A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Article abstract of DOI:10.1016/j.phytochem.2008.04.012

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1′Z)-12-(1′-hexenyloxy)-9,11-dodecadienoic [(ω5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of α-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1′Z,3′Z)-12-(1′,3′-hexadienyloxy)-9,11-dodecadienoic [(ω5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, ¹H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.

Full text of DOI:10.1016/j.phytochem.2008.04.012

Phytochemistry 69 (2008) 2008–2015
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.)
leaves
Ivan R. Chechetkin a, Alexander Blufard a, Mats Hamberg b, Alexander N. Grechkin a,
*
a Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Lobachevsky Street 2/31, P.O. Box 30, 420111 Kazan, Russia
b Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, SE-171 77 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to
the formation of a divinyl ether fatty acid, i.e. (9Z,11E,10Z)-12-(10-hexenyloxy)-9,11-dodecadienoic
[(x5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The
13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of a-linolenic
acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,10Z,30Z)-12-(10,30-hexadienyloxy)-9,11-
dodecadienoic [(x5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substan-
tiated by their UV, mass-, 1H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl
ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase
activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in
all incubations.
Received 23 March 2008
Received in revised form 16 April 2008
Available online 5 June 2008
Keywords:
Linum usitatissimum L.
Linaceae
Biosynthesis
Lipoxygenase pathway
Oxylipins
Ó 2008 Elsevier Ltd. All rights reserved.
Divinyl ether synthase
(x5Z)-Etherolenic acid
(x5Z)-Etheroleic acid
1. Introduction
(Fammartino et al., 2007) have been cloned. Divinyl ethers are not
as well studied as jasmonates with respect to their physiological
The plant lipoxygenase cascade provides a large diversity of
oxylipins. Many of these products are involved in plant cell signal-
ling and defence (Blée, 1998; Grechkin, 1998). The existing diver-
sity of oxylipins is largely established by enzymes belonging to
the CYP74 family of cytochrome P450s (Blée, 1998; Grechkin,
1998). Divinyl ether synthases (DESs) along with allene oxide syn-
thases (AOSs) and hydroperoxide lyases (HPLs) belong to this fam-
ily (Blée, 1998; Grechkin, 1998, 2002; Stumpe and Feussner, 2006).
Not many DESs are described compared to other CYP74 members,
including AOSs and HPLs. At the same time, DES products were dis-
covered in phylogenetically distant species, including brown (Pro-
teau and Gerwick, 1993) and red (Jiang and Gerwick, 1997) algae,
as well as higher plant species of families Ranunculaceae (Hamberg,
1998, 2002, 2004, 2005), Solanaceae (Galliard and Phillips, 1972;
Galliard et al., 1973; Galliard and Mathew, 1975) and Alliaceae
(Grechkin et al., 1995, 1997; Grechkin and Hamberg, 1996; Stumpe
et al., 2008). DES genes of some Solanaceae species including toma-
to (Itoh and Howe, 2001), potato (Stumpe et al., 2001) and tobacco
importance. At the same time, there are increasing data demon-
strating the involvement of divinyl ethers in plant resistance
towards pathogens (Weber et al., 1999; Göbel et al., 2001; Granér
et al., 2003; Cowley and Walters, 2005).
The present work reports the existence of a lipoxygenase-DES
pathway in flax (Linaceae, Eurosids I). The DES was found to specif-
ically utilize 13-hydroperoxides of a-linolenic and linoleic acids. Of
the several linolenic acid-derived divinyl ether fatty acids
previously described, the flax DES specifically generated
(9Z,11E,10Z,30Z)-12-(10,30-hexadienyloxy)-9,11-dodecadienoic [(x5Z)-
etherolenic] acid.
2. Results
2.1. Biosynthesis of oxylipins in vitro in flax leaves
For the preliminary characterization of the lipoxygenase path-
way, the 15,000g supernatant of flax leaf homogenate was
incubated with linoleic acid. GC–MS analysis of products upon
their methylation/hydrogenation/trimethylsilylation has revealed
that the predominant hydroxy acid was 13-hydroxystearic acid
(result not illustrated). This indicates that the major oxygenation
product was 13-hydroperoxide of linoleic acid (13-HPOD). Thus,
13-lipoxygenase activity predominates in flax leaves. Along with
Abbreviations: DES, divinyl ether synthase; 13-HPOT, (9Z,11E,13S,15Z)-13-
hydroperoxy-9,11,15-octadecatrienoic acid; 13-HPOD, (9Z,11E,13S)-13-hydroper-
oxy-9,11-octadecadienoic acid; AOS, allene oxide synthase; HPL, hydroperoxide
lyase; 12-oxo-PDA, (15Z)-12-oxo-10,15-phytodienoic acid.
* Corresponding author. Tel.: +7 843 292 75 35; fax: +7 843 292 73 47.
E-mail address: grechkin@mail.knc.ru (A.N. Grechkin).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.04.012

