Biosynthesis of jasmonic acid in a plant pathogenic fungus, Lasiodiplodia theobromae

Article abstract of DOI:10.1016/j.phytochem.2010.09.013

Jasmonic acid (JA) is a plant hormone that plays an important role in a wide variety of plant physiological processes. The plant pathogenic fungus, Lasiodiplodia theobromae also produces JA; however, its biosynthesis in this fungus has yet to be explored. Administration of [1-¹³C] and [2- ¹³C] NaOAc into L. theobromae established that JA in this fungus originates from a fatty acid synthetic pathway. The methyl ester of 12-oxo-phytodienoic acid (OPDA) was detected in the culture extracts of L. theobromae by GC-MS analysis. This finding indicates the presence of OPDA (a known intermediate of JA biosynthesis in plants) in L. theobromae. ²H NMR spectroscopic data of JA produced by L. theobromae with the incorporation of [9,10,12,13,15,16-²H₆] linolenic acid showed that five deuterium atoms remained intact. In plants, this is speculated to arise from JA being produced by the octadecanoid pathway. However, the observed stereoselectivity of the cyclopentenone olefin reduction in L. theobromae was opposite to that observed in plants. These data suggest that JA biosynthesis in L. theobromae is similar to that in plants, but differing in the facial selectivity of the enone reduction.

Full text of DOI:10.1016/j.phytochem.2010.09.013

Phytochemistry 71 (2010) 2019–2023
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biosynthesis of jasmonic acid in a plant pathogenic fungus, Lasiodiplodia theobromae
⇑
Kohei Tsukada, Kosaku Takahashi, Kensuke Nabeta
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2010
Received in revised form 5 September 2010
Accepted 21 September 2010
Available online 15 October 2010
Jasmonic acid (JA) is a plant hormone that plays an important role in a wide variety of plant physiological
processes. The plant pathogenic fungus, Lasiodiplodia theobromae also produces JA; however, its biosyn-
thesis in this fungus has yet to be explored. Administration of [1-13C] and [2-13C] NaOAc into L. theobro-
mae established that JA in this fungus originates from a fatty acid synthetic pathway. The methyl ester of
12-oxo-phytodienoic acid (OPDA) was detected in the culture extracts of L. theobromae by GC–MS anal-
ysis. This finding indicates the presence of OPDA (a known intermediate of JA biosynthesis in plants) in L.
theobromae. 2H NMR spectroscopic data of JA produced by L. theobromae with the incorporation of
[9,10,12,13,15,16-2H6] linolenic acid showed that five deuterium atoms remained intact. In plants, this
is speculated to arise from JA being produced by the octadecanoid pathway. However, the observed ste-
reoselectivity of the cyclopentenone olefin reduction in L. theobromae was opposite to that observed in
plants. These data suggest that JA biosynthesis in L. theobromae is similar to that in plants, but differing
in the facial selectivity of the enone reduction.
Keywords:
Lasiodiplodia theobromae
Jasmonic acid
Biosynthesis
Octadecanoid pathway
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
thesis and its regulatory mechanism, has been thoroughly investi-
gated (Wasternack, 2007). Recently, the presence of JA (7) and the
Lasiodiplodia theobromae is a pathogenic fungus that infects
plants in tropical and subtropical regions of the world, causing
considerable damage to crops during storage. This fungus is known
to produce a variety of bioactive compounds (Aldridge et al., 1971).
