Second bioluminescence-activating component in the luminous fungus Mycena chlorophos

Article abstract of DOI:10.1002/bio.3165

Mycena chlorophos is an oxygen-dependent bioluminescent fungus. The mechanisms underlying its light emission are unknown. A component that increased the bioluminescence intensity of the immature living gills of M. chlorophos was isolated from mature M. chlorophos gills and chemically characterized. The bioluminescence-activating component was found to be trans-3,4-dihydroxycinnamic acid and its bioluminescence activation was highly structure-specific. ¹³C- and ¹⁸O-labelling studies using the immature living gills showed that trans-3,4-dihydroxycinnamic acid was synthesized from trans-4-hydroxycinnamic acid in the gills by hydroxylation with molecular oxygen as well as by the general metabolism, and trans-3,4-dihydroxycinnamic acid did not produce hispidin (detection-limit concentration: 10 pmol/1 g wet gill). Addition of 0.01 mM hispidin to the immature living gills generated no bioluminescence activation. These results suggested that the prompt bioluminescence activation resulting from addition of trans-3,4-dihydroxycinnamic acid could not be attributed to the generation of hispidin. Copyright

Full text of DOI:10.1002/bio.3165

Research article
Received: 01 March 2016,
Revised: 24 April 2016,
Accepted: 01 May 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.3165
Second bioluminescence-activating component
in the luminous fungus Mycena chlorophos
Katsunori Teranishi*
ABSTRACT – Mycena chlorophos is an oxygen-dependent bioluminescent fungus. The mechanisms underlying its light emission
are unknown. A component that increased the bioluminescence intensity of the immature living gills of M. chlorophos was
isolated from mature M. chlorophos gills and chemically characterized. The bioluminescence-activating component was found
to be trans-3,4-dihydroxycinnamic acid and its bioluminescence activation was highly structure-specific. 13C- and 18O-labelling
studies using the immature living gills showed that trans-3,4-dihydroxycinnamic acid was synthesized from trans-
4-hydroxycinnamic acid in the gills by hydroxylation with molecular oxygen as well as by the general metabolism, and trans-
3,4-dihydroxycinnamic acid did not produce hispidin (detection-limit concentration: 10 pmol/1 g wet gill). Addition of 0.01
mM hispidin to the immature living gills generated no bioluminescence activation. These results suggested that the prompt
bioluminescence activation resulting from addition of trans-3,4-dihydroxycinnamic acid could not be attributed to the
generation of hispidin. Copyright © 2016 John Wiley & Sons, Ltd. –
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: bioluminescence; fungus; metabolism; Mycena chlorophos; trans-3,4-dihydroxycinnamic acid
possessed the following characteristics: a stable complex compris-
ing both protein and non-protein components, long-term light
emission in vitro, and activation with H2O2 and NADPH (11). Re-
cently, Purtov et al. reported that hispidin and 3-hydroxyhispidin
Introduction
Bioluminescence is the production of light by living organisms.
The bioluminescence mechanisms of various organisms such as
fireflies, jellyfishes, and bacteria have been elucidated previously
acted as luciferin precursor and luciferin, respectively, for the
(1). Fungal bioluminescence is also widely found on land. Chew
mycelia bioluminescence of N. nambi, Mycena citricolor, Panellus
et al. collected specimens from three new species of biolumines-
stipticus, and Armillaria borealis (12).
cent fungi in Malaysia, bringing the total number of documented
Mycena chlorophos, which is primarily distributed in Southeast
species of luminous fungi in 2014 to 77 (2). Fungal biolumines-
Asia, is a species of oxygen-dependent bioluminescent fungus
cence, which varies among species, occurs in the basidiocarp,
(13). The fungus emits light spontaneously and continuously from
mycelium, and/or spores (3–5). In spite of the wide popularity of
the mycelium and the pileus. In our cultivation system, the light
fungal bioluminescence, the molecular mechanisms underlying
emitted by the mycelium, when grown on a solid medium, is so
this phenomenon have been subjected to far less scientific inves-
weak that a few minutes of dark adaptation is required to see it.
tigation than bioluminescence from other sources.
