Arachidonic acid-dependent carbon-eight volatile synthesis from wounded liverwort (Marchantia polymorpha)

Article abstract of DOI:10.1016/j.phytochem.2014.08.008

Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort Marchantia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within 40 min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6^KO) lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 volatiles in M. polymorpha. When des6^KO thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acetylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally formed from completely disrupted thalli, while it was formed as the most abundant product in partially disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conversion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxidoreductase was involved in the conversion.

Full text of DOI:10.1016/j.phytochem.2014.08.008

Phytochemistry 107 (2014) 42–49
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Arachidonic acid-dependent carbon-eight volatile synthesis
from wounded liverwort (Marchantia polymorpha)
Hirotomo Kihara a, Maya Tanaka a,b, Katsuyuki T. Yamato c, Akira Horibata c, Atsushi Yamada c,
Sayaka Kita c, Kimitsune Ishizaki d, Masataka Kajikawa e, Hideya Fukuzawa e, Takayuki Kohchi e,
Yoshihiko Akakabe b, Kenji Matsui a,
⇑
a Graduate School of Medicine (Agriculture), Yamaguchi University, Japan
b Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan
c Department of Biology-Oriented Science and Technology, Kinki University, Japan
d Department of Biology, Graduate School of Science, Kobe University, Japan
e Graduate School of Biostudies, Kyoto University, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 March 2014
Received in revised form 25 July 2014
Available online 28 August 2014
Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found
among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort March-
antia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-
octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within
40 min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption
resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase
Keywords:
Common liverwort
Marchantia polymorpha
Marchantiaceae
Bryophyte
Acetylation
Oxidoreduction
1-Octen-3-ol
showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6KO
)
lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally
observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 vol-
atiles in M. polymorpha. When des6KO thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the
alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-
3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acet-
ylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally
formed from completely disrupted thalli, while it was formed as the most abundant product in partially
disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conver-
sion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted
by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxido-
reductase was involved in the conversion.
1-Octen-3-yl acetate
Octan-3-one
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
(Combet et al., 2006). Since some vector insects carrying human
disease are attracted by C8 volatiles, there is the potential for
Eight-carbon (C8) volatiles, such as 1-octen-3-ol (5), octan-3-
one (1), and octan-3-ol (4) (Fig. 1), are ubiquitously found among
fungi and bryophytes (Combet et al., 2006; Croisier et al., 2010;
Asakawa et al., 2013). C8 volatiles are important flavor constitu-
ents in mushrooms (Combet et al., 2006), while they are also
responsible for off-flavors in beverages like wine (Steel et al.,
2013). C8 volatiles attract, or in some cases repel, arthropods
chemo-attraction. This raises the expectation that controlling the
behavior of insects with C8 volatiles could lead to novel strategies
to control certain diseases (Carlson and Carey, 2011). Recently, it
has been reported that 5 may cause dopamine neuron degenera-
tion in Drosophila that might result in Parkinson’s disease
(Inamdar et al., 2013). C8 volatiles are also known to possess bio-
activity properties toward plants, including induction of oxidative
burst, inhibition of growth, and induction of a subset of defense
genes in Arabidopsis thaliana (Splivallo et al., 2007; Kishimoto
et al., 2007). Conidiation of Trichoderma spp. was induced by 5, 1,
and 4 (Nemcovic et al., 2008), but these volatiles also inhibited ger-
mination of Penicillium paneum and Aspergillus nidulans conidia at
high concentrations (Chitarra et al., 2004; Herrero-Garcia et al.,
⇑
Corresponding author. Address: Department of Biological Chemistry, Faculty of
Agriculture, and Department of Applied Molecular Bioscience, Graduate School of
Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. Tel.:
+81 83 933 5850; fax: +81 83 933 5820.
E-mail address: matsui@yamaguchi-u.ac.jp (K. Matsui).
http://dx.doi.org/10.1016/j.phytochem.2014.08.008
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

H. Kihara et al. / Phytochemistry 107 (2014) 42–49
43
In this study, the metabolic pathway responsible for the forma-
tion of C8 volatiles was examined in Marchantia polymorpha (liver-
wort). M. polymorpha is an emerging model plant suitable for
molecular genetics, as it can be used for the preparation of isogenic
lines for molecular and biochemical experiments, is easily trans-
formed, and its genome is currently being sequenced (Kubota
et al., 2013).
1
2
3
4
5
2. Results
Fig. 1. Structures for compounds 1–5.
2.1. Emission of volatiles after mechanical wounding of thalli grown in
the field
2011). Because of these bioactivities, it has been assumed that C8
volatiles perform a signaling function when in the vapor phase.
Oxygenation of polyunsaturated fatty acids and subsequent
rearrangement of the resulting hydroperoxides (HPOs) lead to
the formation of a large family of oxygenated products, collec-
tively known as oxylipins (Feussner and Wasternack, 2002). C8
volatiles are oxylipins formed from the di-oxygenation of linoleic
acid (6) or arachidonic acid (7) to yield the respective HPO fol-
lowed by cleavage (Combet et al., 2006; Croisier et al., 2010).