Previous studies in our laboratory have demonstrated the presence
of various potato-tuber-inducing substances in a culture of L. theo-
bromae: (À)-JA (7) (see Fig. 1), theobroxide (and related com-
pounds), and lasiodiplodins (Nakamori et al., 1994; Matsuura
et al., 1998a,b; Yang et al., 2000; Li et al., 2006; Takei et al.,
2006). Among these compounds, theobroxide exhibits the most
interesting biological activity, which effects not only potato-tu-
ber-inducing activity in potato (Solanum tuberosum), but also in-
duces flower bud formation in morning glory (Pharbitis nil)
(Yoshihara et al., 2000). Encouraged by the unique biological activ-
ities that these compounds display, the biosynthesis of theobrox-
ide (Li et al., 2006) and lasiodiplodins (Kashima et al., 2009a,b)
were elucidated. Among the compounds described above, (À)-JA
(7) was first isolated from the culture of L. theobromae as a sub-
stance used to accelerate plant senescence in the 1970s (Aldridge
et al., 1971). It has since been established that JA (7) is a plant hor-
mone that controls responses to environmental stresses and devel-
opmental events in flowering plants. It plays an important role in
coordinating plant defense responses with physiological stresses
associated with herbivores and microbial pathogens. JA (7) biosyn-
activity of allene oxide synthase (AOS), with respect to JA (7) bio-
synthesis, was demonstrated in a model moss Physcomitrella patens
(Oliver et al., 2009; Bandara et al., 2009).
In plants, JA (7) is biosynthetically produced by the octadeca-
noid pathway as shown in Fig. 1 (Schaller and Stinzi, 2009). The
first step in the octadecanoid pathway is the lipoxygenase (LOX)-
catalyzed oxygenation of
of JA (7) biosynthesis, hydroperoxidation to intermediated (2)
takes place at C-13 of -linolenic acid (1), and is effected by
a-linolenic acid (1). In the specific case
a
13-LOX. The resulting hydroperoxide [13(S)-hydroperoxyoctadec-
atrienoic acid (13-HPOT)] (2) can be metabolized by AOS into an
unstable allene oxide [12,13(S)-epoxyoctadecatrienoic acid
(12,13-EOT) 3], in which cyclization is facilitated by allene oxide
cyclase (AOC) to provide 12-oxo-phytodienoic acid (OPDA) (4).
The later is reduced by OPDA reductase 3 (OPR3) to yield 3-oxo-
2-[(Z)-pent-2-enyl]-cyclopentane-1-octanoic acid (OPC-8:0) (5).
Three subsequent b-oxidation steps afford (+)-iso-JA (6) [(3R,7R)-
configuration], which is further epimerized at C-7 to provide (À)-
JA (7) [(3R,7S)-configuration]. Due to keto-enol interconversion,
the cis-isomer, (+)-iso-JA (6), is readily converted into the more sta-
ble trans-isomer, (À)-JA (7), for steric reasons.
As referred to above, the detailed mechanism of JA (7) signaling
and biosynthesis in plants has been elucidated. However, there is a
little information about the biosynthetic pathway and functions of
JA (7) in L. theobromae. Thakkar et al. (2004) reported that develop-
ment of systemic acquired resistance (SAR) was restricted in the
plants infected with L. theobormae due to deficiency of salicylic
⇑
Corresponding author.
E-mail address: knabeta@chem.agr.hokudai.ac.jp (K. Nabeta).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.09.013

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K. Tsukada et al. / Phytochemistry 71 (2010) 2019–2023
OOH
12
13
O
13
12 13 15 16
10
9
13-LOX
AOS
12
COOH
12,13-EOT (3)
COOH
COOH
13-HPOT (2)
α-linolenic acid (1)
O
O
12
13
12
13
9
AOC
OPR3
11
11
COOH
COOH
9
10
10
12-OPDA (4)
OPC-8:0 (5)
COOH
O
O
6
9
10
7
3
epimerization
β oxidation ( x 3 )
5
COOH
4
(+)-iso-jasmonic acid (6)
(-)-jasmonic acid (7)
Fig. 1. The octadecanoid pathway.
acid (SA). Inhibition of SA biosynthesis in the infected plants,
which was caused by JA (7) released from L. theobromae, might
contribute to the infection of this fungus to plants. In the case of
Pseudomonas syringae, coronatine, a phytotoxin functioning like
JA (7), was shown to induce suppression of SA-mediated defense
system, and disease development in tomato (Uppalapati et al.,
2007).
