipoxygenase pathway in garlic (Allium sativum L.) bulbs: detection of the novel divinyl ether oxylipins. FEBS Lett 371, 159–162. 24 Grechkin AN & Hamberg M (1996) Divinyl ether syn- thase from garlic (Allium sativum L.) bulbs: subcellular localization and substrate regio- and stereospecificity. FEBS Lett 388, 112–114. 25 Grechkin AN, Ilyasov AV & Hamberg M (1997) On the mechanism of biosynthesis of divinyl ether oxylipins by enzyme from garlic bulbs. Eur J Biochem 245, 137– 142. 26 Jiang ZD & Gerwick WH (1997) Novel oxylipins from the temperate red alga Polyneura latissima: evidence for an arachidonate 9(S)-lipoxygenase. Lipids 32, 231–235. 27 Hamberg M (1998) A pathway for biosynthesis of divinyl ether fatty acids in green leaves. Lipids 33, 1061–1071. 28 Hamberg M (2002) Biosynthesis of new divinyl ether oxylipins in Ranunculus plants. Lipids 37, 427–433. 29 Hamberg M (2004) Isolation and structures of two divinyl ether fatty acids from Clematis vitalba. Lipids 39, 565–569. 30 Hamberg M (2005) Hidden stereospecificity in the biosynthesis of divinyl ether fatty acids. FEBS J 272, 736–743. 31 Ogorodnikova AV, Latypova LR, Mukhitova FK, Mukhtarova LS & Grechkin AN (2008) Detection of divinyl ether synthase in lily-of-the-valley (Convallaria majalis) roots. Phytochemistry 69, 2793–2798. 32 Itoh A & Howe GA (2001) Molecular cloning of a divinyl ether synthase. Identification as a CYP74 cytochrome P450. J Biol Chem 276, 3620–3627. 33 Stumpe M, Carsjens J-G, Gobel C & Feussner I (2008) Divinyl ether synthesis in garlic bulbs. J Exp Bot 59, 907–915. 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 4471 34 Weber H, Chetalat A, Caldelari D & Farmer EE (1999) Divinyl ether fatty acid synthesis in late blight-diseased potato leaves. Plant Cell 11, 485–494. 35 Stumpe M, Kandzia R, Go ¨ bel C, Rosahl S & Feussner I (2001) A pathogen-inducible divinyl ether synthase (CYP74D) from elicitor-treated potato suspension cells. FEBS Lett 507, 371–376. 36 Go ¨ bel C, Feussner I, Schmidt A, Scheel D, Sanchez- Serrano J, Hamberg M & Rosahl S (2001) Oxylipin profiling reveals the preferential stimulation of the 9-lipoxygenase pathway in elicitor-treated potato cells. J Biol Chem 276, 6267–6273. 37 Grane ´ r G, Hamberg M & Meijer J (2003) Screening of oxylipins for control of oilseed rape (Brassica napus) fungal pathogens. Phytochemistry 63, 89–95. 38 Cowley T & Walters D (2005) Local and systemic effects of oxylipins on powdery mildew infection in barley. Pest Manag Sci 61, 572–576. 39 Fammartino A, Cardinale F, Go ¨ bel C, Me ` ne-Saffrane ´ L, Fournier J, Feussner I & Esquerre ´ -Tugaye ´ M-T (2007) Characterization of a divinyl ether biosynthetic pathway specifically associated with pathogenesis in tobacco. Plant Physiol 143, 378–388. 40 Chechetkin IR, Blufard A, Hamberg M & Grechkin AN (2008) A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves. Phytochemistry 69, 2008–2015. 41 Stelmach BA, Muller A, Hennig P, Gebhardt S, Schubert-Zsilavecz M & Weiler EW (2001) A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O- (hexadecatrienoyl)-monogalactosyl diglyceride, from Arabidopsis thaliana. J Biol Chem 276 , 12832–12838. 42 Hisamatsu Y, Goto N, Hasegawa K & Shigemori H (2003) Arabidopsides A and B, two new oxylipins from Arabidopsis thaliana. Tetrahedron Lett 44, 5553–5556. 43 Hisamatsu Y, Goto N, Hasegawa K & Shigemori H (2006) Senescence-promoting effect of arabidopside A. Z Naturforsch [C] 61, 363–366. 44 Hisamatsu Y, Goto N, Sekiguchi M, Hasegawa K & Shigemori H (2005) Oxylipins arabidopsides C and D from Arabidopsis thaliana. J Nat Prod 68, 600–603. 45 Andersson MX, Hamberg M, Kourtchenko O, Brunnstro ¨ m A, McPhail KL, Gerwick WH, Go ¨ bel C, Feussner I & Ellerstro ¨ m M (2006) Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-phytodienoic acid-containing galactolipid, arabidopside E. J Biol Chem 281, 31528– 31537. 46 Kourtchenko O, Andersson MX, Hamberg M, Brunnstro ¨ mA,Go ¨ bel C, McPhail KL, Gerwick WH, Feussner I & Ellerstro ¨ m M (2007) Oxo-phytodienoic acid-containing galactolipids in Arabidopsis: jasmonate signaling dependence. Plant Physiol 145, 1658–1669. 47 Buseman CM, Tamura P, Sparks AA, Baughman EJ, Maatta S, Zhao J, Roth MR, Esch SW, Shah J, Williams TD et al. (2006) Wounding stimulates the accu- mulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves. Plant Physiol 142, 28–39. 48 Weiler EW (2003) Sensory principles of higher plants. Angew Chem Int Ed 42, 392–411. 49 Fauconnier ML, Williams TD, Marlier M & Welti R (2003) Potato tuber phospholipids contain colneleic acid in the 2-position. FEBS Lett 538, 155–158. 50 Bell KS, Sebaihia M, Pritchard L, Holden MTG, Hyman LJ, Holeva MC, Thomson NR, Bentley SD, Churcher LJC, Mungall K et al. (2004) Genome sequence of the enterobacterial phytopathogen Erwi- nia carotovora subsp. atroseptica and characterization of virulence factors. Proc Natl Acad Sci USA 101, 11105–11110. 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, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) 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 . Unprecedented pathogen-inducible complex oxylipins from flax – linolipins A and B Ivan R. Chechetkin, Fakhima K. Mukhitova, Alexander S. Blufard, Andrey. galactolipid molecular species from flax leaves. Total galactolipids were extracted from flax leaves, sepa- rated and purified as described in Materials and