I.R. Chechetkin et al. / Phytochemistry 69 (2008) 2008–2015
2009
4 (M+ at m/z 314), which had a characteristic mass fragmentation
pattern (Fig. 1c). This data enabled us to identify the hydrogenation
product 4 as 13-oxa-nonadecanoic acid. Purified compound 1a
possessed a specific UV absorbance with kmax at 250 nm which is
typical for divinyl ethers like etheroleic acid (Grechkin et al.,
1995, 1997; Hamberg; 1998). The 1H NMR spectrum (Table 1)
was recorded for final identification and verification of double
bond geometry of compound 1a. The 1H NMR spectrum (Table 1)
supported by 2D–COSY data (not illustrated) demonstrated that
compound 1a possessed a butadienyl vinyl ether partial structure
having the (1Z,3E,10Z) configuration of double bonds. Coupling con-
stant value (6.2 Hz) unequivocally shows that 10-double bond has
cis configuration. On the basis of the data mentioned, compound
1 was identified as (9Z,11E,10Z)-12-(10-hexenyloxy)-9,11-dodecadi-
enoic acid, i.e. (x5Z)-etheroleic acid.
2.3. Identification of compound 2
Compound 2 (retention time 45.9 min) exhibited UV absor-
bance with kmax at 267 nm (the spectrum was recorded on line
during the normal phase HPLC separation, solvent hexane – diethyl
ether 99.4:0.6). The electron impact mass spectrum of the methyl
ester of 2 (Fig. 1d) showed a molecular ion at m/z 306 and a frag-
mentation pattern similar to those of the methyl esters of ethero-
lenic (Grechkin et al., 1995, 1997) and (x5Z)-etherolenic acid
(Hamberg, 1998). Catalytic hydrogenation of compound 2a
resulted in the formation of the above described product 4, 13-
oxa-nonadecanoic acid, which was identified by its electron impact
mass spectrum (Fig. 1c). Thus, compound 2a had four double
bonds. Their position and geometry were elucidated by 1H NMR
(Table 1, Fig. 2b) and COSY (Fig. 2a) spectral data. Thus, the
obtained data enabled us to identify compound
2
as
(9Z,11E,10Z,10Z)-12-(10,30-hexadienyloxy)-9,11-dodecadienoic acid,
i.e. (x5Z)-etherolenic acid.
Recently a new a-linolenic acid analogue, 3-oxa-a-linolenic
acid, was synthesized and described as a useful precursor in stud-
ies of oxylipin biosynthesis (Hamberg et al., 2006). Here we used
the lipoxygenase-generated 13-hydroperoxide of 3-oxa-a-linolenic
acid (3-oxa-13-HPOT) for further confirmation of the in vitro DES
activity and (x5Z)-etherolenic acid biosynthesis in flax leaves.
The 15,000g supernatant was incubated with 3-oxa-13-HPOT.
Analyses of incubation products (as methyl esters) by GC–MS
enabled us to detect (along with the endogenous unlabelled com-
pound 2, detected in methylated form 2a) the appearance of the
new products 5 and 6 detected as their methyl esters 5a and 6a
(Fig. 3a). The mass spectral data for compound 5a (M+ at m/z
308, Fig. 3b) enabled us to propose a fragmentation scheme
depicted in Fig. 3b. Catalytic hydrogenation of compound 5a affor-
ded product 7, which had a characteristic MS fragmentation
patterns (Fig. 3c). This result demonstrates that compound 7 is
3,13-dioxa-nonadecanoic acid, thus substantiating the identifica-
tion of compound 5 as 3-oxa-(x5Z)-etherolenic acid.
hydro(pero)xy fatty acids, compounds 1a, 2a and 3a were detected
during the GC–MS analyses of the extracted oxylipins. The same
oxylipins were detected after incubations with 13-HPOD, as de-
scribed below.
For further characterization of metabolic pathways, we incu-
bated the 15,000g supernatant with 13-HPOD. Direct analyses of
products as trimethylsilylated methyl esters by GC–MS enabled
us to detect oxylipins 1a, 2a and 3a (Fig. 1a). For identification of
molecular structures of oxylipins 1a, 2a and 3a, their mass spectra
and spectra of their derivatives were recorded. Moreover, oxylipins
1a and 2a were separated and purified by micropreparative HPLC
and their 1H NMR spectra were recorded.
The observations with 3-oxa-13-HPOT confirmed both the
in vitro DES activity and the presence of endogenous (x5Z)-ethero-
lenic acid (2) in the leaves. Integration of total ion chromatograms
revealed that the content of endogenous (x5Z)-etherolenic acid in
leaves reached up to 10% of the extracted total free fatty acids.
2.2. Identification of compound 1
2.4. Identification of compound 3
Compound 1a (retention time 14.8 min, see Fig. 1a) was the ma-
jor product of linoleic acid conversion in vitro. Product 1a pos-
sessed a mass spectral fragmentation pattern (Fig. 1b) identical
to that described previously for the divinyl ethers (9Z,11E,10E)-
12-(10-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid (Grech-
kin et al., 1995, 1997) and (x5Z)-etheroleic acid (Hamberg,
1998). Catalytic hydrogenation of compound 1a afforded product
As mentioned above, compound 3 belonged to the most abun-
dant oxylipins detected after the incubations with cell-free prepa-
rations from flax leaves. The mass spectrum of methyl ester 3a
(Fig. 4a) exhibited a characteristic fragmentation pattern identical
to that earlier recorded on the methyl ester of cis-12-oxo-10,15-
phytodienoic acid (12-oxo-PDA) (Hamberg, 1998). Catalytic hydro-

