Biosynthesis of Jasmonates from Linoleic Acid by the Fungus Fusarium oxysporum. Evidence for a Novel Allene Oxide Cyclase

Article abstract of DOI:10.1002/lipd.12180

Fusarium oxysporum f. sp. tulipae (FOT) secretes (+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile) to the growth medium together with about 10 times less 9,10-dihydro-(+)-7-iso-JA-Ile. Plants and fungi form (+)-JA-Ile from 18:3n-3 via 12-oxophytodienoic acid (12-OPDA), which is formed sequentially by 13S-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase (AOC). Plant AOC does not accept linoleic acid (18:2n-6)-derived allene oxides and dihydrojasmonates are not commonly found in plants. This raises the question whether 18:2n-6 serves as the precursor of 9,10-dihydro-JA-Ile in Fusarium, or whether the latter arises by a putative reductase activity operating on the n-3 double bond of (+)-JA-Ile or one of its precursors. Incubation of pentadeuterated (d₅) 18:3n-3 with mycelia led to the formation of d₅-(+)-JA-Ile whereas d₅-9,10-dihydro-JA-Ile was not detectable. In contrast, d₅-9,10-dihydro-(+)-JA-Ile was produced following incubation of [17,17,18,18,18-²H₅]linoleic acid (d₅-18:2n-6). Furthermore, 9(S),13(S)-12-oxophytoenoic acid, the 15,16-dihydro analog of 12-OPDA, was formed upon incubation of unlabeled or d₅-18:2n-6. Appearance of the α-ketol, 12-oxo-13-hydroxy-9-octadecenoic acid following incubation of unlabeled or [¹³C₁₈]-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid confirmed the involvement of AOS and the biosynthesis of the allene oxide 12,13(S)-epoxy-9,11-octadecadienoic acid. The lack of conversion of this allene oxide by AOC in higher plants necessitates the conclusion that the fungal AOC is distinct from the corresponding plant enzyme.

Full text of DOI:10.1002/lipd.12180

Lipids (2019)
DOI 10.1002/lipd.12180
ORIGINAL ARTICLE
Biosynthesis of Jasmonates from Linoleic Acid by the Fungus
Fusarium oxysporum. Evidence for a Novel Allene Oxide Cyclase
Ernst H. Oliw1 · Mats Hamberg2
Received: 26 February 2019 / Revised: 23 June 2019 / Accepted: 1 July 2019
© 2019 AOCS
Abstract Fusarium oxysporum f. sp. tulipae (FOT) secretes
(+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile) to the growth
medium together with about 10 times less 9,10-dihydro-
(+)-7-iso-JA-Ile. Plants and fungi form (+)-JA-Ile from
18:3n-3 via 12-oxophytodienoic acid (12-OPDA), which is
formed sequentially by 13S-lipoxygenase, allene oxide
synthase (AOS), and allene oxide cyclase (AOC). Plant AOC
does not accept linoleic acid (18:2n-6)-derived allene oxides
and dihydrojasmonates are not commonly found in plants.
This raises the question whether 18:2n-6 serves as the precur-
sor of 9,10-dihydro-JA-Ile in Fusarium, or whether the latter
arises by a putative reductase activity operating on the n-3
double bond of (+)-JA-Ile or one of its precursors. Incubation
of pentadeuterated (d5) 18:3n-3 with mycelia led to the forma-
tion of d5-(+)-JA-Ile whereas d5-9,10-dihydro-JA-Ile was not
detectable. In contrast, d5-9,10-dihydro-(+)-JA-Ile was pro-
duced following incubation of [17,17,18,18,18-2H5]linoleic
acid (d5-18:2n-6). Furthermore, 9(S),13(S)-12-oxophytoenoic
acid, the 15,16-dihydro analog of 12-OPDA, was formed
upon incubation of unlabeled or d5-18:2n-6. Appearance of
the α-ketol, 12-oxo-13-hydroxy-9-octadecenoic acid follow-
ing incubation of unlabeled or [13C18]-labeled 13(S)-
hydroperoxy-9(Z),11(E)-octadecadienoic acid confirmed the
involvement of AOS and the biosynthesis of the allene oxide
12,13(S)-epoxy-9,11-octadecadienoic acid. The lack of con-
version of this allene oxide by AOC in higher plants
necessitates the conclusion that the fungal AOC is distinct
from the corresponding plant enzyme.
Keywords Allene oxide cyclase ꢀ Cytochrome P-450 ꢀ
Jasmonates ꢀ Lipids/oxidation ꢀ Lipoxygenase ꢀ Mass
spectrometry ꢀ Methods/HPLC
Lipids (2019).
Abbreviations
(−)-JA
(+)-JA
(−)-jasmonic acid
(+)-7-iso-jasmonic acid
(+)-JA-Ile (+)-7-iso-jasmonoyl-(S)-isoleucine
11S-
11(S)-hydroperoxy-7(Z),9(E),13(Z)-
hexadecatrienoic acid
12,13(S)-epoxy-9(Z),11-octadecadienoic acid
HPHeTrE
12,13S-
EOD
12,13S-
EOT
12,13(S)-epoxy-9(Z),11,15(Z)-
octadecatrienoic acid
12-OPDA
12-OPEA
13S-
HPODE
13S-
HPOTrE
13S-LOX
AOC
12-oxo-10,15(Z)-phytodienoic acid
12-oxo-10-phytoenoic acid
13(S)-hydroperoxy-9(Z),11(E)-
octadecadienoic acid
13(S)-hydroperoxy-9(Z),11(E),15(Z)-
octadecatrienoic acid
13S-lipoxygenase
allene oxide cyclase
AOS
CP
allene oxide synthase
chiral phase
* Ernst H. Oliw
ernst.oliw@farmbio.uu.se
CYP
cytochrome P450
1
Division of Biochemical Pharmacology, Department of
Pharmaceutical Biosciences, Uppsala University, Husargatan 3,
Box 591, SE-751 24, Uppsala, Sweden
d5-18:2n-6 [17,17,18,18,18-2H5]linoleic acid
d5-18:3n-3 [17,17,18,18,18-2H5]α-linolenic acid
DOX
FOT
2
dioxygenase
F. oxysporum f. sp. tulipae
Department of Medical Biochemistry and Biophysics, Karolinska
Institutet, Solnavägen 1, SE-171 77, Stockholm, Sweden
Lipids (2019)

Lipids
synthase (AOS) to an allene oxide, 12,13(S)-epoxy-9(Z),11,15
(Z)-octadecatrienoic acid (12,13S-EOT) (Wasternack and
Feussner, 2018). The latter is converted by allene oxide
cyclase (AOC) to 12-oxo-10,15(Z)-phytodienoic acid
(12-OPDA) in plastids (Wasternack and Feussner, 2018).
Reduction of the ring double bond and three steps of
β-oxidation in the peroxisomes will convert 12-OPDA to
(+)-JA, which is finally conjugated with Ile (Wasternack and
Feussner, 2018; Wasternack and Strnad, 2016).
Recent work demonstrates that the biosynthesis of
jasmonates by F. oxysporum likely follows the plant path-
way with sequential transformation of 18:3n-3 into
12-OPDA by three enzymes, 13S-LOX, AOS, and AOC
(Oliw and Hamberg, 2017). The biosynthesis of JA-Ile by
plants and fungi and 9,10-dihydro-JA by fungi is outlined
in Fig. 1a. 9,10-Dihydro-JA could be formed either directly
from linoleic acid (18:2n-6) or from 18:3n-3 (Miersch
et al., 1999).
iso-OPDA 12-oxo-9(13),15(Z)-phytodienoic acid
KODE
KOTrE
NP
ketooctadecadienoic acid
ketooctadecatrienoic acid
normal phase
PDB
RP
potato dextrose broth
reversed phase
TIC
total ion current
α-ketol
12-oxo-13-hydroxy-9(Z)-octadecenoic acid,
12-oxo-13-hydroxy-9(Z),15(Z)-
octadecadienoic, or 10-oxo-11-hydroxy-7(Z),
13(Z)-hexadecadienoic acids
Introduction
Fusarium oxysporum is a pathogenic fungus, which infects
nearly all plants, the exception being grasses (Dean et al.,
2012; Ma et al., 2013). A special form (forma specialis
(f. sp.) of F. oxysporum designates a strain with capacity to
infect a specific host. F. oxysporum f. sp. cubense is per-
haps the most infamous strain. It causes Panama disease of
banana, which is a constant threat to this important com-
modity (Ploetz, 2015). Another strain, F. oxysporum f. sp.
tulipae (FOT), infects tulip bulbs and costs tulip farmers
approximately 10 million € annually.1 FOT is of scientific
interest for this reason and also for its capacity to form
large amounts of both jasmonoyl-isoleucine (JA-Ile) and
9,10-dihydro-JA-Ile (Cole et al., 2014; Oliw and Hamberg,
2017), which it shares with at least three other strains of
F. oxysporum (Cole et al., 2014).
Biosynthesis of 9,10-dihydro-JA from 18:2n-6 via a single
cis isomer of 12-oxophytoenoic acid (12-OPEA), as outlined
The biologically relevant form of JA is (+)-7-iso-
jasmonic acid ((+)-JA). It was first identified in lemon
(Demole et al., 1962) and later, 20–30 years ago, in the
growth media of three fungi (Lasiodiplodia theobromae
(Miersch et al., 1987), F. fujikoroi (Miersch et al., 1992)
and F. oxysporum f. sp. matthiola (Miersch et al., 1999)).
However, the first description of JA-Ile and 9,10-dihydro-
JA-Ile produced by F. fujikoroi and L. theobromae dates
back to 1970–71 (Aldridge et al., 1971; Cross and Web-
ster, 1970).
The plant biosynthesis of JA has been investigated in detail
due to the well-documented biological importance of
(+)-JA and (+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile)
(Wasternack and Feussner, 2018; Wasternack and Strnad,
2016). In addition, many of the biosynthetic enzymes have
been expressed and crystallized (Wasternack and Feussner,
2018). 13S-lipoxygenases (13S-LOX) of plants oxidize 18:3n-
Fig. 1 Overview of biosynthess of jasmonoyl-(S)-isoleucine (JA-Ile)
in plants and fungi, possible pathways to 9,10-dihydro-JA in fungi,
and four stereoisomers of the cyclopentane ring of jasmonates
(a) Left, biosynthesis of (+)-7-iso-JA-Ile in plants and fungi. Right,
possible pathways to 9,10-dihydro-(+)-7-iso-JA in fungi indicated by
broken arrows with question marks. The plant allene oxide cyclase
(AOC) does not metabolize 12,13(S)-epoxy-9(Z),11-octadecadienoic
acid (12,13S-EOD), but the fungal AOC might accept it as a substrate
as marked by the broken vertical arrow. Alternatively, the n-3 double
bond of (+)-7-iso-JA-Ile (or 12-oxophytodienoic acid [12-OPDA])
might be reduced as illustrated by the broken horizontal arrows.
(b) Four stereoisomers of the cyclopentane ring of JA: a, the cis iso-
mer (+)-7-iso-JA (the biologically important stereoisomer); b, the
trans isomer
3
to 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic
acid (13S-HPOTrE), which is dehydrated by allene oxide
1 https://www.narcis.nl/research/RecordID/OND1335463: Fusarium
oxysporum f.sp. tulipae, causal agent of the devastating dry rot in tulip
bulbs; unraveling the interaction.
Lipids (2019)