Within fungi, a C18 fatty acid such as 6 is converted into its
10(S)-HPO and then cleaved to yield (R)-5 and the C10 oxo acid
(Wurzenberger and Grosch, 1982). Very recently, it was shown
that a cyanobacterium (Nostoc punctiforme) forms 5 from 6 via
cyclooxygenase- and catalase-related enzymes (Brash et al.,
2014). The diatom (Thalassiosira rotula) also has an ability to
dioxygenize and subsequently cleave fatty acids to form 6-
hydroxy-7-octenoic acid (8), which is structurally related to 5,
from hexadecatrienoic acid (Barofsky and Pohnert, 2007). Bryo-
phytes have pathways to form oxylipins from either C18 or C20
fatty acids (Croisier et al., 2010). In the moss Physcomitrella pat-
ens, the precursor 7 was converted to 5 through the formation
of its respective 12-HPO (Wichard et al., 2005). In spite of these
observations, the enzymes involved in 5 formation have not yet
been identified with the exceptions of the cyclooxygenase/cata-
lase from N. punctiforme and a lipoxygenase (LOX) from P. patens.
When recombinant LOX derived from P. patens was expressed in
Escherichia coli, 5 as well as (Z)-2-octen-1-ol (9) were formed
from 7 in vitro (Senger et al., 2005). Furthermore, little is known
about the pathways employed to form C8 volatiles other than 5,
such as 1-octen-3-yl acetate (3), 1, 1-octen-3-one (2), or 4.
It has been reported that some liverworts emit carbon 8 (C8)
volatiles as well as terpenoids (Suire et al., 2000; Asakawa et al.,
2013). However, it is not known whether the C8 volatiles are emit-
ted when liverworts are grown in their natural habitat. In this
study, volatile compounds in the headspace of thalli from M. poly-
morpha grown in its natural habitat were collected using a head-
space SPME-GC/MS method.
Only minimal emission of volatiles was detected from the intact
thalli of M. polymorpha (Fig. 2). When the thalli were mechanically
wounded with forceps to form wounds on ca. 10% of the thalli,
massive formation of volatiles was observed. Compounds 1 and 5
were the predominant C8 volatiles, while 2, 3, and 4 were detected
as minor C8 volatiles. The MS profiles of other peaks with retention
times ranging from 12 to 16 min suggested that they were C15 ses-
quiterpenoids with the chemical formulae C15H24 (m/z 204, with
peaks a–j) and C15H22 (m/z 202, with the peak j); (Asakawa et al.,
2013; Suire et al., 2000, Supplemental Fig. S1); however, these
were not identified. The amounts of C8 volatiles were quantified
by using calibration curves made with standards (inset of Fig. 2).
The amounts of 1 and 5 increased ca. three fold after mechanical
wounding and reached approximately 15 nmol mÀ2 hÀ1
.
The time course of 1 and 5 emission from the thalli after
mechanical wounding was followed. Because solid-phase microex-
traction (SPME) collection is rather static and unsuitable for esti-
mation of dynamic changes of volatiles, a dynamic charcoal
collection system was used that involved pulling air through a
charcoal cartridge using an air pump. The amounts of 1 and 5
increased within 40 min after mechanical wounding; thereafter,
the values gradually decreased, and returned back to their original
levels after 160 min (Fig. 3).
20
*
**
g
15
10
5
1.0 x 105
f
tr
0
Octan-3-one 1-Octen-3-yl 1-Octen-3-ol
c
(1)
acetate (3)
(5)
j
e
d
h
5
1
b
i
4
2
3
a
20.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Retention time (min)
Fig. 2. Effect of mechanical wounding on volatiles emitted from field-grown M. polymorpha thalli. A mat of intact thalli (58.1 cm2) was gently covered with a plastic cup, and
volatiles accumulated in the headspace were collected using a SPME fiber for 30 min (intact, lower trace). The thalli were wounded with forceps, and then covered with the
cup for SPME collection (wounded, upper trace). The inset shows the amounts of volatiles from intact (white bars) and wounded (black bars) thalli. Average s.e. (n = 5) is
shown. Asterisks indicate significant differences for the noted compound (Student’s t-test, ⁄P < 0.05, ⁄⁄P < 0.01). 1: octan-3-one, 2: 1-octen-3-one, 3: 1-octen-3-yl acetate, 4:
octan-3-ol, 5: 1-octen-3-ol. Putative C15 sesquiterpenoids with the chemical formulas C15H24 (m/z 204, with peaks a–i) and C15H22 (m/z 202, with the peak j) were tentatively
identified based on their MS profiles (Supplemental Fig. S1); however, they are not yet identified.

44
H. Kihara et al. / Phytochemistry 107 (2014) 42–49
1.0
0.8
0.6
0.4
0.2
0.0
40
30
20
10
0
B
BC
1
AC
ab
5
b
***
A
a
A
ab
A
a
a
nd
3
5
0
40
80
120
160
180
Time after mechanical wounding (min)
Fig. 5. Effect of incubation after tissue disruption on C8 volatile formation. The
thalli were frozen and the volatiles were extracted (white bars), or the frozen thalli
were thawed, and incubated for 5 min at 24 °C to facilitate the enzyme reaction
(gray bar). Average s.e. (n = 4) is shown. Asterisks indicate significant differences
for the noted compound (Student’s t-test, ⁄⁄⁄P < 0.001).
Fig. 3. Time course of emission of 1 and 5 after mechanical wounding of M.
polymorpha thallus grown in a field. Average s.e. (n = 6) is shown. Different letters
for each compound refer to significant differences (ANOVA, Bonferroni, P < 0.01).