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I.R. Chechetkin et al. / Phytochemistry 69 (2008) 2008–2015
Fig. 1. GC–MS analyses of oxylipins extracted after incubations of 13-HPOD with 15,000g supernatant of flax leaf homogenate. Products were extracted, purified with
aminopropyl cartridges, methylated, trimethylsilylated and analyzed by GC–MS as described in the Experimental section. (a), Total ion chromatogram of products; (b), (c), (d),
mass spectra and fragmentation schemes of compounds 1a; 4, and 2a, respectively.
genation of compound 3a over Pt catalyst resulted in the formation
of compound 8. Its mass spectral data (Fig. 4c) enabled us to
respectively); (2) difference of mass of fragment [M+–CH2CH =
CHCH2CH3] (240 and 238, respectively). The obtained data
enabled us to identify compound 6 as 3-oxa-12-oxo-PDA, i.e.
12-oxo-PDA derivative biosynthesized from 3-oxa-13-HPOT.
Catalytic hydrogenation of product 6a afforded compound 9. Its
mass spectrum and fragmentation scheme (Fig. 4d) enabled one to
identify compound
8 as 12-oxo-phytonoic acid, the fully
hydrogenated analogue of 12-oxo-PDA. Thus, the data obtained
enabled one to identify compound 3a as cis-12-oxo-PDA methyl
ester.
When 15,000g supernatant of leaf homogenate was incubated
with 3-oxa-13-HPOT, a new product 6 appeared along with com-
pound 3 and divinyl ethers. The mass spectrum of methyl ester
6a (M+ at m/z 308) and the proposed fragmentation scheme
(insert) are presented in Fig. 4b. In general the spectral patterns
of compound 6a were similar to those of compound 3a with two
exceptions: (1) difference of molecular mass (308 and 306,
identify compound
the obtained data demonstrated that the exogenously added
3-oxa-13-HPOT served as precursor of cyclopentenone 6,
3-oxa-12-oxo-PDA. Notably, along with compound 6a, significant
levels of endogenous 12-oxa-PDA 3 (as methyl ester 3a) were
detectable along with products (analyzed as methyl esters) of incu-
bations with 3-oxa-13-HPOT (Fig. 3a).
9 as 3-oxa-12-oxo-phytonoic acid. Thus,
a