Lipids
in Fig. 1a (right), may seem to be a plausible pathway. It
lacks, however, precedence in plants. For example, homoge-
nates of sunflower converts 13(S)-hydroperoxy-9(Z),11(E)-
octadecadienoic acid (13S-HPODE) via an allene oxide to
two cis isomers of 12-OPEA (Grechkin et al., 2007;
Ogorodnikova et al., 2015); this also applies to tomato
CYP74C (Grechkin et al., 2008; Itoh et al., 2002). 9S-LOX-
derived allene oxides in plants can also form two cis
cyclopentenone derivatives (Hamberg, 2000; Itoh et al.,
2002). Maize root is an exception. Its soluble cyclase pro-
duces 9(S),13(S)-10-oxo-11-phytoenoic acid (Ogorodnikova
et al., 2015).
Plant AOC has been investigated in detail using allene
oxides derived from 13S-LOX (Grechkin, 1994; Ziegler
et al., 1999, 2000). The n-3 double bond of 12,13S-EOT is
required for efficient transformation (Grechkin, 1994; Ziegler
et al., 1999). 12,13(S)-epoxy-9(Z),11-octadecadienoic acid
(12,13S-EOD) does not serve as a substrate for plant AOC; it
is transformed at an insignificant rate, about 0.1% or less,
compared to 12,13S-EOT (Grechkin, 1994; Hamberg and
Hughes, 1988; Vick and Zimmerman, 1979; Ziegler et al.,
1999). The conspicuous fungal biosynthesis of 9,10-dihydro-
JA-Ile suggests that the mechanism of biosynthesis of the
key intermediate 12-OPEA could be catalyzed by a fungal
AOC with different catalytic properties from plant AOC.
However, there could also be another alternative involving
the reduction of the n-3 double bond of one of the cyclized
products formed from 18:3n-3 (Fig. 1a, left) (Miersch et al.,
1999). Whether 9,10-dihydro-JA-Ile originates from 18:3n-3
or 18:2n-6 has not yet been determined.
Allene oxides can differ in the geometry of the double
bond (Z or E), which can affect cyclization. For example,
the 10Z isomer of the 9S-HPODE-derived allene oxides
cyclizes to two cis isomers of a cyclopentenone derivative
in aprotic solvents, but the 10E-isomer is not reactive
(Brash et al., 2013; Hebert et al., 2016). This difference
may apply to 11Z and 11E isomers of 13S-LOX-derived
allene oxides (González-Pérez et al., 2017), but flaxseed
AOS (CYP74A) forms allene oxides with 11E configura-
tion (Medvedeva et al., 2005). Plant AOC accelerates cycli-
zation and forms only one of the four possible side chain
stereoisomers. For comparison, the four side-chain stereo-
isomers of JA are shown in Fig. 1b. The biologically rele-
vant form of JA with (+)-7-iso configuration is marked “a,”
and it may isomerize nonenzymatically to the trans isomer
(marked “b”) (Holbrook et al., 1997).
The enzymatic mechanism of formation of
cyclopentenone derivatives from allene oxides has been
studied intensively (Hofmann et al., 2006; Ziegler et al.,
1999, 2000), and in recent years also by quantum physics
(González-Pérez et al., 2017; Hebert et al., 2016). It was
originally proposed that the cyclopentenone was formed
via an ionic mechanism (Grechkin et al., 2002). Density-
functional and other calculations now suggest that the low-
est energy pathway represents homolytic cleavage of the
epoxide and formation of an oxyallyl diradical. The latter is
rearranged to the observed cyclopentenone with cis config-
uration, preferentially from allene oxides with Z configura-
tion (González-Pérez et al., 2017; Hebert et al., 2016).
The main goal of the present investigations was to ana-
lyze the mechanism of biosynthesis of 9,10-dihydro-JA-Ile
and, for comparison, JA-Ile. We used FOT as a model for
the strains of F. oxysporum with capacity to form
jasmonates. We report that 9,10-dihydro-(+)-JA is formed
from 18:2n-6, presumably by an AOC with a broader sub-
strate specificity than the plant AOC, but likely with a simi-
lar reaction mechanism.
Materials and Methods
Materials
Cartridges with C18 silica (SepPak classic C18) were from
Waters (Baltimore, MD, USA). Fatty acids and hydroperox-
ides were dissolved in ethanol and stored in stock solutions at
−20 ꢁC. 18:2n-6 (99%) and C20 fatty acids were from Sigma-
Aldrich (Darmstadt, Germany) and Larodan (Solna, Sweden).
18:3n-3 (99%), [13C18]18:2n-6 (98%), [17,17,18,18,18-2H5]
18:3n-3 (d5-18:3n-3), JA-Ile, 12-OPDA, and 12-oxo-
13-hydroxy-9(Z)-octadecenoic acid (α-ketol) were also from
Larodan. 12-Oxo-9(13),15(Z)-phytodienoic acid (iso-OPDA)
was prepared as recently described for its donor homolog
(Monte et al., 2018). 13S-HPODE was from Larodan, but
also prepared from 18:2n-6 and [13C18]18:2n-6 with soybean
LOX-1
(Lipoxidase,
Sigma-Aldrich).
13S-HPOTrE,
[17,17,18,18,18-2H5]-13S-HPOTrE (d5-13S-HPOTrE), 13R-
HPOTrE and 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic
acid (9S-HPODE), and 15S-hydroperoxides of 20:2n-6,
20:3n-3 and 20:4n-6 and 11S-hydroperoxide of 16:3n-3 were
prepared as described (Chen et al., 2017; Oliw and Hamberg,
2017). (−)-JA, isoleucine, and potato dextrose broth (PDB)
were from Sigma-Aldrich. FOT (NRRL 26954) was from
ARS Culture Collection (Peoria, IL, USA), imported with per-
mission from the Swedish Board of Agriculture, and chosen
due to its capacity to form jasmonates (Cole et al., 2014; Oliw
and Hamberg, 2017). FOT was stored on potato dextrose
agar plates at +4 ꢁC. It was recultivated for 7–10 days at
room temperature and usually used for inoculation within
2 months.
[2,2-2H2]JA and [2,2-2H2]JA-Ile
Sodium (184 mg; 8 mmol) was added to CH3O2H (50 mL)
followed by the ethylene ketal of (ꢂ)-jasmonic acid methyl
Lipids (2019)