In this work, it is demonstrated that JA (7) is synthesized via a
fatty acid synthetic pathway in L. theobromae, which is supported
by 13C labeling experiments. The incorporation of a synthetic 2H-
labeled linolenic acid (1) into iso-JA indicates that JA (7) biosynthe-
sis in L. theobromae is similar to that of plants, differing only in the
OPDA (4) in this fungus has not been reported. In an attempt to test
this hypothesis, the acetone extract of mycelia of L. theobromae
was partially purified by preparative TLC, and subsequently sub-
jected to GC–MS analysis. In conclusion, OPDA (4) was not detected
in free form; however, the OPDA methyl ester was observed in the
selected ion mode monitored at m/z 306 ([M]+), 275 ([MÀOCH3]+),
and 238 (Fig. 3) (Laudert et al., 1996). The retention time associ-
ated with all of the selected ion peaks in this analysis was
87.2 min, which was the same as that of the molecular ion peaks
of an authentic (+)-trans-OPDA methyl ester prepared (i.e.,
CH2N2, Et2O) from natural OPDA (4). These data strongly suggested
that L. theobromae produces natural occurring OPDA (4). JA (7) pro-
duction by way of a fatty acid synthetic pathway, and the presence
of OPDA (4) in L. theobromae, suggests that this fungus produces JA
(7) via OPDA (4). As previously stated, OPDA (4) is an intermediate
facial selectivity of the cyclopentenone reduction (i.e., a- vs. b-hy-
dride attack); the facial selectivity observed in plants is posited on
the basis of the X-ray crystal structure data of tomato OPR3 (Bre-
ithaupt et al., 2006).
Table 1
2. Results and discussion
Incorporation of 13C-labeled sodium acetate ([1-13C] and [2-13C]) into jasmonic acid
(7).
L. theobromae was statically incubated in 1% potato-glucose
medium at 29 °C for 7 days. 13C-labeled sodium acetate ([1-13C]
and [2-13C]) was administered to the culture at a concentration
of 10 mM. The culture was filtered to separate the mycelia and
supernatant after an additional 10 days of incubation. The aqueous
layer was extracted with ethyl acetate (150 ml) and subsequently
purified by chromatography to provide 3.9 mg/150 ml of
[1-13C]acetate-derived JA, and 2.8 mg/150 ml of [2-13C]acetate-de-
rived JA, respectively.
Position
dc (ppm)
13C-atom%
[1-13C]acetate
[2-13C]acetate
1
2
3
4
5
6
7
8
172.4
38.8
38.0
25.5
37.7
219.0
54.0
27.2
124.9
134.1
20.6
14.1
51.6
2.41
0.69
2.63
0.86
3.00
0.65
1.30
1.06
2.16
0.63
5.68
1.27
1.11
1.80
5.49
1.73
4.62
1.19
7.81
1.17
5.89
0.73
4.45
1.79
5.70
1.11
The 13C{1H}-NMR spectrum of [1-13C]acetate-derived JA (7)
showed enhanced signals at C-1, -3, -5, -7, -9, and -11. Meanwhile,
the 13C{1H} NMR spectrum of JA (7) derived from [2-13C]acetate
had intensified signals at C-2, -4, -6, -8, -10, and -12 (Table 1 and
Fig. 2). Biosynthetically 13C-labeled JA (7) was converted into the
methyl ester with ethereal diazomethane, and then its specific
incorporation ratio was calculated by the relative intensity of the
methyl ester signal normalized to 1.11% (natural abundance). The
specific incorporation of 13C generally observed was 1.3–5.7 atom%
for [1-13C]acetate-derived carbons and 4.5–7.8 atom% for
[2-13C]acetate-derived carbons. These data provide clear evidence
that JA (7) is produced through a fatty acid biosynthetic pathway
in L. theobromae.