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2011
Table 1
1H NMR spectral data for compounds 1a and 2a (400 MHz, C2H3CN, 296 K)
Proton
Compound 1a
Compound 2a
Chemical shift,
d (ppm)
Multiplicity
(number of protons)
Coupling constants
(Hz)
Chemical shift,
d (ppm)
Multiplicity
(number of protons)
Coupling constants
(Hz)
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
H10
H20
H30
2.32
1.59
1.31
1.31
1.31
1.31
2.13
5.30
5.90
6.02
6.69
6.30
4.67
2.13
t (2)
7.5 (H3)
2.31
1.59
1.31
1.31
1.31
1.31
2.07
5.34
5.90
6.11
6.75
6.35
5.56
6.29
t (2)
7.5 (H3)
m (2)
m (2)
m (2)
m (2)
m (2)
m (2)
dt (1)
dd (1)
dd (1)
d (1)
m (2)
m (2)
m (2)
m (2)
m (2)
m (2)
dt (1)
dd (1)
ddd (1)
d (1)
7.5 (H8); 10.6 (H10)
11.1 (H11)
11.9 (H12)
7.6 (H8); 10.7 (H10)
11.1 (H11)
11.9 (H12); 1.1 (H9)
dt (1)
dt (1)
m (2)
6.2 (H20); 1.2 (H30)
7.4 (H30)
d (1)
ddd (1)
dddt (1)
6.2 (H20)
11.6 (H30); 1.2 (H40)
11.1 (H40); 1.2 (H10);
1.2 (H50)
H40
1.31
1.31
0.91
3.63
m (2)
m (2)
t (3)
5.45
2.12
1.01
3.63
m (1)
m (2)
t (3)
7.5 (H50);
H50
H60
6.9 (H50)
7.6 (H50)
H(OMe)
s (3)
s (3)
Fig. 2. 1H NMR data for compound 2a, including the 2D–COSY plot (a) and the olefinic region of 1H NMR spectrum (400 MHz, C2H3CN, 298 K).

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Fig. 3. GC–MS analyses of oxylipins extracted after incubations of 3-oxa-13-HPOT with 15,000g supernatant of flax leaf homogenate. Products were extracted, purified with
aminopropyl cartridges, methylated, trimethylsilylated and analyzed by GC–MS as described in the Experimental section. (a), Total ion chromatogram of products; (b), (c),
mass spectra and fragmentation schemes of compounds 5a and 7, respectively.
2.5. Biosynthesis of oxylipins in vitro in flax roots
(x5Z)-etherolenic and (x5Z)-etheroleic acids (respectively), and
its localization in leaves, are properties similar to those of Ranun-
culaceae DESs (Hamberg, 1998, 2002, 2004). (x5Z)-Etherolenic acid
and related divinyl ethers also occur in the brown alga Laminaria
sinclairii (Proteau and Gerwick, 1993). The presence of a cis double
bond at one side of the ether bridge is a distinctive feature of divi-
nyl ethers produced by enzymes of flax, Ranunculaceae as well as
the brown and red algae. Mechanistic studies have suggested that
even single mutations at the active site of DESs may be sufficient to
change the double bond configuration of the divinyl ether products
(Hamberg, 2005). However, the extent of homology in the family of
DES enzymes can be revealed only after cloning and characteriza-
tion of the genes involved.
Divinyl ethers are involved in plant defence against pathogens,
including fungi attacking potato leaves (Weber et al., 1999;
Stumpe et al., 2001; Göbel et al., 2001; Cowley and Walters,
2005; Fammartino et al., 2007). DES genes are constitutively ex-
pressed in the roots of tomato (Itoh and Howe, 2001) and tobacco
(Fammartino et al., 2007), but DES activity is significantly in-
creased in the infected roots (Fammartino et al., 2007). DES genes
in the leaves and other organs (except roots) of Solanaceae plants
are specifically expressed in response to pathogen attack (Weber
et al., 1999; Fammartino et al., 2007). This is in contrast to the sit-
uation in leaves of flax (present study) and Ranunculaceae (Ham-
berg, 1998, 2002, 2004) and in garlic bulbs (Grechkin et al., 1995,
The 15,000g supernatant of flax root homogenate was incu-
bated with linoleic acid. GC–MS analysis of products (methyl esters
TMSi derivatives) revealed the presence of a-ketol as a predomi-
nant product as well as a small amount of 12-oxo-PDA methyl
ester (3a). The mass spectral data for a-ketol Me ester TMSi deriv-
ative possessed the following prominent ions, m/z [ion attribution]
(relative intensity, %): 398 [M+] (0.1); 383 [M+ – CH3] (2); 367
[M+ – CH3O] (0.5); 270 [M+ – C12/C18 + TMSi] (18); 173 [M+ – C1/
C12] (100); 129 (3); 103 [CH2 = O+ – SiMe3] (14); 73 [Si(CH3)3]+
(67), supported identification of the a-ketol as (9Z)-12-oxo-13-hy-
droxy-9-octadecenoic acid. No divinyl ethers were detectable after
in vitro incubations with roots.
3. Discussion
The results obtained demonstrate that flax extends the number
of plant species which possess DES activity and contain divinyl
ethers. This is of particular interest since flax (Linaceae, Malpighi-
ales, Eurosids I) is phylogenetically distant from other higher
plants known to possess DESs, i.e. Ranunculaceae (basal Eudicots),
Solanaceae (Euasterids I) and Alliaceae (monocots), see Fig. 5. The
specificity of flax DES, converting 13-HPOT and 13-HPOD into