Lipids
ester (1 g). The mixture was refluxed for 23 h, acidified
and extracted with diethyl ether. An aliquot (215 mg) was
dissolved in acetone (10 mL) containing 0.5 mL 2 M
hydrochloric acid. After stirring at 22 ꢁC for 45 min, the
deprotected labeled methyl jasmonate was isolated by
extraction with diethyl ether. The product was dissolved in
acetone (4 mL) and 0.1 M potassium phosphate buffer
pH 8.0 (36 mL) was added. Ester hydrolysis was accom-
plished by stirring this mixture with porcine liver esterase
(2500 units) at 22 ꢁC for 4 h. The product (159 mg) was
subject to reversed phase HPLC (RP-HPLC) using a sol-
vent system of acetonitrile/water/acetic acid (35:65:0.005,
v/v/v) yielding a mixture of dideuterated (ꢂ)-JA and
(ꢂ)-7-iso-JA in proportions about 92:8 (w/w). This mixture
was subject to the normal phase HPLC (NP-HPLC) using a
solvent system of ethanol/hexane/acetic acid (1:99:0.025,
v/v/v) to afford a fraction of pure [2,2-2H2](ꢂ)-JA as well
as a fraction consisting of a mixture of [2,2-2H2](ꢂ)-JA and
[2,2-2H2](ꢂ)-7-iso-JA in proportions 1.7:1 (w/w). The lat-
ter mixture of labeled cis and trans isomers (11 mg) was
used for the biological experiments. The mass spectrum of
the methyl ester derivative of [2,2-2H2](ꢂ)-JA showed
prominent ions at m/z 226 (M+), 208 (M+ − H2O),
195 (M+ − OCH3), 151 (M+ − CD2 COOCH3), and
83 (M+ − (CD2 COOCH3 + CH2 CH CH CH2 CH3)
+ H). The sample consisted of 97.1% dideuterated, 2.6%
monodeuterated, and 0.3% undeuterated molecules.
[2,2-2H2]-(ꢂ)JA-Ile was then synthesized from 5 mg
[2,2-2H2] (ꢂ)-7-iso-JA as described (Oliw, 2018) and puri-
obtained. An aliquot of this material was saponified and sub-
ject to final purification by RP-HPLC (solvent system,
acetonitrile/water/acetic acid, 80:20:0.01 (v/v/v)) affording
the title compound (purity, >98%; >99.5% deuterated
molecules).
(ꢂ)-trans-12-OPEA and (ꢂ)-cis-12-OPEA
The title compounds were prepared by six-carbon chain
elongation of 9,10-dihydro-JA followed by dehydrogena-
tion of the cyclopentanone using the methodology recently
described (Monte et al., 2018). Briefly, (ꢂ)-9,10-dihydro-
JA (4.7 mmol, 1.0 g) and methyl hydrogen suberate
(10.6 mmol, 2.0 g) were subject to anodic coupling (1 A
for 1.7 h). Extractive isolation followed by silica gel chro-
matography afforded about 90% pure 12-oxophytonoic acid
methyl ester (0.47 g, yield, 32%). An aliquot of the sample
was dehydrogenated by treatment with palladium(II) ace-
tate, diethyl allyl phosphate, and Na2CO3 in dry N,N-
dimethylformamide at 80 ꢁC for 18 h (Monte et al., 2018).
Extraction with diethyl ether followed by RP-HPLC (sol-
vent system, acetonitrile/water (75:25, v/v) afforded
12-OPEA methyl ester, an aliquot of which was hydrolyzed
to the free acid by treatment with porcine liver esterase
(250 U) in 5 mL of 0.1 M tris buffer (pH 8.0)/acetone, 9:1
(v/v) at 23 ꢁC for 15 h. NP-HPLC (column, 250 × 4.6 mm
of Nucleosil 50-4; mobile phase, 2-propanol/hexane/acetic
acid, 1:99:0.005 (v/v/v)) separated the material into side
chain trans and cis isomers, that is (ꢂ)-trans-12-OPEA
(94%, 24.0 mL effluent) and (ꢂ)-cis-12-OPEA (6%,
29.8 mL), both obtained as colorless viscous oils. In agree-
ment with previous work (Hamberg and Hughes, 1988),
their UV spectra showed λmax (EtOH) = 221 nm. The mass
spectrum of the methyl ester derivative of (ꢂ)-trans-
12-OPEA showed prominent ions at m/z 308 (M+, 4%),
277 (M+—OCH3, 19), 238 (M+—(CH2)4CH3 H, 30),
206 (238—CH3OH, 23), 178 (M+—(CH2)5COOCH3 + H,
20), 165 (M+—(CH2)6COOCH3, 14), 151 (M+—
(CH2)7COOCH3, 38), 109 (56), 96 (100), and 82 (53), in
full accord with the spectrum previously published
(Hamberg and Hughes, 1988).
fied by RP-HPLC using
a
solvent system of
methanol/water/acetic acid, 700:300:0.1; all aliquot of each
fraction was analyzed with LC–MS by direct injection and
those with [2,2-2H2] (ꢂ)-7-iso-JA-Ile were combined.
[17,17,18,18,18-2H5]18:2n-6 (d5-18:2n-6)
18:2n-6, deuterated in the ω1 and ω2 positions, was prepared
by coupling of acetylenic fragments followed by partial
hydrogenation of triple bonds. Briefly, the tetrahydropyranyl
derivative of propargyl alcohol (1.19 g, 8.5 mmol) was
treated with butyllithium and coupled to [4,4,5,5,5-2H5]
1-bromopentane (1 g, 6.4 mmol) (CDN Isotopes, Pointe-
Claire, Quebec, Canada). The resulting tetrahydropyranyloxy
derivative of 2-octyn-1-ol was converted into 1-bromo-
2-octyne by treatment with triphenylphosphine dibromide in
dichloromethane. Following purification on a silica gel col-
umn, the labeled bromooctyne (934 mg, 4.8 mmol) was sub-
ject to copper-promoted coupling (Caruso and Spinella,
2003) to methyl 9-decynoate (910 mg, 5 mmol). The
resulting acetylenic ester (1.34 g, 4.5 mmol) was semi-
hydrogenated using Lindlar catalyst/quinoline in ethyl
acetate. Following purification on a column of silica gel,
methyl [17,17,18,18,18-2H5]linoleate (1.1 g, 3.7 mmol) was
Fungal Cultures
A volume of 50–60 mL of PDB (2.4%; w/v) or PDB/20%
glucose or maltose (1:2, v/v) in 250 mL baffled flasks was
inoculated with FOT on agar (0.5–1 cm2) from a culture on
PDA plates and grown with shaking (100 rpm) at 28 ꢁC in
the dark (Oliw and Hamberg, 2017). For estimation of
jasmonates secreted to the medium, the loss of water by
evaporation was estimated by weight and replenished with
water. The growth media were centrifuged (3100 x g) and
the supernatant was then assayed for jasmonates. After
Lipids (2019)

Lipids
about a week, the culture was colored increasingly red,
likely due to bikavarin (Limon et al., 2010). Aliquots of the
growth medium were harvested after 4 up to 21 days. An
aliquot (1 mL) of the medium was extracted on a cartridge
of C18 silica (SepPak/C18), washed with water (2 mL), and
lipids were eluted with ethyl acetate (4–5 mL). The organic
extract was evaporated under a stream of N2, dissolved in
ethanol (100 μL), and 10 μL was then analyzed for JA-
conjugates by LC–MS/MS as described (Oliw and
Hamberg, 2017).
The reddish mycelia of 2.5–6 mL of liquid culture (2–3
weeks old) were collected by centrifugation (3100 ×g;
10–20 min), suspended in 2.5–6 mL 0.1 M KHPO4 buffer
(pH 7.5)/2 mM EDTA/0.04% Tween-20 (v/v/v), recentr-
ifuged, and finally suspended in the same volume of fresh
buffer in 15 mL tubes. Fatty acids were added to a final
concentration of about 100–120 μM, and again after
2 and/or 4 h. The tubes were incubated at room temperature
with tilting (Duomax 1030, Heidolph) for 23 or 30 h at
22 ꢁC. An aliquot of the mycelia was then precipitated by
centrifugation (3100 ×g; 10 min) after 4, 23, and 30 h. The
supernatant was diluted with water and extracted with C18
silica (SepPak/C18). The extracts were reduced to dryness
under a stream of N2 and dissolved in ethanol for analysis.
In some experiments, the fatty acids were added either
directly to mycelia, which had been disrupted by sonication
(Bioruptor Next Gen, 10 × 30 s, 4 ꢁC), or to the low-speed
supernatant after centrifugation (17, 000 ×g, 20 min;
+4 ꢁC). These incubations were performed on ice.
buffer by sonication (0.2 mg protein/ml (UV analysis;
280 nm)). Incubation of an aliquot with 100 μM 13S-
HPOTrE (30 min; +4 ꢁC) showed that the AOS activity
was present in the microsomal fraction. A volume of
0.5 mL of the latter was incubated with 20 μM 13S-
HPOTrE, 13S-HPODE, or 11S-HPHeTrE (11(S)-
hydroperoxy-7(Z),9(E),13(Z)-hexadecatrienoic acid) for
30 min on ice (n = 4). An amount of 2 μg of the internal
standard (iso-OPDA) was added before extractive isolation
(SepPak/C18). The biosynthesis of the three α-ketols was
estimated during LC–MS/MS analysis from the signal
intensities of m/z 197, 197, and 169 (197–28), respectively,
and the intensity of the internal standard (iso-OPDA; m/
z 165).
LC–MS/MS Analysis
RP-HPLC with MS/MS analysis was performed with a Sur-
veyor MS pump (ThermoFisher, Hägersten, Sweden) and
an analytical octadecyl silica column (5 μm; 2.0 ×
150 mm, Phenomenex, Torrance, CA, USA). The column
was eluted at 0.25–0.35 mL/min with /water/acetic acid,
650:350:0.05, 700:300:0.05, or 750:250:0.05 (v/v/v). The
effluent was subject to electrospray ionization in a linear
ion trap mass spectrometer (LTQ, ThermoFisher). The
heated transfer capillary was set at 310 ꢁC, the ion isolation
width at 1.5 amu, the collision energy at 35 (arbitrary
scale), and the tube lens at −52 V. Samples were
injected manually (Rheodyne 7510, Bensheim, Germany)
or by an auto sampler (Surveyor Autosampler Plus,
ThermoFisher).
0.1–0.3 mM 13S-HPODE and [13C18]13S-HPODE were
incubated with the suspended mycelia (2–4 mL) as above
for 2 h at room temperature in the KHPO4 buffer. Mycelia
were precipitated by centrifugation and the supernatant was
analyzed as above. HPODE and HPOTrE (100 μM) were
also incubated with low-speed supernatant of sonicated
mycelia on ice (45 min) before extractive isolation as above.
The extracts were dissolved in hexane and hydroperoxides
were reduced to alcohols with triphenylphosphine before
LC–MS analysis of α-ketols and epoxyalcohols (m/z
311 ! full scan or m/z 309 ! full scan, respectively) and
analysis of 13-ketooctadecadienoic acid (13-KODE)/
13-ketooctadecatrienoic acid (13-KOTrE) and 12-OPEA/
12-OPDA (m/z 293 ! full scan and m/z 291 ! full scan,
respectively).
NP-HPLC with in-line MS/MS analysis was performed
with a silica column (5 μm; 2 × 250 mm, Kromasil 100 SI,
Dalco Chromtech, Märsta, Sweden), which was eluted with
isopropyl alcohol/hexane/acetic acid, 1.5–4.0:98.5–
96.0:0.05 (v/v/v) at 0.5 mL/min (Constametric 3200,
LDC/MiltonRoy, Riviera Beach, FL, USA). The eluate was
mixed in-line with isopropyl alcohol/water (3:2 (v/v);
0.3 mL/min) from the Surveyor MS pump (ThermoFisher).
The combined effluents were introduced by electrospray
ionization into the ion trap mass spectrometer above.
Enantiomers of α-ketols and 12-OPEA were analyzed in
the same way by chiral phase (CP)-HPLC (Reprosil
Chiral AM; 5 μm; 250 × 2 mm; Dr. Maisch, Ammerbuch,
Germany)), eluted with ethanol/hexane/acetic acid,
5:95:0.05 (v/v/v), at 0.2 mL/min. The eluate was mixed
with isopropyl alcohol/water (3:2 (v/v); 0.15 mL/min) as
above.
Preparation of the Microsomal Fraction
Nitrogen powder of FOT was homogenized in 0.1 M
KHPO4 buffer (pH 7.5)/2 mM EDTA/0.01% Tween-20
(10 passes) and centrifuged for 10 min at 30,000 ×g
(+4 ꢁC; Sorvall Evolusion RC). Microsomes were prepared
from the low-speed supernatant by centrifugation for 1 h
(+4 ꢁC, 75,000 ×g). The microsomes were suspended in
Hydrogenation of [2H2]JA-Ile was performed with H2
and catalytic amounts of Pd/C in methanol for 2 min.
Hydroperoxides were reduced to alcohols in hexane with
5 μL triphenylphosphine (10 mg/mL cyclohexane).
Lipids (2019)