Airth et al. have demonstrated that the cell-free light emitting
system for the bioluminescence of Armillaria mellea required
When young fruiting bodies grew, the pileus contracted, the stipe
began to elongate at 20°C, and at approximately 90% relative hu-
midity, bioluminescence was weak (Fig. 1, stage 1). Subsequently,
luciferase, luciferin, molecular oxygen, and reduced pyridine nucle-
the pileus expanded horizontally; bright green light was emitted
otide (6). In 2009, Oliveira and Stevani attested that the mechanism
from the whole pileus and was continuously emitted for approxi-
proposed by Airth and Foerster for fungal bioluminescence is
mately 1 day at 20°C and at approximately 90% relative humidity
correct using four species of bioluminescent fungi (7). Thereafter,
cross-reaction experiments with luciferin and luciferase extracts
(Fig. 1, stage 2). Few chemical and biochemical studies on M.
chlorophos bioluminescence have been reported to date
from bioluminescent species, performed by Oliveira et al.,
(12,14–17). Mori et al. suggested that the bioluminescence of M.
supported the hypothesis that all known bioluminescent fungal
lineages share the same type of luciferin and luciferase and that
chlorophos involves enzymatic reaction (s) in the cell membrane
(14). Purtov et al. reported that adding hispidin to a mixture of
there is a single luminescence mechanism in all the biolumines-
NADPH and a crude cold water-extract solution prepared from
cent fungi (8). However, the luciferase and luciferin were not
isolated and their structures and luminescent properties were
not determined. With respect to the bioluminescence system of
the mycelium of the bioluminescent fungus Neonothopanus
nambi, in 2011 and 2013, Bondar et al. suggested the presence
the bioluminescent fruiting body of M. chlorophos enhanced the
in vitro luminescence (12). However, they did not state whether
hispidin acted as luciferin precursor or luciferin for the biolumines-
* Correspondence to: Katsunori Teranishi, Graduate School of Bioresources, Mie
University, 1577 Kurimamachiya, Tsu, Mie 514 8507, Japan. Email address:
teranisi@bio.mie-u.ac.jp
of Mn-peroxidase (s) and detected a low-molecular-weight, heat-
stable component, which increased the luminescence in the myce-
lium (9,10). However, these components have not been isolated
and their chemical structures have not yet been identified. In
2014, they extracted an N. nambi luminescence system, which
Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu,
Mie, Japan
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Copyright © 2016 John Wiley & Sons, Ltd.

K. Teranishi
synthesized from trans-3,4,5-trimethoxycinnamic acid according to
a literature method (18).
Fruiting bodies of M. chlorophos. A mycelium strain of M.
chlorophos (Berkeley & M.A. Curtis) Saccardo was purchased from
the National Institute of Technology and Evaluation’s Biological Re-
source Center in Japan. Fruiting bodies of M. chlorophos were cul-
tivated from the mycelium at 20°C and at approximately 90%
relative humidity in the dark, as previously described (14), and
were then carefully harvested at stages 1 and 2. The fruiting bodies
were immediately used for bioluminescence assay after harvesting
or were stored in liquid nitrogen for extraction.
Extraction. The pilei at stage 2 (10 g) stored in liquid nitrogen
were homogenized with 10 mL water using a Vibracell ultrasonic
disintegrator (Sonics & Materials Inc, Newtown, CT, USA) in an ice
bath for 30 sec, then centrifuged at 12,000 rpm at 5°C for 10 min.
After separating the supernatant, the pellet was extracted twice,
using 10 mL water each time. All the supernatants were combined
and 10 mL methanol was added; the resulting mixture was stored
in an ice bath for 10 min and centrifuged at 12,000 rpm at 5°C for
10 min. The supernatant was evaporated under reduced pressure
to approximately 5 mL. The concentrate was dissolved in water to
10 mL and centrifuged at 12,000 rpm at 5°C for 10 min. The super-
natant was stored at À80°C until it was used for isolation of the
bioluminescence-activating component. This extraction process
was also performed with 5 kg of wet pileus.
Figure 1. Growth and bioluminescence of M. chlorophos. The upper and lower
photographs were taken under white light and in the dark, respectively, using a digital
camera (Lumix; Panasonic, Osaka, Japan) at 20°C and approximately 90% relative
humidity using the following settings: ISO, 400; aperture, f2.8; and exposure time,
1.3 sec as a representative of many fruiting bodies.
High-performance liquid chromatography photo-diode array
detection (HPLC-PDA)-MS analysis of the extract was performed
using the following procedure. A mixture of fresh gills (2.0 g) at
stage 2 and 10 mM phosphate buffer at pH 7.0 (8 mL) were
homogenized using a Vibracell ultrasonic disintegrator in an ice
bath for 30 sec, then centrifuged at 12,000 rpm at 5°C for 10 min.