9
10
11
12
OMe
O
9
7
11
O
5
3
ONa
1
O
OH
In plants, a-linolenic acid (1) is transformed into JA (7) through
the octadecanoid pathway. We have been interested in whether
OPDA (4)—a key intermediate of the octadecanoid pathway—is
present in the culture of L. theobromae; however, the presence of
Fig. 2. Labeling patterns of jasmonic acid (7) following incorporation of 13C-labeled
sodium acetate into L. theobromae. Filled square and circle symbols represent
[1-13C] and [2-13C] in 13C-labeled sodium acetate, respectively.

K. Tsukada et al. / Phytochemistry 71 (2010) 2019–2023
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incubated for an additional 10 days. Chromatographic separation
of a residue from the culture filtrate of L. theobromae gave iso-
JA (6a) incorporating [2H6]-LA (1a) (1.5 mg). After methyl ester
formation, the 2H NMR spectrum of the resulting iso-JA methyl
ester was immediately taken. The 2H NMR spectroscopic data
established the presence of five 2H signals corresponding to H-
3, -4a, -7, -9, and -10 (Fig. 4) (Seto et al., 1999). Analysis of the
EI-MS data by comparison of [M+4]+ ion peak (m/z = 228, [2H4]-
JA (7a)) and [M+5]+ ion peak (m/z = 229, [2H5]-iso-JA (6a)) with
the methyl ester of biosynthetically labeled and natural JA (7)
showed that 6.98% and 2.96% of the peak intensities were en-
hanced, respectively. Considering the isomerization, 9.94% of 2H
was enriched in the iso-JA (6a) derived from [2H6]-LA (1a). This
incorporation demonstrates that
a-linolenic acid (1) is a precur-
sor in JA (7) biosynthesis of L. theobromae (Fig. 5). The presence
of iso-JA (6) suggests that it is produced first and subsequently
isomerized to JA (7) in L. theobromae. Moreover, it is interesting
that the positions of 2H incorporation in [2H6]-LA (1a) derived
iso-JA (6a) are in accordance with the positions speculated by
the cyclization mechanism involved in reactions mediated by
AOS and AOC in plants (Schaller and Stinzi, 2009). Accordingly,
OPDA (4) is most likely metabolized to JA (7) through reduction
of the cyclopentenone moiety and subsequent b-oxidation of
the side-chain. While the reduction occurs prior to the b-oxida-
tion in plants, the order of events (i.e., reduction or b-oxidation)
are unknown in L. theobromae.
From a stereochemical perspective, the reductase could facili-
tate hydride attack from either the
a- or b-face, which would
determine the configuration at C-4 in [2H6]-LA derived iso-JA
(6a). In plants, the X-ray crystal structural data of tomato OPR3
suggests that the reduced flavin cofactor is placed on the opposite
Fig. 3. Segment of selected ion monitored chromatograms of GC–MS spectral data
of OPDA methyl ester produced by L. theobromae. Column; a chiral b-DEX fused
silica capillary column (0.25 mm i.d. Â 30 m).
side of the OPDA (4) side-chain (i.e.,
a-face) (Breithaupt et al.,
2006). The attack of a hydride from the
a-face would result in
derived from
pathway.
a
-linolenic acid (1) by way of the known plant
the original proton attached at C-10 in OPC-8:0 to be in a b-orien-
tation. On the other hand, the 2H NMR spectrum of the [2H6]-LA
Next, it was investigated as to whether
also
a-linolenic acid (1) is
(1a) derived iso-JA methyl ester indicated an a
-orientation of 2H-
a
precursor of JA (7) in L. theobromae; therefore,
4. This supports the contention that the hydride attack from the
reductase occurs on the b-face of the cyclopentenone plane at C-
4, which is in contrast to that in plants. These data suggest that
the cyclopentenone reduction mechanism in L. theobromae is dif-
ferent from that in plants.
[9,10,12,13,15,16-2H6]linolenic acid ([2H6]-LA) (1a) was synthe-
sized according to Hungerford and Kitching (1998). [2H6]-LA
(1a) was supplemented to a 7-day-old culture of L. theobromae,
reaching a final concentration of 1 mM, and the fungus was
Fig. 4. 1H NMR spectrum of natural jasmonic acid (7) (A) and 2H NMR spectrum of biosynthetically labeled iso-jasmonic acid (6a) derived from [9,10,12,13,15,16-2H6]lin-
olenic acid (1a) (B).