2.2. Volatiles in tissues
The peaks k–p, and the peaks q and r were tentatively identified
as sesquiterpenoids and oxidized sesquiterpenoids, respectively,
based on their MS profiles (Supplemental Fig. S2); however, these
were not investigated further. Oxidized sesquiterpenoids were
probably present because solvent extraction was used as a separa-
tion method instead of SPME-GC/MS (Fig. 4). Among the C8 vola-
tiles, only 3 was detected in intact thalli. When frozen thalli
were thawed and incubated for 5 min at 24 °C to disrupt the tis-
sues, the amount of 3 decreased while that of 5 increased (Figs. 4
and 5). The amounts and compositions of putative sesquiterpe-
noids and oxidized sesquiterpenoids, however, remained essen-
tially unchanged from those detected from intact thalli after
incubation of freeze-disrupted thalli at room temperature.
Rapid formation of volatile oxylipins after mechanical wound-
ing is similar to C6 green leaf volatile formation from vascular
plants (Matsui, 2006; Matsui et al., 2012). In vascular plants, the
amounts of green leaf volatiles in intact tissues are usually low,
but after disruption of the plant tissues, they are rapidly formed
from endogenous substrates such as free fatty acids and glyceroli-
pids through successive catalysis by LOXs and HPO lyases
(Nakashima et al., 2013). Accordingly, it was assumed that C8 vol-
atiles are formed de novo from C18 or C20 fatty acids through oxy-
genation and subsequent cleavage after disruption of the tissues
(Wichard et al., 2005; Combet et al., 2006). On the other hand, it
should be noted that liverworts contain oil bodies that most prob-
ably accumulate diversified mixtures of terpenoids and/or aro-
matic compounds (Suire et al., 2000; He et al., 2013). Thus, it is
also possible that the C8 volatiles accumulate in certain cells or
organelles such as oil bodies, and that they are simply released
because the walls of the reservoirs are disrupted (Tissier, 2012).
To distinguish between these two possibilities, volatiles from
intact thalli were extracted with an organic solvent, which would
help to eliminate any enzymatic reactions that might be promoted
after mechanical damage to the plant tissues. In the intact thalli,
the accumulation of compounds was found with the retention
times ranging from 21 to 28 min and from 29 to 35 min (Fig. 4).
2.3. Hydrolysis of 3 to 5
It was expected that formation of 5 after mechanical wounding
would be dependent on the hydrolysis of 3 stored in the thalli. To
confirm this, crude enzyme extract from the thalli were prepared
and added to 3, and it was efficiently hydrolyzed to yield 5. The
highest activity was observed from pH 7.0 to 8.0, and the amount
of 5 formed was dependent on the amount of crude enzyme soln.
added to the reaction mixture (Supplemental Fig. S3). Furthermore,
the activity disappeared when the crude extract was heat-dena-
tured. These results suggested that the thalli possess an esterase
q
5
3
1.0 x 104
16.0
17.0
18.0
o
5.0 x 106
Retention time (min)
n
r
l
m
k
p
Intact
Freeze-thawed
10.0
15.0
20.0
25.0
30.0
Retention time (min)
Fig. 4. Effect of incubation after tissue-disruption on the amounts of endogenous volatiles. Volatiles were extracted with methyl tert-butyl ether from intact thalli (lower
chromatogram) or from freeze–thaw treated thalli (upper chromatogram), and analyzed using GC–MS. Inset shows the enlarged chromatogram from 15.8 to 18.4 min. C15
sesquiterpenoids with the chemical formulas C15H24 (m/z 204, with peaks k to p) and C15H26O (m/z 222, with the peaks q and r) were tentatively identified based on their MS
profiles (Supplemental Fig. S2).

H. Kihara et al. / Phytochemistry 107 (2014) 42–49
45
12
15
500 µV
R-(-)-5
14
7
6
S-(+)-5
(20:3)
(20:4)
17
16
Complete mixture
13
17:0
(IS)
(20:3)
des6KO
Without substrate
Without enzyme
Tak-2
RI
10.0
10.5
11.0
11.5
Retention time (min)
20
5
15
10
Retention time (min)
Fig. 6. Resolution of enantiomers of 5 formed from racemic 3. The crude enzyme
extract prepared from des6KO thalli was reacted with racemic 3, and the 5 formed by
enzyme catalyzed hydrolysis was subjected to chiral phase GC analysis (upper
chromatogram). 5 was not detected with the reaction mixture prepared without
substrate (middle chromatogram) or without enzyme (lower chromatogram).
Fig. 7. Fatty acid profiles of thalli from wild type (accession Tak-2 and RI) and
mutant (des6KO) M. polymorpha. Total lipids were extracted from thalli of Tak-2
(Takaragaike-2) (a female parent of des6KO), RI (a cross between Tak-1 and Tak-2),
and des6KO, and their fatty acid compositions were analyzed with GC–MS. The
positions of double bonds in 20:3 and 20:4 fatty acid species detected only with
des6KO thalli (shown in parentheses) were not determined. 6: linoleic acid
capable of hydrolyzing 3 to yield 5. When a racemic mixture of 3
was used as substrate, the esterase showed a slight preference
for the (R)-enantiomer, and yielded (R)-5 with an enantiomeric
excess (e.e.) value of 64% (Fig. 6). When the frozen powder was
thawed in the absence of oxygen (in the presence of Ar), the
amount of 5 formed was almost equivalent to the amount formed
in the presence of oxygen. Thus, 5 formation after tissue disruption
was independent of molecular oxygen.