After separating the supernatant, the pellet was extracted one
more time using 10 mL water. All the supernatants were combined
and 20 mL methanol was added. The resulting mixture was stored
in an ice bath for 10 min and centrifuged at 12,000 rpm at 5 °C for
10 min. The supernatant was evaporated under reduced pressure
to approximately 2 mL. The concentrate was centrifuged at 12,000
rpm at 5°C for 10 min. The supernatant was evaporated under
reduced pressure to dryness, and the concentrate was dissolved
in water to 0.2 mL and centrifuged at 12,000 rpm at 5°C for 10
min. The resulting supernatant (0.02 mL) was analyzed using the
HPLC–PDA-MS system equipped with an MD-910 detector ( JASCO
Corp, Tokyo, Japan), a COSMOSIL 5PBr column (4.6 mm × 250 mm;
Nacalai Tesque, Inc, Kyoto, Japan), and a ZQ 4000 MS spectrometer
(Waters Corporation, Milford, MA, USA) using a subsequent mobile
phase system-1. The HPLC mobile phase was a mixture of 10 mM
aqueous formic acid (A) and CH3CN (B). A linear gradient was
achieved starting with 0% B, reaching 50% B in 30 min, and subse-
quently reaching 100% B in 10 min at a flow rate of 0.8 mL/min
(see Fig. 2). MS analysis was carried out using electrospray ioniza-
tion (ESI) in negative mode.
cence of the M. chlorophos fruiting body or whether hispidin was
detected in the fruiting bodies of M. chlorophos. Recently, Teranishi
indicated that trans-4-hydroxycinnamic acid, in the gills of M.
chlorophos structure specifically, increased the bioluminescence
activity in the living gills (17). At present, little chemical information
is available concerning the bioluminescence of M. chlorophos, and
the chemical mechanism underlying this phenomenon is unknown.
In the present study, we discovered a second bioluminescence-
activating component, which played a more significant role in
activating bioluminescence in living M. chlorophos gills at stage 1
than did trans-4-hydroxycinnamic acid, from the mature M.
chlorophos gills, at stage 2. This paper describes the isolation,
chemical structure, and metabolism of the second
bioluminescence-activating component.
Experimental
Chemicals
trans-4-Hydroxycinnamic acid; trans-3,4-dihydroxycinnamic acid; and
trans-ferulic acid were purchased from TCI chemicals (Tokyo, Japan).
trans-2,4-Dihydroxycinnamic acid; trans-2,3-dimethoxycinnamic acid;
trans-3,5-dimethoxycinnamic acid; 3,4-dihydroxyhydrocinnamic
acid; 1,2,3–13C3-trans-4-hydroxycinnamic acid (99 atom % 13C);
hispidin; and 1 M BBr3 in CH2Cl2 were purchased from Sigma Aldrich
Co. (St. Louis, MO, USA). trans-3,4-Dihydroxycinnamamide was
purchased from Ark Pharm Inc. (Libertyville, IL, USA). 1,2,3–13C3-
trans-3,4-dihydroxycinnamic acid (98.7 atom % 13C) was purchased
from Toronto Research Chemicals Inc. (Toronto, ON, Canada). 18O2
(97 atom % 18O) was purchased from Taiyo Nippon Sanso Co.
(Tokyo, Japan). trans-3,4,5-Trimethoxycinnamic acid and other
chemicals were purchased from Wako Pure Chemical Industries
Ltd. (Osaka, Japan). trans-3,4,5-Trihydroxycinnamic acid was
Isolation. The extract solution stored at À80°C was centrifuged at
12,000 rpm at 5°C for 10 min. After separating the supernatant, a
mixture of the supernatant (1 L) from pilei (1 kg) and 0.1 M aque-
ous phosphoric acid (100 mL) was adsorbed on a column (45
mm × 170 mm) containing Chromatorex DM1020T ODS gel (Fuji
Silysia, Aichi, Japan) prepared with 10 mM aqueous phosphoric
acid. The elution was conducted by adding, stepwise, 0, 10, 20,
30, 40, 50 and 100% of 10 mM phosphoric acid/methanol to the
mixture (each volume = 1 L). The bioluminescence-activating
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Bioluminescence of Mycena chlorophos
Figure 2. HPLC chromatogram of an aqueous extract (0.1 mL/1 g wet gill) from M. chlorophos gills at stage 2.
component existed in 20% of a 10 mM phosphoric acid/methanol
eluent. The eluent was neutralized to pH = 7.0 with aqueous NaOH
and evaporated to 100 mL under reduced pressure in the dark.