Lipids
Fig. 2 Normal phase HPLC (NP-HPLC) separation and MS/MS analysis of jasmonoyl-(S)-isoleucine (JA-Ile) and 9,10-dihydro-JA-Ile with MS2
spectra of the three major jasmonate conjugates in the growth medium (PDB). (a) NP-HPLC-MS/MS analysis. Top trace, total ion current (TIC).
Peak I, 9,10-dihydro-JA-Ile; peaks II and III, JA-Ile. (b) MS2 spectrum of the main isomer of JA-Ile (peak II), (+)-7-iso-JA-Ile. (c) MS2 spectrum
of the minor isomer of JA-Ile (peak III). (d) MS2 spectrum of the main isomer of 9,10-dihydro-JA-Ile (peak I), 9,10-dihydro-(+)-7-iso-JA-Ile.
The inserts in b–d indicate MS/MS fragmentation ions. The cis and trans isomers differ in their signal intensities of m/z 172 compared to the base
peak (m/z 130); compare m/z 172 in the MS2 spectra in b and d with m/z 172 in the MS2 spectrum in C (with a different region for magnification).
TIC, total ion current
Results
Ile and 9,10-dihydro-JA-Ile eluted with peak III of the deu-
terated standards, and the minor isomer with peak IV. By
using a synthetic standard of (−)-JA-Ile containing a few
percent of (+)-JA-Ile, it was found that the former eluted
with peak IV and the latter with peak III. The fungus thus
formed (+)-JA-Ile and 9,10-dihydro-(+)-JA-Ile as the main
jasmonates.
On RP-HPLC, the elution order was reversed in compar-
ison with NP-HPLC as illustrated by separation of
[2,2-2H2]-9,10-dihydro-JA-Ile (Fig. 3c). 9,10-Dihydro-
(+)-JA-Ile eluted as the second peak (marked III) and the
(−) isomer as the first peak (marked IV, cf. Fig. 3b, c).
JA-Ile and 9,10-Dihydro-JA-Ile in the Growth Medium
JA-Ile and 9,10-dihydro-JA were assayed in the growth
medium by RP-HPLC or NP-HPLC with MS/MS analysis.
The separation of JA-Ile and 9,10-dihydro-JA by NP-HPLC
is shown in Fig. 2a. (+)-JA-Ile was found in peak II,
(−)-JA-Ile in peak III, and 9,10-dihydro-JA-Ile in peak I.
The MS2 spectra (m/z 322 ! full scan) of JA-Ile in
peaks I and II are shown in Fig. 2b, c, respectively. The
main difference between the two MS2 spectra is the signal
intensity of m/z 172 compared to the base peak (m/z 130).
The MS2 spectrum of 9,10-dihydro-JA (m/z 324 ! full
scan) in peak I is shown in Fig. 2d. The fragmentation with
a relatively weak signal at m/z 172 was similar to the MS2
spectrum in Fig. 2b with its weak signal at m/z 172.
We next examined the biological products by NP-
HLPLC–MS/MS after admixture with [2,2-2H2]JA-Ile and
[2,2-2H2]-9,10-dihydro-JA-Ile during NP-HPLC–MS/MS.
The dideuterated standards contain two chiral centers
(cf. Fig. 1b), resulting in four isomers of each (I-IV). They
were resolved by NP-HPLC as shown in Fig. 3a (JA-Ile)
and Fig. 3b (9,10-dihydro-JA-Ile). The main isomer of JA-
Biosynthesis of JA-Ile from 18:3n-3 by Mycelia
The separation by RP-HPLC of JA-Ile and 9,10-dihydro-
JA-Ile released to the growth medium is shown in
Fig. 4a. We next examined whether mycelia transformed
d5-18:3n-3 into d5-(+)-JA-Ile) and into d5-9,10-dihydro-
(+)-JA-Ile. To this respect, mycelia were incubated with d5-
18:3n-3 for 4–30 h. In addition to the unlabeled (+)-JA-Ile
and 9,10-dihydro-(+)-JA-Ile from endogenous substrates,
mycelia transformed d5-18:3n-3 into d5-JA-Ile as shown in
Lipids (2019)

Lipids
Fig. 3 LC-MS analysis of jasmonoyl-(S)-isoleucine (JA-Ile) and 9,10-dihydro-JA-Ile in the growth medium (PDB). The main metabolites were
identified as cis and trans isomers of JA-Ile and 9,10-dihydro-JA-Ile. (a) Elution of the biologically formed JA-Ile (top trace, marked a and b) in
comparison with [2,2-2H2]JA-Ile (bottom trace) on NP-HPLC with MS/MS analysis (eluted with 3% isopropropyl alcohol in hexane,
0.5 mL/min). Authentic standards showed that the cis isomer, (+)-7-iso-JA-Ile, eluted with peak III and the trans isomer, (−)-JA-Ile, with peak
IV. (b) Elution of the biologically formed 9,10-dihydro-JA-Ile and [2,2-2H2]9,10-dihydro-JA-Ile on NP-HPLC. The two biological stereoisomers
(marked a and b) were identified as the cis (9,10-dihydro-(+)-7-iso-JA-Ile) and trans (9,10-dihydro-(−)-JA-Ile) isomers in peaks III and IV,
respectively. (c) Reversed phase HPLC (RP-HPLC)-MS/MS analysis of [2,2-2H2]9,10-dihydro-JA-Ile. Separation of the four stereoisomers of
[2,2-2H2]9,10-dihydro-JA-Ile on RP-HPLC (65% methanol in water, 0.3 mL/min). The peaks marked I-IV likely contain the compounds in peaks
I-IV of (b) as judged from co-elution with the two biologically formed stereoisomers and their MS2 spectra with marked different signal intensi-
ties of m/z 172
Fig. 4b. Importantly, d5-9,10-dihydro-(+)-JA-Ile could not
be detected as a metabolite of d5-18:3n-3.
Biosynthesis of 12-OPDA from 18:3n-3
Cochromatography of biological JA-Ile with [2,2-2H2]-
JA-Ile showed that the latter was resolved by RP-HPLC
only into two major peaks and later eluting the third peak.
(+)-JA-Ile (and (−)-JA-Ile) co-eluted with a peak, the sec-
ond peak of [2,2-2H2]-JA-Ile.
As previously reported, d5-18:3n-3 was converted to d5-
12-OPDA (and labeled α-ketol) after incubation with
mycelia in buffer for 1 h at room temperature (Oliw and
Hamberg, 2017).
We found that the transformation of d5-18:3n-3 into
d5-12-OPDA was undetectable in cultures grown for
only 4 days, clearly detectable after 8 days of growth,
We conclude that d5-18:3n-3 is not converted to signifi-
cant amounts of d5-9,10-dihydro-JA-Ile under these
conditions.
and appeared to be maximal after 2–3 weeks.
A
Fig. 4 Reversed phase HPLC (RP-HPLC)-MS/MS analysis of jasmonoyl-(S)-isoleucine (JA-Ile) and 9,10-dihydro-JA-Ile in the growth medium
(PDB) of Fusarium oxysporum f. sp. tulipae (FOT) and transformation of d5-18:n-3 to d5-JA-Ile by mycelia in phosphate buffer. (a) RP-HPLC-
MS/MS analysis and separation of JA-Ile and 9,10-dihydro-JA-Ile. (b) RP-HPLC-MS/MS analysis of JA-Ile and d5-JA-Ile formed during incuba-
tion of mycelia of FOT with d5-18:3n-3 in phosphate buffer. The arrow shows that d5-18:3n-3 was not converted to d5-9,10-dihydro-JA-Ile in
detectable amounts
Lipids (2019)