(18:2
D
D a-linolenic acid (18:3D
9.12), 7: arachidonic acid (20:4 5,8,11,14), 12: 9,12,15),
5,8,11,14,17), 14: palmitic acid (16:0), 15: hexadecatrie-
7,10,13), 16: stearic acid (18:0), 17: oleic acid (18:1 9).
13: arachidonic acid (20:5
D
noic acid (16:3
D
D
Table 1
Effect of exposing des6KO thalli to 5 on the amounts of C8 volatiles produced.
Control
Exposure of 5
(nmol g FWÀ1
)
2.4. Effect of the absence of arachidonic acid (7)
1
2
3
5
nd*
nd
nd
nd
27.3 5.34
nd
18.7 3.97
5.48 2.82
It has been reported that linoleic acid is the substrate from
which
5 is formed in mushrooms, fungi, and cyanobacteria
(Wurzenberger and Grosch, 1982; Combet et al., 2006; Brash
et al., 2014). However, it was also recently reported that 7 was con-
verted into 5 in most bryophytes (Croisier et al., 2010; Senger et al.,
2005). M. polymorpha thalli contain a substantial amount of 7
(3.1 mol% of total fatty acids), as well as 6 (6.4 mol%) (Kajikawa
et al., 2008). Therefore, both 6 and 7 may act as substrates for
the formation of 5 and its derivatives in M. polymorpha. Recently,
The amounts are given as means SE (n = 4).
*
nd: not detected.
to form 3 from 5 was investigated. For this, the des6KO mutant was
used that lacked the ability to form 5 and its derivatives because
conversion of 5 to its acetate could be easily followed in the
absence of endogenous C8 volatiles. After exposing the mutant
thalli to the vapor of 5 for 18 h, accumulation of 3 as well as 5
and 1 was detected while nothing was detected when thalli were
exposed to the carrier solvent (Table 1). This indicated that M. poly-
morpha absorbed 5 from the atmosphere and converted it into 3.
a
D
6-desaturase gene (MpDES6) was knocked out that is involved
in the desaturation steps to form
-linolenic acid (18:3D6,9,12
(10) from 7 and stearidonic acid (18:3 -linole-
6,9,12,15) (11) from
nic acid (18:3
9,12,15) (12), respectively, (Kajikawa et al., 2008)
c
D
)
a
D
through a targeted gene-disruption technique (Ishizaki et al.,
2013; Kubota et al., 2014). In the transgenic line (des6KO), both
the n-6 pathway and n-3 pathway were terminated at the
desaturase step, thus, 6 and 12 accumulated and no formation of
7 and eicosapentaenoic acid (20:5
5,8,11,14,17) (13) was observed
D6-
2.6. NAD(P)H-dependent oxidation/reduction of 5
D
(Fig. 7). When the thalli of the wild type strain was used for volatile
analysis, 30.4 1.08 nmol g fr wtÀ1 of 3 was detected in intact
thalli tissues and 26.3 0.58 nmol g fr wtÀ1 of 5 was detected in
freeze–thawed thalli after solvent extraction. In contrast, these
C8-volatiles were only minimally detected in intact or freeze–
thaw-treated thalli of the des6KO plant (see Table 1). This result
suggested that 7 and/or 13 but not 6 was the substrate for 5 forma-
tion in M. polymorpha thalli.
In the field experiment, partially wounded thalli emitted a sub-
stantial amount of 1 (Fig. 2), whereas 1 was only minimally
detected in intact thalli or completely disrupted thalli (Fig. 3). Par-
tial wounding, but not complete disruption, might be the prerequi-
site to form 1. This was reminiscent of green leaf volatile
metabolism where complete disruption of leaf tissues resulted in
the formation of the primary product, hexenal in this case, while
partial wounding of the leaf tissues resulted in the conversion of
hexenal to the corresponding hexen-1-ol in the presence of NADPH
which served in maintaining intact tissues located alongside the
disrupted tissues (Matsui et al., 2012).
2.5. Conversion of 5 to 3
Because 5 is the primary product of the oxylipin pathway start-
ing from fatty acids (Wurzenberger and Grosch, 1982; Wichard
et al., 2005), its acetate was assumed to be formed from 5 via the
activity of an acetylation enzyme. In the green leaf volatile path-
way in vascular plants, an acetyl CoA transferase is involved in
the formation of (Z)-3-hexen-1-yl acetate from (Z)-3-hexen-1-ol
(D’Auria et al., 2007). Therefore, the ability of M. polymorpha thalli
From intact M. polymorpha thalli, emission of the C8 volatiles
was generally undetectable, but partial mechanical wounding
facilitated the emission of 1 (Fig. 7). When the thalli were com-
pletely homogenized without addition of any cofactors, emission
of 1 was largely suppressed, while 3 and 5 were the prominent
C8 volatiles emitted into the headspace (Fig. 7). When NADH was
added during disruption, the amount of
5 was significantly

46
H. Kihara et al. / Phytochemistry 107 (2014) 42–49
45
30
15
0
b
b
b
b
b
Intact
b
Partial wounding (50%)
Total disruption
b
Total disruption + NADH
Total disruption + NADPH
b
b
c
a
a
a
a
a
a
a
a
a
a
1
3
5
2
Fig. 8. Effect of addition of NAD(P)H on the C8 volatiles emitted from M. polymorpha thalli. C8 volatiles extracted from intact (white bars), partially wounded (50%, gray bars),
totally disrupted (black bars), totally disrupted in the presence of NADH (coarse stripe), and totally disrupted M. polymorpha thalli in the presence of NADPH (dense stripe)
were quantified. The average s.e. (n = 4–5) is shown. Different letters for each compound refer to significant differences (one way ANOVA, Fisher, P < 0.05).
increased while levels of 3 and 5 were largely unchanged. Addition
of NADH or NADPH significantly increased the amount of 2 (Fig. 8).