This chromatography process was also performed for 5 kg of
pileus.
A second purification of the fraction of the component obtained
in the first chromatography experiment (for 50 g of pileus) was
conducted using the HPLC–PDA system and a COSMOSIL 5C18-
PAQ column (20 mm × 250 mm; Nacalai Tesque, Inc.) with a 10
mM aqueous sodium phosphate buffer (pH = 7.0) as the mobile
phase at a flow rate of 5 mL/min. The bioluminescence-activating
component was eluted after 35 and 40 min (Fig. S1A), and the
fraction was evaporated under reduced pressure to near dryness
in the dark. This second HPLC purification was also performed for
all 20% of 10 mM phosphoric acid/methanol fractions obtained
in the first purification.
The final purification of the component fraction derived from
500 g of pileus in the second chromatography run was obtained
using the HPLC–PDA system and a COSMOSIL 5PBr column
(20 mm × 250 mm; Nacalai Tesque, Inc.) with a 1 mM sodium
phosphate buffer (pH = 7.0). The flow rate was set to 5 mL/min to
decrease the phosphate salt concentration. The bioluminescence-
activating component eluted between 33 and 42 min (Fig. S1B),
and the fraction containing the bioluminescence-activating compo-
nent was evaporated until dry under reduced pressure in the dark.
This third HPLC purification was also performed for all fractions
obtained in the second purification. Approximately 0.5 mg of the
bioluminescence-activating component was obtained from 5 kg of
wet pileus. The purity of the isolated bioluminescence-activating
component was analyzed using the HPLC–PDA system and a
COSMOSIL 5PBr column (4.6 mm × 250 mm) with the mobile phase
system-1 (Fig. S1C).
internal standard in approximately 0.2 M sodium phosphate buffer
(pD = 7.0)/D2O; 1H of acetone: 2.04 ppm, 13C of acetone CH3: 31.6
ppm. Chemical shifts and coupling constants are reported as δ/
1
(ppm) and J (Hz), respectively; H NMR: δ = 6.16 (1H, d, J = 15.9
Hz), 6.74 (1H, d, J = 7.9 Hz), 6.88 (1H, dd, J = 1.8 and 7.9 Hz), 6.97
(1H, d, J = 1.8 Hz), 7.10 (1H, d, J = 15.9 Hz) (Fig. S3A), 13C NMR:
δ115.19, 116.93, 122.15, 122.36, 128.73, 141.36, 144.90, 146.52,
176.69 (Fig. S3B).
Measurement
of
bioluminescence
activity. The
gill
bioluminescence-activating capacities of each fraction in the
three-step chromatography processes for the purification of the
bioluminescence-activating
component,
the
isolated
bioluminescence-activating component, and the commercially
available trans-3,4-dihydroxycinnamic acid and its analogues were
measured by the following method. Phosphate buffer (10 mM, pH
= 7.0; 1.5 μL) and a gill section (2 mm × 2 mm) of M. chlorophos at
stage 1 were placed in a black 96-well plate (96/V-PP, Eppendorf
AG, Hamburg, Germany). The bioluminescence intensity was mea-
sured every 1 min for approximately 20 min at 25°C using a Micro-
plate Luminometer Centro (Berthold Technologies GmbH & Co KG,
Bad Wildbad, Germany) to confirm the steadfastness of the biolu-
minescence intensity. Then, 10 μL of the extract solution, each frac-
tion solution, the solution of the isolated bioluminescence-
activating component in 10 mM phosphate buffer (pH = 7.0),
trans-3,4-dihydroxycinnamic acid solution in 10 mM phosphate
buffer (pH = 7.0) or its analogue solution in 10 mM phosphate
buffer (pH = 7.0) was added to the wells, and the bioluminescence
intensity was measured every 1 min for more than 20 min at 25°C
using a Microplate Luminometer Centro. At each time point, the in-
tensities were accumulated for 1 sec. The bioluminescence intensi-
ties are provided as the mean of independent measurements.