Lipids
Fig. 5 Time curve for the relative biosynthesis of d5-12-oxophytodienoic acid (12-OPDA) from d5-18:n-3 by mycelia of Fusarium oxysporum
f. sp. tulipae (FOT) in phosphate buffer in comparison with the biosynthesis of 12-OPDA from endogenous 18:3n-3. (a) Reversed phase HPLC
(RP-HPLC)-MS/MS analysis of labeled and unlabeled 12-OPDA with selective ion monitoring of the characteristic ions, m/z 170 and 165.
(b) Time curve showing the ratio of labeled and unlabeled 12-OPDA
comparison of the release of labeled and unlabeled
12-OPDA over time is shown in Fig. 5. The ratio
between labeled and unlabeled 12-OPDA increased rap-
idly in the first 30 min and then increased slowly for
up to at least 2 h.
The MS2 spectra of [13C18]9,10-dihydro-(−)JA-Ile and
[13C18]9,10-dihydro-(+)-JA-Ile differed in the signal inten-
sities of m/z 174 relative to the base peak, m/z 130, as
shown in Fig. 6c, and by the selected ion chromatograms
of m/z 172 in Fig. 6a, and m/z 174 in Fig. 6b.
The secretion of labeled and unlabeled JA-Ile and
9,10-dihydro-JA-Ile by mycelia, which were harvested after
2, 4, 23, and 30 h, showed that these jasmonates were
detectable at 4 h. However, a second addition of substrate
at 4 h and incubation overnight facilitated analysis of the
labeled metabolites.
Biosynthesis of 12,13S-EOT from 13S-HPOTrE
13S-HPOTrE was transformed by the low-speed supernatant
of sonicated mycelia of FOT to an α-ketol, epoxyalcohols,
and 13-KOTrE, however, only traces of 12-OPDA were
detected. The latter was likely formed from endogenous
18:3n-3 as d5-13S-HPOTrE did not appear to be trans-
formed into d5-12-OPDA. The α-ketol and the d5-labeled
α-ketol were identified as 13-hydroxy-12-oxo-12(Z),15(Z)-
octadecadienoic acid by comparison of the MS2 spectrum of
authentic 13-hydroxy-12-oxo-9(Z)-octadecenoic acid dis-
cussed below. CP-HPLC analysis of the α-ketol showed that
it contained the 13R and 13S stereoisomers in a ratio
of 60:40.
Biosynthesis of 12-OPEA from 18:2n-6
The MS2 spectrum (m/z 293 ! full scan) of 12-OPEA is
illustrated in Fig. 7a. Characteristic signals were noted at
m/z 165 and 150 in addition to the signals due to loss from
the carboxylate anion of water, CO, and CO2 at m/z
275, 265, and 249 (base peak), respectively.
Next, we examined whether 18:2n-6 was oxidized to
12-OPEA by analysis of the products formed by the low-
speed supernatant of mycelia. RP-HPLC–MS/MS analysis
showed that 18:2n-6 was converted to a compound with the
same retention time as synthetic 12-OPEA and with a simi-
lar MS2 spectrum with the three characteristic ions, m/z
249, 165, and 150. Mycelia transformed d5-18:2n-6 into
d5-12-OPEA, and the MS2 spectrum (298 ! full scan) is
shown in Fig. 7b with signals at m/z 249 + 5, 165 + 5, and
150 + 5. The mycelia also formed 12-OPDA from endoge-
nous 18:3n-3, and it was separated from d5-OPEA as
shown in Fig. 7c.
13R-HPOTrE was not transformed by the AOS activities
of mycelia after sonication; epoxyalcohols and 13-KOTrE
were the main products.
Biosynthesis of 9,10-Dihydro-JA-Ile from 18:2n-6
[17,17,18,18,18-2H5]linoleic acid (d5-18:2n-6) was trans-
formed by mycelia into d5-9,10-dihydro-JA-Ile as judged by
RP-HPLC–MS/MS analysis (Fig. 6a). The main product
was d5-9,10-dihydro-(+)-JA-Ile, but significant amounts of
d5-9,10-dihydro-(−)-JA-Ile were also detected. As shown in
Fig. 3c, the four stereoisomers of [2,2-2H2]-9,10-dihydro-
JA-Ile were partly resolved by RP-HPLC, and 9,10-dihydro-
(−)-JA-Ile elutes before 9,10-dihydro-(+)-JA-Ile.
Biosynthesis of 12,13S-EOD from 13S-HPODE
We also confirmed by RP-HPLC–MS/MS analysis that
We then investigated 13S-HPODE and [13C18]13S-HPODE
as substrates for AOS. RP-HPLC–MS/MS analysis showed
[13C18]18:2n-6 was transformed in the same way (Fig. 6b).
Lipids (2019)

Lipids
Fig. 6 Reversed phase HPLC (RP-HPLC)-MS/MS analysis of the
transformation of d5-linoleic acid (18:2n-6), [13C18]18:2n-6, and
endogenous 18:2n-6 to 9,10-dihydro-JA-Ile by mycelia of Fusarium
oxysporum f. sp. tulipae (FOT). (a) RP-HPLC-MS/MS analysis of
9,10-dihydro-JA-Ile and d5-9,10-dihydro-JA-Ile. The major peak at
15 min in the top chromatogram was identified as the cis isomer,
9,10-dihydro-(+)-7-iso-JA-Ile, formed from endogenous 18:2n-6, and
the corresponding peak in the middle chromatogram as d5-9,-
Fig. 7 MS2 spectrum of 12-oxo-10-phytoenoic acid (12-OPEA) and
Reversed phase HPLC (RP-HPLC)-MS/MS analysis of d5-12-OPEA
obtained from incubation of mycelia with [17,17,18,18,18-2H5]lin-
oleic acid (d5-18:2n-6). (a) MS2 spectrum (m/z 293 ! full can) of
synthetic 12-OPEA. (b) MS2 spectrum (m/z 298 ! full can) of d5-
12-OPEA, obtained by incubation of d5-18:2n-6 of with mycelia for
2 h; this culture had been grown for 21 days in PDB. (c) RP-HPLC-
MS/MS analysis of endogenous 12-oxophytodienoic acid (12-OPDA)
and d5-12-OPEA in the experiment described in (b). The RP-HPLC
column was eluted with 75% methanol in water
10-dihydro-(+)-7-iso-JA-Ile. (b) RP-HPLC-MS/MS analysis of
13
9,10-dihydro-JA-Ile and [
C ]9,10-dihydro-JA-Ile. Unlabeled and
18
labeled material in the peak at 15 min was identified as 9,10-dihydro-
(+)-7-iso-JA-Ile. (c) Partial MS2 spectra of the minor (trans; left) and
13
major (cis; right) stereoisomers of [
C ]9,10-dihydro-JA-Ile formed
18
13
from [
C ]18:2n-6 by mycelia and released to the phosphate buffer.
18
Note the different signal intensities of m/z 174 (with two 13C atoms)
compared to the base peak (m/z 130). The signal intensity of m/z
174 was magnified in the MS2 spectrum to the right as indicated by
the bar
those of the authentic α-ketol. CP-HPLC analysis of the
α-ketol showed that it consisted of the 13R and 13S stereoiso-
mers in a ratio of 59:39. The epoxyalcohols, which were pre-
sumably formed nonenzymatically from 13S-HPODE and
eluted between 8 and 11 min showed characteristic signals at
m/z 197, 211, and 227 (11-hydroxy-12,13-epoxyoctadecenoic)
and at m/z 171, 193, 211, and 227 (9-hydroxy-12,-
13-epoxyoctadecenoic acid) (Oliw et al., 2006).
that 0.1–0.3 mM 13S-HPODE and 0.1–03 mM [13C18]13S-
HPODE were transformed by mycelia into 13-KODE,
epoxyalcohols, and an α-ketol, 13-hydroxy-12-keto-9(Z)-
octadecenoic acid. The latter eluted after the epoxyalcohols
(bottom trace, Fig. 8a). The α-ketol was identified by its MS2
(Fig. 8b) and MS3 spectra (Fig. 8c), which were identical to
We also investigated 9S-HPODE as a substrate, but it
was mainly transformed into 9-KODE and epoxyalcohols.
Lipids (2019)