I.R. Chechetkin et al. / Phytochemistry 69 (2008) 2008–2015
2013
1997; Grechkin and Hamberg, 1996) where the DES gene is consti-
tutively expressed.
tinuous oxygen bubbling followed by extraction and purification
by normal phase HPLC. Flax plants (Linum usitatissimum L., cv.
Novotorzhski) were grown in the fields near Kazan in Summer
2007.
4. Experimental
4.1. Materials
4.2. Incubations of cell-free preparation from flax leaves with linoleic
acid, 13-HPOD and 3-oxa-13-HPOT
Unlabelled linoleic acid, as well as linoleic acid and soybean
lipoxygenase type
V was purchased from Sigma. Sodium
Leaves or roots of 35 days old flax plants (5 g) were dispersed in
20 ml cold (+4 °C) 50 mM Tris/HCL buffer, pH 7.5 with Ultra-Turrax
T25. The obtained homogenate was centrifuged at 15,000g for
15 min. The supernatant was decanted and used as enzyme prep-
aration for the following incubations immediately. The enzyme
preparation (20 ml) was incubated with 1 mg of linoleic acid, 13-
HPOD or 3-oxa-13-HPOT 40 min at the room temperature.
borohydride as well as the silylating reagents were purchased from
Fluka (Buchs, Switzerland). (9Z,11E,13S)-13-Hydroperoxy-9,11-
octadecadienoic acid (13-HPOD) and (9Z,11E,15Z,13S)-13-hydro-
peroxy-3-oxa-9,11,15-octadecatrienoic acid (3-oxa-13-HPOT)
were prepared by incubation of linoleic or 3-oxa-a-linolenic acids,
respectively, with soybean lipoxygenase at 0 °C, pH 9.0, under con-
Fig. 4. Mass spectra and fragmentation schemes (inserts) for compounds 3a (a); 6a (b); 8 (c) and 9 (d). Compounds 8 and 9 are the products of catalytic hydrogenation of
products 3a and 6a, respectively.

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I.R. Chechetkin et al. / Phytochemistry 69 (2008) 2008–2015
and 2D–COSY spectra of purified compounds were recorded with
Bruker Avance 400 instrument, 400 MHz, C2H3CN, 296 K.
Acknowledgements
The authors thank Dr. Faina K. Mukhitova for mass spectral
records. This work was supported by Grant 06-04-48430 from
Russian Foundation for Basic Research and
a Grant from
Russian Academy of Sciences (program ‘‘Molecular and Cell
Biology”) and from the Swedish Research Council for Environ-
ment, Agricultural Sciences and Spatial Planning (Project No.
2001-2553).
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Alternatively, the methyl esters of products were preliminarily
separated by normal phase HPLC on two serially connected Sepa-
ron SIX columns (150 ꢀ 3.2 mm; 5 lm) eluted with hexane –
Et2O 99.4:0.6 (by volume), flow rate 0.6 ml/min.
4.5. Spectral studies
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UV spectra of isolated products were recorded with Perkin El-
mer Lambda 25 spectrophotometer. Alternatively, the UV spectra
of oxylipins were recorded on line during the HPLC separations
using an SPD-M20A diode array detector (Shimadzu). GC–MS anal-
yses were performed using a Shimadzu QP5050A mass spectrome-
ter connected to Shimadzu GC-17A gas chromatograph equipped
with an MDN-5 S (5% phenyl 95% methylpolysiloxane) fused capil-
lary column (length, 30 m; ID 0.25 mm; film thickness, 0.25 lm).
Helium at a flow rate of 30 cm/s was used as the carrier gas. Injec-
tions were made in the split mode using an initial column temper-
ature of 120 °C. The temperature was raised at 10 °C/min until
240 °C. Full scan or selected ion monitoring (SIM) analyses were
both performed using ionization energy of 70 eV. The 1H NMR
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a
divinyl ether synthase.
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3627.
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species of Laminaria (brown algae). Lipids 28, 783–787.
Stumpe, M., Carsjens, J.-G., Göbel, C., Feussner, I., 2008. Divinyl ether synthesis in
garlic bulbs. J. Exp. Bot. 59, 907–915.
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