Measurement of bioluminescence spectra. A gill section at stage 1
was placed in a tube, 1.5 μL of 10 mM phosphate buffer (pH = 7.0)
was added, and the bioluminescence spectrum was measured at
25°C using a LumiFL-Spectrocapture (Atto Co., Ltd, Tokyo, Japan).
Then, 0.01 mL of the 0.3 mM trans-3,4-dihydroxycinnamic acid in
10 mM phosphate buffer (pH = 7.0) was added to the tube before
recording luminescence spectra. The intensities were accumulated
for 30 sec.
Chemical structure
HPLC–PDA-MS spectrometry analyses of the bioluminescence-
activating component and commercially available trans-3,4-
dihydroxycinnamic acid were performed at 25°C on an HPLC sys-
tem equipped with a COSMOSIL 5PBr column (4.6 mm × 250
mm). The HPLC mobile phase system-1 was used for the analysis.
MS analysis was carried out using ESI in negative mode. A UV–vis
absorption spectrum was obtained from the HPLC–PDA analysis
of the bioluminescence-activating component (Fig. S4). The
negative ESI-MS spectrum showed 178.98 [M-1]– as a molecular
Metabolism analysis. A pileus at stage 1, was divided into equal
parts. Approximately 20 parts (2.0 g) were then incubated in 0.23
mM 1,2,3–13C3-trans-4-hydroxycinnamic acid in 10 mM phosphate
buffer at pH 7.0 (8.0 mL) under 18O2 at 25°C in the dark for 1.5 h
and then the mixture was kept at À80°C. The mixture was homog-
enized using a Vibracell ultrasonic disintegrator in an ice bath for
30 sec, then centrifuged at 12,000 rpm at 5 °C for 10 min. After
separating the supernatant, the pellet was extracted one more
1
ion (Fig. S2). H and 13C NMR spectra of the bioluminescence-
activating component and commercially available trans-3,4-
dihydroxycinnamic acid were measured with
a JNM-A500
spectrometer ( JEOL, Tokyo, Japan) at 21°C operating at 500 MHz
1
for H NMR and 125 MHz for 13C NMR. Acetone was used as an
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K. Teranishi
time using 10 mL water. All supernatants were combined and 20
mL methanol was added. The resulting mixture was stored in an
ice bath for 10 min and centrifuged at 12,000 rpm at 5°C for 10
min. The supernatant was evaporated under reduced pressure to
approximately 2 mL. The concentrate was centrifuged at 12,000
rpm at 5°C for 10 min. The supernatant was evaporated under re-
duced pressure to dryness, and the concentrate was dissolved in
water to 0.2 mL and centrifuged at 12,000 rpm at 5°C for 10 min.
The resulting supernatant (0.02 mL) was analyzed using the
HPLC–PDA–MS system equipped with a COSMOSIL 5PBr column
(4.6 mm × 250 mm). The HPLC mobile phase system-1 was used
for the analysis (see Fig. 6). MS analysis was carried out using ESI
in negative mode. A mixture of the residual pileus (2.0 g) and 10
mM phosphate buffer at pH 7.0 (8.0 mL) was kept at À80 °C, and
its extract solution was prepared and analyzed using the HPLC–
PDA-MS system in the same manner as mentioned above (see
Fig. 6). For identification of trans-3,4-dihydroxycinnamic acid in
HPLC–PDA–MS analysis, a mixture of extract (10 μL), 0.1 mM
trans-3,4-dihydroxycinnamic acid (5 μL), and 0.1 mM trans-2,4-
dihydroxycinnamic acid (2.5 μL) was analyzed.
1H NMR (500 MHz, DMSO-d6, 20°C) δ/ppm 6.46 (1H, d, J = 15.9
Hz), 6.65 (1H, t, J = 7.9 Hz), 6.82 (1H, d, J = 7.9 Hz), 7.03 (1H, d, J =
7.9 Hz), 7.83(1H, d, J = 15.9 Hz), 9.07 (1H, br. s), 9.67 (1H, br.s),
12.20 (1H, br.s) (Fig. S6A); 13C NMR (125 MHz, DMSO-d6, 21°C) δ/
ppm 116.63, 118.12, 118.62, 119.15, 121.49, 139.83, 145.23,
145.60, 168.10(Fig. S6B); ESI-MS m/z: [M – 1]– found 178.92 (calcu-
lated M for C9H8O4 - H: 179.03); Anal. Found: C, 60.39; H, 5.03%, Cal-
culated for C9H8O4: C, 60.00; H, 4.48%.