Lipids
Fig. 8 Biosynthesis of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid (12,13S-EOD) as detected by the hydrolysis product, the α-ketol 12-oxo-
13-hydroxy-9(Z)-octadecenoic acid, formed after incubation of mycelia with 0.3 mM 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-
13
HPODE) and [
C
]13S-HPODE. (a) Reversed phase HPLC (RP-HPLC)-MS/MS analysis of 13-KODE (m/z 293 ! full scan) and analysis of
18
epoxyalcohols (peaks I and II) and an α-ketol in peak III (m/z 311 ! full scan). Trace amounts of 12-oxo-10-phytoenoic acid (12-OPEA) were
detected in the peak marked by the arrow at the retention time of synthetic 12-OPEA. The RP-HPLC column was eluted with 70% methanol.
(b) MS2 spectrum (m/z 311 ! full scan) of the α-ketol, 12-oxo-13-hydroxy-9(Z)-octadecenoic acid, produced from 13S-HPODE by mycelia of
Fusarium oxysporum f. sp. tulipae (FOT) in phosphate buffer; the α-ketol is likely the hydrolysis product of 12,13S-EOD. (c) MS3 spectrum (m/z
311 ! 293 ! full scan) of the α-ketol, 12-oxo-13-hydroxy-9(Z)-octadececoic acid, in a. (d) MS2 spectrum (m/z 329 ! full scan) of the labeled
13
α-ketol, [
C
]12-oxo-13-hydroxy-9(Z)-octadecenoic acid, formed from [13C18]13S-HPODE
18
The α-ketol, 9-hydroxy-10-oxo-12(Z)-octadecenoic acid,
could not be detected.
In conclusion, 13S-LOX, 13S-AOS, and AOC likely
sequentially form 12-OPEA. 13S-AOS is the only enzyme,
which can be readily detected in cell-free preparations of
FOT with 13S-hydroperoxides of 18:2n-6 and 18:3n-3 as
substrates.
12-OPEA elutes before 13-KODE on RP-HPLC with a
retention time of 14 min. Small amounts of 12-OPEA in
comparison with 13-KODE formed by mycelia could be
identified in the minor peak with this retention time (cf. top
trace, Fig. 8a); this trace amount of 12-OPEA could be
formed from endogenous 18:2n-6 in analogy with 12-OPDA
discussed above.
CP-HPLC Analysis of Fungal 12-OPEA
12-OPEA and 12-OPDA, which were released from the
mycelium of FOT, were analyzed by CP-HPLC-MS/MS
as shown in Fig. 9a. 12-OPEA eluted as a major peak
1 min before 12-OPDA. For comparison, (ꢂ)-cis-
12-OPEA was separated by CP-HPLC into two major
components (peaks I and II), whereas natural 12-OPDA
eluted between the two 12-OPEA isomers, about 1 min
after peak I (Fig. 9b) The first isomer (peak I) of
12-OPEA had the same retention time as 12-OPEA
formed by FOT, whereas the second isomer (peak II)
eluted 1.6 min later.
Sonicated mycelia also transformed 13S-HPODE and
[13C18]13S-HPODE into the α-ketol as a major product; the
MS2 spectrum of the [13C18]-labeled α-ketol is shown in
Fig. 8d. The α-ketol in these experiments appeared to be a
relatively prominent metabolite in comparison with the bio-
synthesis of labeled 9,10-dihydro-JA-Ile from d5- and
[13C18]18:2n-6 by mycelia. Furthermore, neither intact nor
sonicated mycelia appeared to oxidize 18:3n-3 to 13S-
HPOTrE significantly as only small amounts of 9- and
13(S)-hydro(pero)xy-9(Z),11(E)-octadecadienoic acid (13-
H(P)ODE) could be detected by LC-MS analysis. However,
1 mM d5-13S-HPOTrE was converted to trace amounts of
d5-12-OPDA by mycelia of FOT as previously reported
(Oliw and Hamberg, 2017).
We conclude that the fungus likely forms 9S,13S-
12-OPEA, which is the expected precursor of 9,10-
dihdydro-(+)-7-iso-JA-Ile (Fig. 1a, right).
Lipids (2019)

Lipids
Fig. 9 Chiral phase (CP)-HPLC-MS/MS analysis of 12-oxo-
10-phytoenoic acid (12-OPEA) and 12-oxophytodienoic acid
(12-OPDA). (a) CP-HPLC-MS/MS analysis (m/z 291 ! 165) of
12-OPDA and 12-OPEA (m/z 293 ! 165), which were released from
the mycelium of Fusarium oxysporum f. sp. tulipae (FOT). 12-OPDA
and 12-OPEA eluted 1 min apart. (b) CP-HPLC-MS/MS analysis of
natural 12-OPDA (m/z 291 ! 165) and synthetic (ꢂ)-cis-12-OPEA
(m/z 293 ! 165). The first eluting isomer of cis-12-OPEA (peak I)
had the same retention time as the biologically formed isomer in
(a) and eluted 1 min before 12-OPDA. The latter eluted between the
two cis isomers (peaks I and II) of 12-OPEA
Fig. 10 Transformation of linoleic acid (18:2n-6) and 13(S)-
hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPODE) by
Fusarium oxysporum f. sp. tulipae (FOT). (a) Biosynthesis of
9,10-dihydro-(+)-7-iso-JA-Ile from 18:2n-6 by mycelia. Biosynthesis
occurs by transformation of 12,13(S)-epoxy-9(Z),11-octadecadienoic
acid (12,13S-EOD) into 9S,13S-12-OPEA by fungal allene oxide
cyclase (AOC) and then in several steps to the detected end product,
9,10-dihydro-(+)-7-iso-JA-Ile. Whether 12,13S-EOD is formed with
11E (as shown) or with 11Z configuration is unknown.
(b) Transformation of 13S-HPODE by mycelia into the detected
α-ketol, which can be formed by nonenzymatic hydrolysis of
12,13S-EOD
Substrate Specificity of AOS
The biosynthesis of α-ketols (n = 4) by the AOS activity of
the microsomal fraction, estimated with an internal standard
(iso-OPDA), averaged 0.81 ꢂ 0.19 (SD), 0.12 ꢂ 0.05, and
0.13 ꢂ 0.02 with 13S-HPOTrE, 13S-HPODE, and 11S-
HPHeTrE as substrates, respectively. This suggested that
13S-HPODE and 11S-HPHeTrE were isomerized by the
AOS at a rate of only about 15% compared to 13S-
HPOTrE. We, therefore, conclude that 13S-HPOTrE is the
likely precursor of fungal jasmonates. This was further
supported by the lack of conversion of 13S-HPOTrE(n-6)
and the insignificant transformation (<5%) of 15S-
hydroperoxides of 20:2n-6, 20:3n-3, and 20:4n-6 into
allene oxides in comparison with 13S-HPOTrE(n-3).
and, to reduce the nitrogen concentration, PDB/20% malt-
ose, 1:2 (v/v), as bikaverin production is augmented by
nitrogen deficiency (Limon et al., 2010). The weight was
kept constant by the addition of water. The concentration
of JA-Ile and 9,10-dihydro-JA-Ile increased between Days
4 and 8 in parallel in the two media, but then remained rela-
tively stable toward the stationary phase at days 12 and
16 (cf. ref. (Srivastava et al., 2011). The ratio of
9,10-dihydro-JA/JA-Ile averaged 9%, and remained con-
stant. However, the red color of the medium increased
steadily.
Fusarium Homologs of Plant AOS
Comparison of the Biosynthesis of JA-Ile and Bikaverin
Blast analysis of genome of F. oxysporum Fo47 with the
predicted N-terminal 9S-AOS domain of 9S-dioxygenase
(DOX)-AOS of F. oxysporum (GenBank EGU88194; resi-
dues 700–1141) detected only a few cytochrome P450
The release of JA-Ile and 9,10-dihydro-JA-Ile to the growth
media was estimated after 4, 8, 12, and 16 days by LC-MS
analysis of an aliquot. The two growth media were PDB
Lipids (2019)

Lipids
(CYP) sequences, which could be aligned over 139–224
consecutive residues but only with up to 29% sequence
identity. They all contained the heme thiolate in the
ArgXxxCys sequence whereas all DOX-CYP fusion pro-
teins with a functional CYP domain contain the HisXxxCys
sequence. It, therefore, seems unlikely that the CYP
domain of 9S-DOX-AOS is a homolog of 13S-AOS.
Blast analysis of F. oxysporum Fo47 with AOS of Ara-
bidopsis thaliana (GenBank AED94842) and CYP74C of
tomato (GenBank AAN76867) only detected CYP with up
to 30% query cover and only with up to 33% sequence
identities. They did not contain nine amino acids inserted
between the (Phe/Trp)XxxXxxGly motif and the heme
thiolate ligand of plant AOS (CYP74) (Brash, 2009). There
are, therefore, no obvious homologs to plant AOS (CYP74)
in the genome of F. oxysporum Fo47.
We know little about the next enzyme in the cascade,
13S-AOS, but its activity was readily detected in subcellu-
lar fractions of mycelia with 13S-HPOTrE and 13S-HPODE
as substrates. 9S-DOX-AOS of F. oxysporum with 9S-AOS
in the C-terminal domain has been expressed and purified
to homogeneity (Chen et al., 2017; Hoffmann and Oliw,
2013). Amino acids of catalytic importance for the 9S-AOS
activities remain unknown except for the heme thiolate
ligand in the conserved sequence HisXxxCys of all func-
tional fungal DOX-CYP fusion proteins. The sequence of
about nine amino acids, which is inserted before the heme
thiolate ligand of plant AOS (CYP74), is lacking in fungal
8- and 9-AOS domains (Hoffmann et al., 2013; Oliw et al.,
2016). A search for the CYP sequences of F. oxysporum
Fo47 in GenBank with the 9S-AOS sequences detected a
handful CYP, but they aligned with only 23–28% sequence
identity. Two plant AOS were also investigated but without
compelling sequence homology to CYP of F. oxysporum.
13S-AOS, therefore, seems to be unrelated to both 9S-AOS
of F. oxysporum and to plant 13S-AOS.
Previous work has shown that JA biosynthesis of
F. oxysporum is influenced by the growth conditions (Cole
et al., 2014; Oliw and Hamberg, 2017). Comparison of the
expression of CYP mRNA of F. oxysporum under growth
in Czapec-DOX broth under light (with low JA-Ile biosyn-
thesis) with the expression in PDB in the dark (with high
biosynthesis) might indicate potential 13S-AOS candidates
(Oliw and Hamberg, 2017). Whether expressed sequence
tags of mRNA could provide this information might be
worth further investigations.
Fusarium Homologs of Plant AOC
Plant AOC have structural similarities to lipocalins and
dirigent protein 6 of the calycin superfamily (Gasper et al.,
2016; Pickel et al., 2012), but we could not detect homo-
logs of these proteins of A. thaliana in the genomes of the
F. oxyporum complex.
Discussion
The biosynthesis of JA, JA-Ile, and 9,10-dihydro-JA-Ile by
two fungi, F. fujikoroi and L. theobromae, was described in
1970–1971 (Aldridge et al., 1971; Cross and Webster, 1970),
but the biosynthetic pathway from 18:3n-3 to JA-Ile in anal-
ogy with plants was confirmed only recently (Oliw and
Hamberg, 2017; Tsukada et al., 2010). The main finding of
this report is that 9,10-dihydro-JA-Ile is formed by FOT from
18:2n-6 via 9S,13S-12-OPEA (Fig. 10a). This implies that the
allene oxide 12,13S-EOD could be a substrate of AOC of
other fungi. This observation has several consequences.
The first step is likely catalyzed by the 13S-LOX of
F. oxysporum. So far, 13S-LOX activities of mycelia have
only been indirectly confirmed by the biosynthesis
of jasmonates, but the recombinant 13S-LOX of
F. oxysporum f. sp. lycopersici has been characterized
(Brodhun et al., 2013). This enzyme oxidizes 18:3n-3 and
18:2n-6, and the latter is transformed with even higher cata-
lytic efficiency than 18:3n-3 (Brodhun et al., 2013). The
genes of secondary metabolites of fungi are often found in
clusters (Rokas et al., 2018). A catalase gene flanks the
gene of 13S-LOX (Teder et al., 2017). This catalase can be
aligned with 79% amino acid identity to the homolog of
F. graminearum, but the latter lacks AOS activity (Teder
et al., 2017).
A comparison of fungal and plant AOC highlights the
main finding of this report. According to observations by
Vick and Zimmerman (1979) and extended by many later
investigations (Grechkin, 1994; Vick and Zimmerman,
1979; Ziegler et al., 1999), the plant AOC require the n-3
double bond of 18:3n-3; without the n-3 double bond the
efficiency with 18:2n-6 as a substrate is reduced by a factor
of 103 (Grechkin, 1994). The reaction mechanism of plant
AOC has been analyzed from there-dimensional
(3D) structures (Hofmann et al., 2006; Neumann et al.,
2012) and recently by theoretical physical calculations
(González-Pérez et al., 2017; Hebert et al., 2016). As
reported here, fungal AOC transform 12,13S-EOD, but may
nevertheless use the same reaction mechanism. Fungal AOC
may, therefore, catalyze homolytic cleavage of the epoxide
of 12,13S-EOD with formation of an oxyallyl radical, which
rearranges to 9S,13S-12-OPEA (González-Pérez et al.,
2017; Hebert et al., 2016). An outline of the transformation
of 12,13S-EOD into 9S,13S-12-OPEA by AOC and then
into 9,10-dihydro-(+)-7-iso-JA-Ile is shown in Fig. 10a.
An interesting question is whether the very same fungal
enzyme could catalyze both AOS and AOC activities,
which has been reported for CYP74C (Grechkin et al.,
Lipids (2019)