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K. Tsukada et al. / Phytochemistry 71 (2010) 2019–2023
O
D
D
D
D
D
cyclization
D
D D
D
D
D
COOH
COOH
(4a)
[2H5] OPDA
[2H6] linolenic acid
O
(1a)
O
D
D
D
R
D
D
D
H
D
D
R= C6H12COOH
or COOH
D
D
H-
COOH
[2H5] iso-jasmonic acid
(6a)
attack of hydride from β-face
O
H
epimerization
D
D
H
D
D
COOH
[2H4] jasmonic acid
(7a)
Fig. 5. The presumed jasmonic acid biosynthetic pathway in L. theobromae.
culture of L. theobromae was inoculated into a Erlenmeyer flask
(500-ml) containing 150 ml of a 1% potato-glucose medium. Each
medium was statically incubated at 29 °C in the dark. After 7 days
of incubation, [1-13C]NaOAc acetate and [2-13C]NaOAc (99 atom%,
respectively) were dissolved in H2O (1 ml) which was supple-
mented to give a final concentration of 10 mM, and then the incu-
bation was continued for an additional 10 days. The 17-day-old
culture (150 ml) for each administration experiment was filtered,
made alkaline by addition of 5% aqueous NaHCO3, and extracted
with EtOAc (250 ml). The resultant aqueous layer was acidified
by addition of 6 M HCl (30 ml), and extracted with EtOAc
(300 ml). After the EtOAc extract was concentrated in vacuo, the
resultant residue was purified by preparative SiO2 gel TLC (n-hex-
ane:EtOAc:AcOH = 60:40:1, Merck, USA), and subsequently iso-
lated by HPLC (TSK gel ODS80Ts,TOSOH, 20 mm i.d. Â 250 mm,
MeOH–H2O (4:1%, v/v) solution, 5.0 ml/min, retention time = 16.2 -
min). For the calculation of incorporated ratio, JA (7) was converted
to the methyl ester with ethereal diazomethane.
3. Conclusions
This study demonstrates that JA (7) biosynthesis in L. theobro-
mae is similar to that in plants (e.g., cyclization). It is interesting
that the cyclization mechanism in L. theobromae appears to be
identical to that in plants, which was supported by administration
experiments of stable isotope-labeled compounds. Therefore, L.
theobromae potentially has proteins, which have similar enzymatic
activities to 13-LOX, AOS, and AOC. In stark contrast, the reduction
of the cyclopentenone moiety differed between L. theobromae and
plants. Highlighted by the case of gibberellin biosynthesis, there
are large differences between plant and fungal genes involved in
gibberellin biosynthesis. Fungi evolved a gibberellin biosynthetic
pathway independently (Bömke and Tudzynski, 2009). Considering
the clear difference between the observed facial selectivity of the
cyclopentenone reduction that was observed in L. theobromae
and that in plants, it can be suggested that this fungus has an inde-
pendently evolved JA (7) biosynthesis pathway. The detailed mech-
anism of JA (7) biosynthesis in L. theobromae requires further
investigation. Additional studies aimed at determining the mecha-
nism and specific enzymes involved for each biosynthetic step are
required to fully define the JA (7) biosynthetic route in L. theobro-
mae. In addition, these studies could lend new insight into plant–
microbe interactions on a fundamental level.