Addition of either NAD+ or NADP+ did not change the amount and
composition of C8 volatiles emitted from the homogenate.
An enzyme assay was next performed using a crude enzyme
soln. prepared from M. polymorpha thalli with 5 as a substrate. In
the absence of cofactors, formation of 2 from 5 was detected
(Table 2). When either NADH or NADPH was added to the reaction
mixture, formation of 2 was entirely suppressed, while 1 was
formed from 5.
et al., 2001). However, this result does not agree with the compo-
sition of C8 volatiles observed in wounded M. polymorpha thalli,
where (Z)-2-octen-1-ol was undetectable. Three LOX genes,
MpLOX1 to 3, have been examined to date and shown to form the
15(S)-HPO of 7 (Kanamoto et al., 2012). Nine additional genes
homologous to LOX were identified in the EST database of M. poly-
morpha. Further examination of the substrate and product specific-
ities of the MpLOXs other than MpLOX1 to 3 should be conducted
to identify the enzyme(s) involved in 5 formation from 7 and 13 in
M. polymorpha.
Rapid emission of volatile compounds after mechanical wound-
ing is observed with a wide array of plants and fungi (Arimura
et al., 2009). There are two types of responses that induce volatile
emission. One is the up-regulation of genes coding enzymes
involved in the synthesis of volatiles, a process often observed
for terpenoid synthesis induced after mechanical wounding of vas-
cular plants and occurs over several hours (Arimura et al., 2009).
The other is the rapid emission of volatiles without gene up-regu-
lation that occurs within seconds to minutes. In the latter case,
rapid volatile emission is supported by de novo synthesis from
endogenously reserved precursors with pre-existing enzymes, or
via release of volatile compounds indigenous to a special reservoir
in plant tissues. Here, it was found that rapid emission of 5 and 1
occurred after mechanical wounding of M. polymorpha thalli, this
being supported by the observation that hydrolysis of 3 accumulat-
ing in the tissues yielded 5, and that by subsequent oxido-reduc-
tion of a portion of 5 to 1. Tissue disruption of the fruiting bodies
of A. bisporus (Combet et al., 2009) and the mycelium of Penicillium
sp. (Kermasha et al., 2002) promotes rapid formation of 5 from 6
through an oxygenation/cleavage reaction. Formation of green leaf
volatiles is also induced in a similar way through the oxygenation/
cleavage of fatty acids upon disruption of vascular plant tissues
(Matsui, 2006). In contrast, soybean seeds (Matsui et al., 2011)
and some plants belonging to the Lamiaceae accumulated 5 in their
tissues and released their volatiles upon tissue disruption. Even
though rapid emission of 5 after mechanical wounding is rather
common among fungi and plants, the mechanisms by which it
occurs are diversified, which suggests that the mechanism
employed by liverworts was acquired independently and probably
in a convergent manner from the ones used by fungi and vascular
plants. The prompt formation of 5 might enhance the ability of M.
polymorpha to defend itself against enemies in a low light and high
humidity habitat. Some flowering plants lack the ability to form 5
and thus, it is likely that in these cases, the emission of 5 is not
essential for plant survival.
3. Discussion
The results described herein indicated that in M. polymorpha
thalli (R)-5 is formed from 7 or 13. (R)-5 is stored in the tissues
as 3 after acetylation. Upon tissue wounding, the acetate was
hydrolyzed to yield (R)-5. A portion of 5 remaining in the tissues
was converted into 1 via 2 (Fig. 8). As a result, (R)-5 and 1 were
released from the wounded thalli of M. polymorpha (Fig. 9).
Even though 5 is widely distributed among plant and fungal
kingdoms, the enzyme system involved in its biosynthesis has
not yet been identified. In mushrooms (Agaricus bisporus), 6 was
converted to (R)-5 via a LOX-like reaction from its 10(S)-HPO and
in turn, the subsequent HPL-like reaction on the 10(S)-HPO
(Wurzenberger and Grosch, 1982). In the moss P. patens, 7 was
converted into (R)-5 through formation of its 12(S)-HPO
(Wichard et al., 2005). By using a knock-out mutant lacking the
desaturase enzyme essential to form 7 and 13, it can be suggested
that these fatty acids are the substrates used for the formation of 5
in the liverwort M. polymorpha. One of the LOXs identified in P. pat-
ens, PpLOX1, catalyzed the formation of 5 and (Z)-2-octen-1-ol
from 7 in vitro. Based on this, it was assumed that PpLOX1 was pri-
marily responsible for the formation of 5 in the moss (Senger et al.,
2005). This agrees with the known chemistry of the b-scission
reaction toward the alkoxy radical derived from the 12-HPO of 7,
where both 5 and (Z)-2-octen-1-ol are the products (Delcarte
Table 2
Effect of NADH and NADPH on the conversion of 5 to 1 or 2 in the presence of crude
enzyme prepared from M. polymorpha (wild type) thalli.
1
2
(nmol mLÀ1 minÀ1
)
No addition
NADH
NADPH
nd*
1.94 0.244
2.39 0.380
1.14 0.414
nd*
nd*
Because of the range of bioactivities reported for 5, especially
against arthropods, it is assumed that rapid formation of 5 at
wound sites in mosses and liverworts is necessary for defense
The amounts are given as means SE (n = 4).