Lipids
Brash, A. R. (2009) Mechanistic aspects of CYP74 allene oxide
synthases and related cytochrome P450 enzymes. Phytochemistry,
70:1522–1531.
Brash, A. R., Boeglin, W. E., Stec, D. F., Voehler, M.,
Schneider, C., & Cha, J. K. (2013) Isolation and characterization of
two geometric allene oxide isomers synthesized from 9S-
hydroperoxy-linoleic acid by P450 CYP74C3: Stereochemical
assignment of natural fatty acid allene oxides. The Journal of Bio-
logical Chemistry, 288:20797–20806. https://doi.org/10.1074/jbc.
M113.482521
Brodhun, F., Cristobal-Sarramian, A., Zabel, S., Newie, J.,
Hamberg, M., & Feussner, I. (2013) An iron 13S-lipoxygenase with
an alpha-linolenic acid specific hydroperoxidase activity from
Fusarium oxysporum. PLoS One, 8:e64919. https://doi.org/10.
1371/journal.pone.0064919
Caruso, T., & Spinella, A. (2003) Promoted coupling reactions for the
preparation of skipped diynes. Tetrahedron, 59:7787–7790.
Chen, Y., Jernerén, F., & Oliw, E. H. (2017) Purification and site-
directed mutagenesis of linoleate 9S-dioxygenase-allene oxide
synthase of Fusarium oxysporum confirms the oxygenation mecha-
nism. Archives of Biochemistry and Biophysics, 625-626:24–29.
https://doi.org/10.1016/j.abb.2017.05.007
2008). This seems unlikely as the AOS activities of
F. oxysporum with exogenous 13S-HPODE and 13S-
HPOTrE as substrates were relatively prominent, but the
AOC activities were not detected in cell free preparations.
13S-AOS is the only enzyme in the fungal biosynthesis of
12-OPDA and 12-OPEA, which so far can be demonstrated
with confidence. The enzyme is present in the microsomal
fraction and metabolizes 13S-HPOTrE rapidly compared to
both 13S-HPODE and the 11S-hydroperoxide of 16:3n-3.
Plant and fungal AOC likely bind allene oxides in a
hydrophobic environment and thereby exert stereochemical
control of the cyclic products (Hofmann et al., 2006; Neu-
mann et al., 2012). Homologs of plant AOC could not be
identified in the genome of strains of F. oxysporum
sequenced so far. It is possible that proteins with a similar
β-barrel structure may possess AOC activities in
F. oxysporum (cf. (Pickel et al., 2012)).
How can the low 13S-LOX and AOC activities be
explained? It is likely that 13S-LOX, 13S-AOS, and AOC
may be functionally linked in an enzyme complex. This
complex accepts exogenous 18:3n-3 and 18:2n-6 as sub-
strates and converts them via 12-OPDA and 12-OPEA in a
tightly controlled process to JA-Ile and 9,10-dihydro-JA-Ile,
respectively. This occurs without significant accumulation
of hydroperoxides or α-ketols in either the growth medium
(Miersch et al., 1987, 1999; Oliw and Hamberg, 2017;
Tsukada et al., 2010) or in cell-free preparations. In contrast,
added 13S-HPODE and 13S-HPOTrE are readily trans-
formed into allene oxides, but not to cyclopentenone fatty
acids (cf. Fig. 10b) (Oliw and Hamberg, 2017). This obser-
vation lends further support for a tightly controlled process
from 18:2n-6 and 18:3n-3 to the jasmonate end products.
In conclusion, 18:2n-6 is oxidized to 12-OPEA and
converted to 9,10-dihydro-JA-Ile by fungi in analogy with
the oxidation and transformation of 18:3n-3 via 12-OPDA
into JA-Ile in fungi and plants. This implies that fungal
AOC has broader substrate specificity than plant AOC, but
it may form 12-OPDA and 12-OPEA from allene oxides by
the same diradical reaction mechanism.
Cole, S. J., Yoon, A. J., Faull, K. F., & Diener, A. C. (2014) Host per-
ception of jasmonates promotes infection by Fusarium oxysporum
formae speciales that produce isoleucine- and leucine-conjugated
jasmonates. Molecular Plant Pathology, 15:589–600. https://doi.
org/10.1111/mpp.12117
Cross, B. E., & Webster, G. R. B. (1970) New metabolites of
Gibberella fujikuroi. Journal of the Chemical Society C: Organic:
1838–1842.
Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E.,
Di Pietro, A., Spanu, P. D., … Foster, G. D. (2012) The top 10 fun-
gal pathogens in molecular plant pathology. Molecular Plant
Pathology, 13:414–430. https://doi.org/10.1111/j.1364-3703.2011.
00783.x
Demole, E., Lederer, E., & Mercier, D. (1962) Isolement et determi-
nation de la structure du jasmonate de methyle, constituant odorant
caracteristique de l’essence de jasmin. Helvetica Chimica Acta, 45:
675–685. https://doi.org/10.1002/hlca.19620450233
Gasper, R., Effenberger, I., Kolesinski, P., Terlecka, B.,
Hofmann, E., & Schaller, A. (2016) Dirigent protein mode of action
revealed by the crystal structure of AtDIR6. Plant Physiology, 172:
2165–2175. https://doi.org/10.1104/pp.16.01281
González-Pérez, A. B., Grechkin, A., & de Lera, A. R. (2017)
Rearrangement of vinyl allene oxide geometric isomers to
cyclopentenones. Further computational insights with biologically
relevant model systems. Organic & Biomolecular Chemistry, 15:
2846–2855. https://doi.org/10.1039/c6ob02791a
Grechkin, A. N. (1994) Cyclization of natural allene oxide fatty acids.
The anchimeric assistance of beta, gamma-double bond beside the
oxirane and the reaction mechanism. Biochimica et Biophysica
Acta, 1213:199–206.
Acknowledgements This work was supported by Vetenskapsrådet
Grant K2013-67X-06523-31-3, The Knut and Alice Wallenberg
Foundation Grant KAW 2004.0123, and by Uppsala University. We
thank Ms. Julia Swan for linguistic control of the text.
Grechkin, A. N., Chechetkin, I. R., Mukhtarova, L. S.,
&
Hamberg, M. (2002) Role of structure and pH in cyclization of
allene oxide fatty acids: Implications for the reaction mechanism.
Chemistry and Physics of Lipids, 120:87–99.
Conflict of Interest The authors declare that they have no conflict
of interest.
Grechkin, A. N., Mukhtarova, L. S., Latypova, L. R., Gogolev, Y.,
Toporkova, Y. Y., & Hamberg, M. (2008) Tomato CYP74C3 is a
multifunctional enzyme not only synthesizing allene oxide but also
catalyzing its hydrolysis and cyclization. Chembiochem, 9:
2498–2505.
References
Grechkin, A. N., Ogorodnikova, A. V., Gnezdilov, O. I.,
&
Mukhtarova, L. S. (2007) Detection of a pathway from linoleate to
a novel cyclopentenone: cis-12-oxo-10-phytoenoic acid in sun-
flower roots. Chembiochem, 8:2275–2280. https://doi.org/10.1002/
cbic.200700393
Aldridge, D., Galt, S., Giles, D., & Turner, W. (1971) Metabolites of
lasiodiplodia theobromae. Journal of the Chemical Society C:
Organic:1623–1627.
Lipids (2019)