Compound 2. 89% yield; mp. 220°C decomposition; λmax /nm
(ε) (10 mM phosphate buffer pH 7.0) 220 (24500), 280 (18600); IR
(KBr) νmax 3349, 1659, 1616, 1483, 1433, 1348, 1290, 1253, 1167,
1013 cm–1; 1H NMR (500 MHz, DMSO-d6, 20°C) δ/ppm 6.28 (1H, d,
J = 15.9 Hz), 6.29 (1H, d, J = 2.4 Hz), 6.46 (2H, d, J = 2.4 Hz), 7.38
(1H, d, J = 15.9 Hz), 9.46 (2H, br.s), 12.36 (1H, br.s) (Fig. S7A); 13C
NMR (125 MHz, DMSO-d6, 27 °C) δ/ppm 104.64, 106.11, 118.62,
135.81, 144.45, 158.68, 167.55 (Fig. S7B); ESI-MS m/z: [M – 1]– found
A pileus at stage 1, was divided into three equal parts. Approxi-
mately 30 parts (2.0 g) were then incubated in 0.23 mM 1,2,3–13C3-
trans-3,4-dihydroxycinnamic acid in 10 mM phosphate buffer at
pH 7.0 (8.0 mL) under 18O2 at 25 °C in the dark for 1.5 h and then
the mixture was kept at À80 °C. The residual parts (2.0 g) were kept
in 0.23 mM 1,2,3–13C3-trans-3,4-dihydroxycinnamic acid in 10 mM
phosphate buffer at pH 7.0 (8.0 mL) under nitrogen at 25°C in
the dark for 1.5 h and then the mixture was kept at À80 °C. In
addition, the residual parts (2.0 g) were kept in 8 mL 10 mM phos-
phate buffer (pH 7.0) at À80°C. Extract solutions for these mixtures
were prepared and analyzed in the same manner as mentioned
above (FigS5).
trans-4-Hydroxycinnamic acid and trans-3,4-dihydroxycinnamic
acid were detected at 310 nm and hispidin was detected at 370
nm during HPLC-PDA analysis. The retention time of hispidin in
the HPLC analytical system was approximately 27 min, and the
detection-limit concentrations of hispidin in HPLC–PDA and
HPLC–MS analyses in our analysis method were 100 pmol/1 g
wet gill and 10 pmol/1 g wet gill, respectively.
Synthesis
trans-2,3-Dihydroxycinnamic acid (1) and trans-3,5-
dihydroxycinnamic acid (2). trans-2,3-Dimethoxycinnamic acid
(100 mg, 0.56 mmol) or trans-3,5-dimethoxycinnamic acid (100
mg, 0.56 mmol) was added to 1 M BBr3 in CH2Cl2 (20 mL, 20 mmol)
at À80°C. The reaction mixture was kept at 20°C for 3 h; it was then
poured into water and CH2Cl2 (20 mL) was added to it. The organic
layer was dried over anhydrous Na2SO4 and evaporated. The resi-
due obtained was crystallized with AcOEt to afford the desired
demethylated products as a white solid. Melting points were de-
termined using an ASONE ATM-01 and are uncorrected. UV–vis ab-
sorption and IR spectra were obtained using a V-530 UV-Vis
spectrometer ( JASCO Corp., Tokyo, Japan) and an FT/IR 410 spec-
trometer ( JASCO Corp, Tokyo, Japan), respectively. 1H and 13C
NMR spectra were measured with a JNM-A500 spectrometer. MS
analyses were carried out using a ZQ 4000 MS spectrometer in
ESI negative mode. Elemental analyses were performed with a
Yanaco CHN CORDER MT-3 instrument.
Figure 3. Effect of trans-3,4-dihydroxycinnamic acid on gill bioluminescence of M.
chlorophos at stages 1 (A) and 2 (B). Gills in which the original bioluminescence inten-
sity was approximately 5000 were used for (A) (gills used: n > 16).
Compound 1. 82% yield; mp. 210–212°C; λmax /nm (ε) (10 mM
phosphate buffer pH 7.0) 218 (17300), 278(15900); IR (KBr) νmax
3459, 3282, 1678, 1624, 1588, 1484, 1373, 1333, 1254, 1193 cm–1;
Figure 4. Bioluminescence spectra after the addition of 0.3 mM trans-3,4-
dihydroxycinnamic acid to M. chlorophos gills at stage 1.