*
nd: not detected.

H. Kihara et al. / Phytochemistry 107 (2014) 42–49
47
Arachidonic acid (7)
Oxygenation
12-Hydroperoxy-5,8,10,14-(Z,Z,E,Z)-
eicosatetraenoic acid
Cleavage
reaction
Oxidation
Reduction
Hydrolysis
Acetylation
1-Octen-3-one (2)
1-Octen-3-ol (5)
1-Octen-3-yl acetate (3)
NAD(P)H
Reduction
Octan-3-one (1)
Fig. 9. Proposed biosynthetic pathway to form C8 volatiles in M. polymorpha thalli.
against herbivores and pathogens (Wichard et al., 2005). However,
4. Experimental
it is unknown what the advantage is for M. polymorpha to metab-
olize the beneficial volatile compound 5. Here it was found that
5 in the vapor phase was absorbed by the intact thalli of M. poly-
morpha, and converted into its acetate probably intracellularly,
where the other substrate, acetyl-CoA, was available. In plant
leaves, (Z)-3-hexenol exogenously supplied as a vapor was effi-
ciently converted into (Z)-3-hexenyl acetate by acetyl CoA:(Z)-3-
hexen-1-ol acetyltransferase (D’Auria et al., 2007; Matsui et al.,
2012). This acetylation is believed to be important to abrogate
the bioactivity of (Z)-3-hexenol, which induces defense responses
in plants (Matsui et al., 2012). 5 is also able to induce an oxidative
burst and expression of a subset of defense genes in plants
(Splivallo et al., 2007; Kishimoto et al., 2007). Therefore, it might
be important to modulate the bioactivities of 5 through acetylation
at the expense of acetyl-CoA, which is a precious carbon source
that could otherwise be used for growth and development.
Complete disruption of the thalli tissues resulted in predomi-
nant formation of 5, while partial disruption facilitates conversion
of 5 to 1. The intact tissues surrounding the wounded and dis-
rupted tissues should help reduce any secondary bioactivities of
5. In this case, 5 participates in oxido-reduction rather than acety-
lation. The genotoxic potential of 1 for mammalian cells is lower
than that of 5 (Kreja and Seidel, 2002). 1 is also much less reactive
4.1. Plant materials
M. polymorpha L. collected at the Yoshida campus (E131°29’,
N34°11’) of Yamaguchi University, Yamaguchi, Japan, was used in
all experiments except for the experiment with des6KO plant. For
some experiments, the harvested thalli were further grown in a
growth chamber at 21 °C under continuous fluorescent light sup-
plemented with light from an incandescent bulb (16–
33
l
mol mÀ2 sÀ1) or on a half strength Gamborg-B5 plate at
21 °C under continuous fluorescent light (43–77
l
mol mÀ2 sÀ1).
Under low intensity light, the formation of gemma, archegonio-
phore, and antheridiophore was suppressed. The des6KO strain
was prepared through a targeted gene-disruption procedure with
F1 sporelings derived from crosses between male and female
accessions, Takaragaike-1 (Tak-1) and Takaragaike-2 (Tak-2),
respectively (Ishizaki et al., 2013; Kubota et al., 2014), and was
aseptically grown on a half strength Gamborg-B5 plate at 21 °C
under continuous fluorescent light.
4.2. Volatile collection in the field
than 2, which harbors an a,b-unsaturated carbonyl structure and is
Volatiles emitted from thalli in their natural habitat at the cam-
pus of Yamaguchi University were collected on December 5 and 19,
2012. It was occasionally cloudy, and essentially no wind was
noted on those dates. Collection was done from 10:00 to 17:00.
The average temperature was 6.3 to 6.7 °C and the relative humid-
ity was 43–70%. To induce mechanical wounding, the thalli were
pinched with forceps on ca. 10% of the area. For the SPME collec-
tion, a lawn of thalli was gently covered with a round-shaped poly-
styrene cup (basal area, 58.1 cm2; height, 5 cm). A SPME fiber (50/
prone to participate in a Michael addition reaction (Schultz et al.,
2005). Accordingly, 1 is the most inert C8 volatile, and, contrary
to 3, it does not accumulate in thallus tissues. It is assumed that
5 formed in the wounded tissues subsequently diffuses into the
neighboring intact tissues where it is converted into 1 to suppress
its bioactivities, and thereafter, 1 is discarded into the atmosphere.
Addition of NAD(P)H made the C8 metabolism in the completely
disrupted tissues partly similar to that observed in partially dis-
rupted tissues, by converting 5 to 2 and 1. Oxidation of the sec-
alcohol, which may be catalyzed by alcohol dehydrogenase, is
reversible (Edegger et al., 2006), while reduction of the double
bond, which may be catalyzed by alkenal reductase (Yamauchi
et al., 2011; Wu et al., 2013), is irreversible. Thus, the overall reac-
tion can be determined by the rate of reduction of the double bond
that is dependent on NAD(P)H. However, increase in the amounts
of 2 during complete disruption of thalli in the presence of
NAD(P)H could not be explained only with this proposal. Involve-
ment of other metabolic pathway could be suggested, and further
study is definitely needed in order to dissect the C8 volatile metab-
olism completely in wounded thallus of M. polymorpha.