Lipids
Hamberg, M. (2000) New cyclopentenone fatty acids formed from
linoleic and linolenic acids in potato. Lipids, 35:353–363.
Grechkin, A. N. (2015) Stereospecific biosynthesis of (9S,13S)-
10-oxo-phytoenoic acid in young maize roots. Biochimica et Bio-
physica Acta, 1851:1262–1270. https://doi.org/10.1016/j.bbalip.
2015.05.004
Hamberg, M., & Hughes, M. A. (1988) Fatty acid allene oxides III.
Albumin-induced
cyclization
of
12,13(S)-epoxy-9
(Z),11-octadecadienoic acid. Lipids, 23:469–475.
Oliw, E. H. (2018) Product specificity of fungal 8R- and 9S-
dioxygenases of the peroxidase-cyclooxygenase superfamily with
amino acid derivatized polyenoic fatty acids. Archives of Biochem-
istry and Biophysics, 640:93–101. https://doi.org/10.1016/j.abb.
2017.12.018
Oliw, E. H., Aragó, M., Chen, Y., & Jernerén, F. (2016) A new class
of fatty acid allene oxide formed by the DOX-P450 fusion proteins
of human and plant pathogenic fungi, C. immitis and Z. tritici.
Journal of Lipid Research, 57:1518–1528. https://doi.org/10.1194/
jlr.M068981
Oliw, E. H., Garscha, U., Nilsson, T., & Cristea, M. (2006) Payne
rearrangement during analysis of epoxyalcohols of linoleic and
alpha-linolenic acids by normal phase liquid chromatography with
tandem mass spectrometry. Analytical Biochemistry, 354:111–126.
Hebert, S. P., Cha, J. K., Brash, A. R., & Schlegel, H. B. (2016)
Investigation into 9(S)-HPODE-derived allene oxide to
cyclopentenone cyclization mechanism via diradical oxyallyl inter-
mediates. Organic & Biomolecular Chemistry, 14:3544–3557.
https://doi.org/10.1039/c6ob00204h
Hoffmann, I., Jernerén, F., & Oliw, E. H. (2013) Expression of fusion
proteins of Aspergillus terreus reveals a novel allene oxide
synthase. The Journal of Biological Chemistry, 288:11459–11469.
https://doi.org/10.1074/jbc.M113.458257
Hoffmann, I., & Oliw, E. H. (2013) Discovery of a linoleate 9S-
dioxygenase and an allene oxide synthase in a fusion protein of
Fusarium oxysporum. Journal of Lipid Research, 54:3471–3480.
https://doi.org/10.1194/jlr.M044347
Hofmann, E., Zerbe, P., & Schaller, F. (2006) The crystal structure of
Arabidopsis thaliana allene oxide cyclase: Insights into the
oxylipin cyclization reaction. Plant Cell, 18:3201–3217. https://doi.
org/10.1105/tpc.106.043984
Holbrook, L., Tung, P., Ward, K., Reid, D. M., Abrams, S.,
Lamb, N., … Moloney, M. M. (1997) Importance of the chiral cen-
ters of jasmonic acid in the responses of plants (Activities and
antagonism between natural and synthetic analogs). Plant Physiol-
ogy, 114:419–428.
Itoh, A., Schilmiller, A. L., McCaig, B. C., & Howe, G. A. (2002)
Identification of a jasmonate-regulated allene oxide synthase that
metabolizes 9-hydroperoxides of linoleic and linolenic acids. The
Journal of Biological Chemistry, 277:46051–46058. https://doi.org/
10.1074/jbc.M207234200
Limon, M. C., Rodriguez-Ortiz, R., & Avalos, J. (2010) Bikaverin
production and applications. Applied Microbiology and Biotechnol-
ogy, 87:21–29. https://doi.org/10.1007/s00253-010-2551-1
Ma, L. J., Geiser, D. M., Proctor, R. H., Rooney, A. P.,
Oliw, E. H.,
& Hamberg, M. (2017) An allene oxide and
12-oxophytodienoic acid are key intermediates in jasmonic acid
biosynthesis by Fusarium oxysporum. Journal of Lipid Research,
58:1670–1680. https://doi.org/10.1194/jlr.M077305
Pickel, B., Pfannstiel, J., Steudle, A., Lehmann, A., Gerken, U.,
Pleiss, J., & Schaller, A. (2012) A model of dirigent proteins
derived from structural and functional similarities with allene oxide
cyclase and lipocalins. The FEBS Journal, 279:1980–1993. https://
doi.org/10.1111/j.1742-4658.2012.08580.x
Ploetz, R. C. (2015) Fusarium wilt of banana. Phytopathology, 105:
1512–1521. https://doi.org/10.1094/PHYTO-04-15-0101-RVW
Rokas, A., Wisecaver, J. H., & Lind, A. L. (2018) The birth, evolution
and death of metabolic gene clusters in fungi. Nature Reviews.
Microbiology, 16:731–744. https://doi.org/10.1038/s41579-018-
0075-3
Srivastava, S., Pathak, N., & Srivastava, P. (2011) Identification of
limiting factors for the optimum growth of Fusarium oxysporum in
liquid medium. Toxicology International, 18:111–116. https://doi.
org/10.4103/0971-6580.84262
Teder, T., Boeglin, W. E., Schneider, C., & Brash, A. R. (2017) A
fungal catalase reacts selectively with the 13S fatty acid hydroper-
oxide products of the adjacent lipoxygenase gene and exhibits 13S-
hydroperoxide-dependent peroxidase activity. Biochimica et Bio-
physica Acta - Molecular and Cell Biology of Lipids, 1862:
706–715. https://doi.org/10.1016/j.bbalip.2017.03.011
O’Donnell, K., Trail, F.,
… Kazan, K. (2013) Fusarium
pathogenomics. Annual Review of Microbiology, 67:399–416.
https://doi.org/10.1146/annurev-micro-092412-155650
Medvedeva, N. V., Latypov Sh, K., Balandina, A. A.,
Mukhtarova, L., & Grechkin, A. N. (2005) Geometrical configura-
tion of 12,13-epoxy-9,11-octadecadienoic acid, a product of the
reaction catalyzed by flaxseed allene oxide synthase (CYP74A).
Bioorganicheskaia Khimiia, 31:659–660.
Miersch, O., Bettina Brücknera, B., Jürgen Schmidt, J.,
Sembdner, G. (1992) Cyclopentane fatty acids from Gibberella
fujikuroi. Phytochemistry, 31:3835–3837.
&
Tsukada, K., Takahashi, K., & Nabeta, K. (2010) Biosynthesis of
jasmonic acid in
a plant pathogenic fungus, Lasiodiplodia
theobromae. Phytochemistry, 71:2019–2023.
Miersch, O., Bohlmann, H., & Wasternack, C. (1999) Jasmonates and
related compounds from Fusarium oxysporum. Phytochemistry, 50:
517–523.
Vick, B. A., & Zimmerman, D. C. (1979) Substrate specificity for the
synthesis of cyclic fatty acids by a flaxseed extract. Plant Physiol-
ogy, 63:490–494.
Wasternack, C., & Feussner, I. (2018) The oxylipin pathways: Bio-
chemistry and function. Annual Review of Plant Biology, 69:
363–386. https://doi.org/10.1146/annurev-arplant-042817-040440
Wasternack, C., & Strnad, M. (2016) Jasmonate signaling in plant
stress responses and development - active and inactive compounds.
New Biotechnology, 33:604–613. https://doi.org/10.1016/j.nbt.
2015.11.001
Miersch, O., Preiss, A., Sembdner, G., & Schreiber, K. (1987)
(+)-7-Iso-jasmonic acid and related compounds from
Botryodiblodia theobromae. Phytochemistry, 26:1037–1039.
Monte, I., Ishida, S., Zamarreno, A. M., Hamberg, M., Franco-
Zorrilla, J. M., Garcia-Casado, G., … Solano, R. (2018) Ligand-
receptor co-evolution shaped the jasmonate pathway in land plants.
Nature Chemical Biology, 14:480–488. https://doi.org/10.1038/
s41589-018-0033-4
Ziegler, J., Stenzel, I., Hause, B., Maucher, H., Hamberg, M.,
Grimm, R., … Wasternack, C. (2000) Molecular cloning of allene
oxide cyclase. The enzyme establishing the stereochemistry of
octadecanoids and jasmonates. Journal of Biological Chemistry,
275:19132–19138. https://doi.org/10.1074/jbc.M002133200
Ziegler, J., Wasternack, C., & Hamberg, M. (1999) On the specificity
of allene oxide cyclase. Lipids, 34:1005–1015.
Neumann, P., Brodhun, F., Sauer, K., Herrfurth, C., Hamberg, M.,
Brinkmann, J., … Ficner, R. (2012) Crystal structures of Phys-
comitrella patens AOC1 and AOC2: Insights into the enzyme
mechanism and differences in substrate specificity. Plant Physiol-
ogy, 160:1251–1266. https://doi.org/10.1104/pp.112.205138
Ogorodnikova, A. V., Gorina, S. S., Mukhtarova, L. S.,
Mukhitova, F. K., Toporkova, Y. Y., Hamberg, M.,
&
Lipids (2019)