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Bioluminescence of Mycena chlorophos
178.92 (calculated M for C9H8O4 - H: 179.03); Anal. Found: C, 60.27;
H, 4.72%, Calculated for C9H8O4: C, 60.00; H, 4.48%.
hydroxycinnamic acid (Fig. 2); the amount of the second
bioluminescence-activating component in the wet gill is men-
tioned in the next section. The second bioluminescence-activating
component (approximately 0.5 mg) was isolated from an aqueous
extract from 5 kg of wet pilei at stage 2 using three-step chroma-
tography (see Experimental and Fig. S1A, B). The high purity of
the isolated bioluminescence-activating component was con-
firmed by analytical HPLC as shown in Fig. S1C.
Results and discussion
Isolation
Recently, we reported a bioluminescence assay of living M.
chlorophos gills at stage 1 in the research of bioluminescence-
activating components (17). In the present study, we found the
presence of a second bioluminescence-activating component in
M. chlorophos gills at stage 2, using the bioluminescence assay
method (see Experimental). HPLC-PDA analysis of an aqueous
extract from the gills at stage 2 showed that the amount of the
second bioluminescence-activating component was lower than
that (4.2 nmol/1 g wet gill used in the present study) of trans-4-
Chemical structure
Negative ESI-MS spectrometry of the second bioluminescence-
activating component indicated a molecular weight of 178.98 [M
1
– 1]– (Fig. S2). H and 13C NMR (Fig. S3) measurements revealed
trans-3,4-dihydroxycinnamic
acid
and
the
HPLC–PDA
Figure 5. Effect of trans-3,4-dihydroxycinnamic acid analogues on the gill bioluminescence of M. chlorophos at stage 1. Gills in which the original bioluminescence intensity was
approximately 5000 were used for this assay ( gills used: n > 8).
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K. Teranishi
Figure 6. HPLC chromatograms of the extracts. (A) Extract (0.1 mL/1 g wet gill, 20 μL) from fresh gills at stage 1. (B) Extract (0.1 mL/1 g wet gill, 20 μL) from gills incubated with
1,2,3–13C3-trans-4-hydroxycinnamic acid under 18O2. (C) Mixture of the extract shown in B (10 μL); 0.1 mM trans-3,4-dihydroxycinnamic acid (5 μL); and 0.1 mM trans-2,4-
dihydroxycinnamic acid (2.5 μL).
chromatogram and PDA spectrum (Fig. S4) of the component
were consistent with those of commercially available trans-3,4-
dihydroxycinnamic acid, indicating that the second
Bioluminescence activation
The addition of 0.01, 0.03, 0.1, and 0.3 mM trans-3,4-
dihydroxycinnamic acid to the living gills at stage 1 promptly
increased the bioluminescence intensity in a concentration-
dependent manner (Fig. 3). The intensity reached a maximum
value in 1 min after the addition. The bioluminescence spectrum,
after activation by the addition of trans-3,4-dihydroxycinnamic
acid, showed a light emission maximum wavelength at approxi-
mately 525 nm, coinciding with the original bioluminescence
spectrum (Fig. 4). The addition of even 1 mM trans-3,4-
dihydroxycinnamic acid to the living gills at stage 2 did not
increase the bioluminescence intensity as well as did trans-4-
hydroxycinnamic acid, as shown in our previous report (17).
Because the contents of trans-4-hydroxycinnamic acid and trans-
3,4-dihydroxycinnamic acid in gills at stage 2 were significantly
higher than those at stage 1, as shown in Figs 2 and 6(A), no influ-
ence of trans-3,4-dihydroxycinnamic acid to the bioluminescence
activities of the gills at stage 2 was attributed to the original
presence of sufficient amount of the bioluminescence-activating
components in the gills.
bioluminescence-activating
component
was
trans-3,4-
dihydroxycinnamic acid. The amount of bioluminescence-
activating component in the aqueous extract from the gills at
stage 2 used in the present study was approximately 1 nmol/1 g
wet gill based on the HPLC–PDA analysis using commercially avail-
able trans-3,4-dihydroxycinnamic acid.