4.3. Identification of OPDA methyl ester in L. theobromae
L. theobromae was statically cultured in a Erlenmeyer flask (500
ml) containing 1% potato-glucose medium (150 ml) at 29 °C for
17 days in the dark. The mycelia were soaked in acetone, and the
extract was concentrated in vacuo. The resulting aqueous solution
was extracted with EtOAc (20 ml). After concentration, the EtOAc
extract (23 mg) was loaded onto silica gel TLC plate (Merck,
USA), which was developed with CHCl3. A band (Rf ꢀ 0.3–0.4)
was scraped, extracted with a mixed solution of MeOH and CHCl3
(3:7), and concentrated in vacuo. The residue (1 mg) was dissolved
4. Experimental
4.1. General
Data were obtained with the following instruments: 1H NMR
(270 MHz) and 13C-NMR (67.8 MHz), Jeol JMN-EX270 FT-NMR
spectrometer; 2H NMR (76.5 MHz), Bruker AMX500 FT-NMR spec-
trometer; EI-MS, Jeol JMS-T100GCV mass spectrometer; GC–MS,
Varian 1200L GC/MS/MS system. The NMR shift values were refer-
enced to residual solvent signals as follows; 1H NMR, CDCl3
(d1H = 7.24 ppm); 2H NMR, CHCl3 (d2H = 7.24 ppm); 13C-NMR, CDCl3
(d13C = 77.0 ppm).
in CHCl3 (100
spectrometry.
ll), and 1 ll of the solution was analyzed by GC–MS
The GC–MS spectrometer was a 1200L GC/MS/MS system (Var-
ian, USA) equipped with a b-DEX fused silica capillary column
(0.25 mm  30 m, 0.2 mm film thickness, Supelco, USA). He was
used as carrier gas at a constant flow rate of 1.0 ml/min. The tem-
perature of the ion source and vaporizing chamber were heated to
200 °C, with an ionization voltage of 70 eV. The temperature gradi-
ent was initiated at 50 °C, was held isothermal for 1 min, and sub-
sequently raised at a 10 °C/min to a final temperature of 190 °C,
which was held for 80 min. The retention times of (+) and
(À)-trans-OPDA methyl esters were 87.6 min and 89.7 min,
4.2. Administration of 13C-labeled sodium acetate in L. theobromae
Spores of L. theobromae were maintained on 1% potato-glucose
agar medium at 30 °C and transferred at intervals of 6 months. A
piece (approximately 1 cm2) of agar bearing the spore-formed

K. Tsukada et al. / Phytochemistry 71 (2010) 2019–2023
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2
4.4. Administration of H-labeled linolenic acid (1a) in L. theobromae
[9,10,12,13,15,16-2H6]linolenic acid (1a) was synthesized
according to the method of Hungerford and Kitching (1998). A 1%
potato-glucose medium (100 ml) was prepared in an Erlenmeyer
(300 ml) flask. Incubation of L. theobromae was carried out as
described above. After 7 days of incubation, [9,10,12,13,15,
16-2H6]linolenic acid (1a) was dissolved in 2 mM NH4OH aqueous
solution (500 ll), which was supplemented to give a final concen-
tration of 1 mM. The incubation was continued for an additional
10 days. The filtrate of the culture, in which an equal volume of
2 M HCl was added, was extracted with EtOAc (300 ml). After con-
centration, the extract (120 mg) was subjected to preparative TLC
(SiO2 gel, n-hexane:EtOAc:AcOH = 60:40:1, Merck, USA). The resul-
tant residue (8.9 mg) was purified by HPLC (TSK gel ODS80Ts,TOS-
OH, 20 mm i.d. Â 250 mm, MeOH–H2O (4:1, v/v), 5.0 ml/min,
retention time = 16.2 min) to give pure iso-JA (6) (1.5 mg). After
methyl ester formation with ethereal diazomethane, the 2H NMR
of the methyl ester of iso-JA was taken immediately. The intensities
of [M+4]+ ion peak (m/z = 228, [2H4]-JA (7a)) and [M+5]+ ion peak
(m/z = 229, [2H5]iso-JA (6a)) with the methyl ester of biosyntheti-
cally labeled JA were given as the percent relative to the intensity
of the molecular ion peak of natural JA (m/z = 224) as 100% in EI-
MS data.
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Acknowledgment
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The phytotoxin coronatine contributes to pathogen fitness and it required for
suppression of salicylic acid accumulation in tomato inoculated with
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We thank Dr. E. Fukushi of our department for measuring EI-MS
spectrometry.
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