30 lm DVB/Carboxen/PDMS, Supelco, Bellefonte, PA, USA) was
exposed to the headspace in the hood for 30 min. After collection,
the fiber was inserted into the insertion port of a GC–MS (QP-5050,
Shimadzu, Kyoto, Japan) equipped with a Stabilwax column (30 m
length  0.25 mm i.d.  0.25
lm film thickness, Restek, Bellefonte,
PA). The column temperature was programmed as follows: 40 °C
for 2 min, increasing by 10 °C minÀ1 to 200 °C for 2 min. The carrier
gas (He) was delivered at 86.1 kPa. The glass insert was an SPME
Sleeve (Supelco). Splitless injection with a sampling time of
1 min was used. The fiber was held in the injection port for
10 min to fully remove any compounds from the matrix. The
temperatures of the injector and interface were 240 and 200 °C,

48
H. Kihara et al. / Phytochemistry 107 (2014) 42–49
respectively. The mass detector was operated in the electron
impact mode with an ionization energy of 70 eV.
crude enzyme soln. The reaction was initiated by mixing the crude
enzyme soln. (200 L) suspended in
L) with 10 mg mLÀ1 3 (10
l
l
To identify each compound, retention indices and MS profiles of
corresponding authentic specimens were used. To construct cali-
bration curves for quantification, a cotton swab containing given
amounts of 1 (Wako Pure Chemicals, Osaka, Japan), 3 (Sigma–
Aldrich, St. Louis, MO), 4 (Tokyo Chemical Industry Co., Tokyo,
Japan), 2 (Tokyo Chemical Industry), 5 (Alfa Aesar, Ward Hill,
MA), and caryophyllene (Sigma–Aldrich) was placed approxi-
mately 2–3 cm above ground with almost the same soil composi-
tion as the collection site but without any plants; then, the
ground was covered with the hood for SPME collection. An aqueous
soln. of each compound was prepared by suspending it in a small
amount of Tween 20. In the absence of addition of the volatile com-
pounds, the emission of the volatiles was not detected.
0.2% Tween 20 in 50 mM Na phosphate, pH 7.0 (total volume of
1 mL) in a shaking water bath at 24 °C for 30 min. After the reac-
tion, the C8 compounds were extracted with MTBE and analyzed
using GC.
Resolution of each enantiomer was carried out using a GC
equipped with a 0.25 mm  30 m
a-DEX 120 column (Supelco).
The column temp. was maintained at 100 °C. Injector and detector
(FID) temperature was set at 200 °C. N2 gas was used as carrier gas
at 20.0 cm sÀ1. Under these conditions, (S)-5 and (R)-5 (Acros
Organics, Geel, Belgium) eluted at 11.0 and 11.2 min, respectively.
4.5. Acetylation activity
To follow the time course after mechanical wounding, the lawn
of thalli was gently covered with a round-shaped polystyrene cup
(basal area, 58.1 cm2; height, 5 cm) immediately after mechanical
wounding to 10% of the surface, and the headspace inside of the
cup was continuously passed through a charcoal cartridge (ORBO
32 Small Activated Coconut Charcoal (20/40), 100/50 mg, Sigma–
Aldrich) at 1000 mL minÀ1 with an air-pump (N86KT.18, KNF Japan
Co., Tokyo, Japan). After collecting volatiles for 40 min, the
adsorbed compounds were eluted three times with CH2Cl2
To examine the acetylation activity responsible for forming 3
from 5, the des6KO mutant strain was used. The des6KO thalli grown
on a half-strength Gamborg-B5 plate were exposed to the vapor of
5 by hanging a sheet (7 Â 7 mm) of filter paper containing
10 mg mLÀ1 5 soln.(20
l
L) in 0.2% Tween 20 for 18 h under contin-
uous light at 21 °C. As a control, 0.2% Tween 20 (20
l
L) was used.
After exposure, the thalli were collected from the plate, and the
volatiles in the tissues were immediately extracted with MTBE as
described in Section 4.3.
(250 lL containing 1 l
g mLÀ1 cyclohexanol). A portion of the
extract was subjected to GC–MS analysis essentially as shown
above. Injection was performed using a split-mode with a ratio
of 10. The column temperature was programmed as follows:
40 °C for 2 min, increasing by 5 °C minÀ1 to 150 °C, by 10 °C minÀ1
to 200 °C, then held at 200 °C for 2 min. The carrier gas (He) was
delivered at 86.1 kPa.
4.6. Oxidoreductase assay
The thalli were collected and incubated on wet paper towel for
3 h at 24 °C to recover from the response caused by wounding dur-
ing collection. The thalli (ca. 0.1 g fr wt) were mixed with an equal
volume of distilled H2O with or without 5 mM NADH or NADPH,
and then were thoroughly homogenized in a mortar. The homoge-
nate was transferred into a glass vial (22 mL, Perkin Elmer, Wal-
tham, MA, USA), sealed tightly with a butyl rubber stopper and a
crimp-top seal (National Scientific, Rockwood, TN, USA), and the
volatiles in the headspace were collected using a SPME fiber at
25 °C for 30 min. To analyze volatiles from partially wounded
thalli, about 50% of the surface area of thalli were mechanically
wounded with forceps, and then placed in the vial for SPME collec-
tion. Intact thalli were used as the control. The volatiles were ana-
lyzed as described in Section 4.2. Aq. solutions of standard
compounds were prepared from 10 mg mLÀ1 suspensions in 0.2%
Tween 20 and used to construct calibration curves.