Of the various trans-3,4-dihydroxycinnamic acid analogues
tested for bioluminescence-activating capacity, using the gills at
stage 1 (Fig. 5), there was no compound that possessed
bioluminescence-activating capacity. Based on the results pre-
sented in Fig. 5 and the previous result, showing that trans-3-
hydroxycinnamic acid had no bioluminescence-activating capacity
(17), trans-alkene and 4-hydroxy groups were necessary for the ac-
tivation of gill bioluminescence, and the 3-hydroxy group in trans-
3,4-dihydroxycinnamic acid reinforced the bioluminescence-
activating capacity of the trans-4-hydroxycinnamic acid molecule.
Figure 7. Negative ESI-MS spectrum of trans-3,4-dihydroxycinnamic acid in the extract
from the gills incubated with 1,2,3–13C3-trans-4-hydroxycinnamic acid under 18O2.
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Bioluminescence of Mycena chlorophos
3-Methoxy; 3,5-dihydroxy; and 2-hydroxy groups substituted in
trans-4-hydroxycinnamic acid completely inhibited its activating
capacity, suggesting that the hydroxyl groups substituted on the
phenyl skeleton of trans-cinnamic acid are strongly recognized
by the bioluminescence system and that trans-4-hydroxycinnamic
acid and trans-3,4-dihydroxycinnamic acid affect the biolumines-
cence system in a highly structure-specific way.
Conclusion
We found a second bioluminescence-activating component, trans-
3,4-dihydroxycinnamic acid in M. chlorophos gills at stage 2. This
component promptly and highly structure-specifically increased
the bioluminescence intensity of the living gills at stage 1.
Moreover, incubation experiments revealed that this component
was synthesized from trans-4-hydroxycinnamic acid in the gills as
well as by the general metabolism and that hispidin (detection-
limit concentration: 10 pmol/1 g wet gill) was not accumulated
via the biosynthesis from trans-3,4-dihydroxycinnamic acid in the
gills in the presence or absence of molecular oxygen. Addition of
0.01 mM hispidin to the living gills at stage 1 generated no biolu-
minescence activation. These results suggested that the prompt
bioluminescence activation resulting from addition of trans-3,4-
dihydroxycinnamic acid could not be attributed to the generation
of hispidin. We are now aiming to elucidate the mechanisms of
bioluminescence activation by trans-3,4-dihydroxycinnamic acid
and trans-4-hydroxycinnamic acid to chemically understand the
bioluminescence phenomenon.
Metabolism
Hispidin is known to be biosynthesized from trans-3,4-
dihydroxycinnamic acid, which is generated from trans-4-
hydroxycinnamic acid in the fungus, Polyporus hispidus (19–21).
In the present study, biosynthesis of trans-3,4-dihydroxycinnamic
acid in M. chlorophos was investigated using the fresh gills at stage
1 with labelled 1,2,3–13C3-trans-4-hydroxycinnamic acid (found m/z
166.13 [M – 1]–) and 18O2. As shown in Fig. 6(A), there was a trace of
trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic
acid in the gills at stage 1. The gills were incubated with 0.23
mM 1,2,3–13C3-trans-4-hydroxycinnamic acid under 18O2 at 25 °C
for 1.5 h in the dark, and the labeled products were analyzed by
HPLC–PDA–MS spectrometry. It was found that the addition of
13C labelled trans-4-hydroxycinnamic acid to the gills at stage 1 in-
creased the detected amount of trans-3,4-dihydroxycinnamic acid
at first (Fig. 6B) and that it carried a significant amount of the
18O label (m/z 182.10 [M-1]–/184.11 [M – 1]– = 1.4:4.5) (Fig. 7).
These results indicated that trans-3,4-dihydroxycinnamic acid
was synthesized from 1,2,3–13C3-trans-4-hydroxycinnamic acid
with molecular oxygen. In contrast, trans-2,4-dihydroxycinnamic
acid, which was expected as an oxidation product of trans-4-
hydroxycinnamic acid, was not generated (Fig. 6B, C). This result
was not inconsistent with the result that trans-2,4-
dihydroxycinnamic acid was not detected in the extract mixture
analyzed by HPLC shown in Fig. 2. In addition, hispidin was not
detected in the extract of the incubated gills: detection-limit
concentrations of hispidin in HPLC–PDA and HPLC–MS analyses
in our analysis method were 100 pmol/1 g wet gill and 10
pmol/1 g wet gill, respectively.
Acknowledgements
We are grateful to Mr Morizono Toshihiro, Iwade Research Institute
of Mycology Co., Ltd, for the cultivation of M. chlorophos.
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