4.3. Solvent extraction
The thalli collected from the campus and grown in a growth
chamber for 3 months were used for solvent extraction. Three
hours before analysis, the thalli were collected and placed on a
wet towel to avoid wound-responses brought about during collec-
tion. The thalli were snap-frozen with liquid N2, and powdered
with a mortar. Volatiles were extracted from a portion (0.2 g fr wt)
with 1 mL of methyl tert-butyl ether (MTBE) containing nonanyl
acetate (1 l
g mLÀ1) (Tokyo Chemical Industry). Another portion
(0.2 g fr wt) was thawed in a water bath, and then incubated for
an additional 5 min at 24 °C to facilitate the enzyme reaction.
The enzyme reaction was terminated by adding MTBE (1 mL) con-
taining nonanyl acetate. The MTBE mixture was dispersed with the
aid of an ultrasonic washer for 10 s, then mixed vigorously. The
mixture was centrifuged at 210Âg for 10 min at 25 °C (8100 Centri-
fuge, Kubota, Tokyo, Japan). A portion of the extract was subjected
to GC–MS analysis essentially as described above. Injection was
performed using a split-mode with a ratio of 2. The column tem-
perature was programmed as follows: 40 °C for 2 min, increasing
by 5 °C minÀ1 to 200 °C for 2 min. The carrier gas (He) was deliv-
ered at 26.7 cm sÀ1. For quantification of 1, 2, 3, 4, and 5, each stan-
dard was suspended in 0.2% Tween 20 at 10 mg mLÀ1, and the
suspension was diluted to prepare a series of aq. solutions,
extracted with MTBE containing nonanyl acetate, and then sub-
jected to GC–MS analysis to construct calibration curves.
To estimate the oxidoreductase activity toward 5, crude enzyme
soln. (100
l
L) prepared as described in Section 4.4. was mixed with
10 mg mLÀ1 5 (10
lL) suspended in 0.2% Tween 20, and incubated
at 27 °C for 5 min in 50 mM sodium phosphate (pH 7.0) with or
without 5 mM NADH or NADPH. After the reaction, the C8 com-
pounds were extracted with 1 mL of MTBE containing nonanyl ace-
tate (1 l
g mLÀ1), and quantitatively analyzed using GC–MS
essentially as described in Section 4.4 but with the modified col-
umn condition [40 °C (5 min) to 200 °C (2 min) at 8 °C minÀ1 with
He as a carrier gas at 26.7 cm sÀ1].
4.7. Fatty acid analysis
Approximately 2 g (FW) of M. polymorpha thalli were ground at
room temperature and extracted with CHCl3: MeOH (9 mL, 1:2 v/v)
for 20 min. After adding 5 mL each of CHCl3 and H2O, the organic
phase was recovered by centrifugation at 1860Âg, at 4 °C for
20 min. After addition of EtOH (À15 mL), the extract was freeze-
dried, and fatty acids were trans-methylated with 10% (v/v)
H2SO4 in MeOH (2.5 mL) at 80 °C for 2 h. Fatty acid methyl esters
were extracted with hexane (2.5 mL), and a portion was analyzed
with GC–MS (Shimadzu GCMS-QP2010 Ultra) equipped with a
quadrupole mass detector (EI, 70 eV) and a DB-23 capillary column
4.4. Esterase assay
The thalli (wild type or des6KO, 5 g fr wt) were homogenized
with 50 mM Na phosphate (10 mL, pH 7.0 containing 2.5% Polyklar
VT (w/v)) (Wako Pure Chemicals) in a mortar with the aid of sea
sand on ice. The homogenized soln. was centrifuged at 10,000Âg
for 30 min at 4 °C, and the resultant supernatant was used as the

H. Kihara et al. / Phytochemistry 107 (2014) 42–49
49
Ishizaki, K., Johzuka-Hisatomi, Y., Ishida, S., Iida, S., Kochi, T., 2013. Homologous
recombination-mediated gene targeting in the liverwort Marchantia
polymorpha L. Sci. Rep. 3 (art. no. 1532).
Kajikawa, M., Matsui, K., Ochiai, M., Tanaka, Y., Kita, Y., Ishimoto, M., Kohzu, Y.,
Shoji, S., Yamato, K.T., Ohyama, J., Fukuzawa, H., Kohchi, T., 2008. Production of
(30 m  0.25 mm, film thickness 0.25
lm; Agilent), the carrier gas:
He (0.89 mL minÀ1), oven temperature: first kept at 160 °C for
1 min, elevated to 210 °C (4 °C minÀ1), kept at 210 °C for 6.5 min.
arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid
desaturase, 6-elongase, and 5-desaturase genes. Biosci. Biotechnol. Biochem.
72, 435–444.
D6-
Acknowledgments
D
D
Kanamoto, H., Takemura, M., Ohyama, K., 2012. Cloning and expression of three
lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli.
Phytochemistry 77, 70–78.
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compounds by hydroperoxide lyase from enzymatic extract of Penicillium sp. J.
Mol. Catal. B Enzym. 19–20, 479–487.
This work was supported by JSPS KAKENHI Grant Number
23580151 and Yobimizu Project from Yamaguchi University, and
also partly by JST, ALCA (to HF). We thank Sakiko Ishida for techni-
cal assistance.
Kishimoto, K., Matsui, K., Ozawa, R., Takabayashi, J., 2007. Volatile 1-octen-3-ol
induces a defensive response in Arabidopsis thaliana. J. Gen. Plant Pathol. 73,
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Appendix A. Supplementary data
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