Research article

Received: 12 February 2016,

Revised: 25 February 2016,

Accepted: 26 February 2016

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.3129

Identification of possible light emitters in the

gills of a bioluminescent fungus Mycena
chlorophos
Katsunori Teranishi*
ABSTRACT: The pileus of Mycena chlorophos actively, spontaneously, and continuously emits green light. Molecular mechanisms underlying this bioluminescence remain unclear. We investigated light emitters in the pileus of M. chlorophos to
determine the underlying mechanisms. High-performance liquid chromatographyfluorescencephotodiode arraymass
detection analyses showed that actively luminescent gills in the pileus exclusively and abundantly possessed riboflavin,
riboflavin 5-monophosphate, and flavin adenine dinucleotide as green-fluorescent components. These components were
localized in the bioluminescent region of the gills at the microscopic level. Fluorescence spectra of these green-fluorescent
components and the gills were identical with the spectrum of gill bioluminescence (maximum emission wavelength,
525 nm). Thus, our results indicated that the possible light emitters in the pileus of M. chlorophos were riboflavin, riboflavin
5-monophosphate, and/or flavin adenine dinucleotide. Copyright 2016 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publishers web site.
Keywords: Bioluminescence; fungus; Mycena chlorophos; mushroom

Introduction
Bioluminescence, visible light emission by living organisms is a
widespread phenomenon in nature and has drawn much
attention. Fungal bioluminescence is widely found on land
environments. Chew et al. reported that the total number of documented species of luminous fungi in 2014 was 77 (1). All known
luminous fungi are saprotrophic and belong to the Agaricales
lineage of Basidiomycota (2). Fungal bioluminescence, which
varies among species, occurs in the basidiocarp, and/or mycelia
(24). Fungi continuously emit light only during a certain period
of their growth with maximum light intensity in the range of
520530 nm (2,57).
Molecular studies on bioluminescence have focused on substances serving as energy sources, enzymes catalyzing bioluminescence reactions, and light emitters. With respect to the light
emitters in fungal bioluminescence, Endo et al. isolated a fluorescent compound ergosta-4,6,8(14),22-en-3-one from the fruiting
bodies of a luminous fungus Lampteromyces japonicus whose fluorescence emission peak was nearly identical to that of its bioluminescence emission peak (maximum emission at 524 nm) (8). Isobe
et al. isolated a fluorescent flavin compound called lampteroflavin
from the fruiting bodies of L. japonicus and concluded that it was
the light emitter in the bioluminescence of L. japonicus based on
the similarities between its fluorescence and bioluminescence
spectra (9). Although results obtained by OKane et al. did not support those reported by Isobe et al., these results did not exclude
the role of lampteroflavin in bioluminescence (10).
Mycena chlorophos, which was used in the present study, is a
species of molecular oxygen-dependent bioluminescent fungus
that is primarily found in Southeast Asia (11). In our cultivation
system, young fruiting bodies grew, the pileus unexpanded, and
the stipe began to elongate at 20 C and at approximately 90% relative humidity (Fig. 1, stage I). Subsequently, the pileus expanded

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to a flat disc, and bright green light was continuously emitted from
the whole pileus and was continuously emitted for approximately
1 day at 20 C and at approximately 90% relative humidity (Fig. 1,
stage II). The bioluminescence intensity gradually decreased while
the white fruiting body turned brown (Fig. 1, stage III), and finally
decayed without luminescence. No light was emitted from the
stipe during any of the growth stages. Light emitted by mycelia
grown on a solid medium was very weak.
Hayashi et al. extracted and purified a luminiferous flavin compound, which was not riboflavin, using high-performance liquid
chromatography (HPLC) from the extracts of fruiting bodies of M.
chlorophos; however, other green-fluorescent components were
not detected (12). They proposed that this flavin compound was
responsible for the M. chlorophos bioluminescence. However, the
chemical structure of this flavin compound is unknown. Recently,
Purtov et al. reported that adding hispidin to a mixture of NADPH
and cold water-extract solution prepared from the bioluminescent
fruiting body of M. chlorophos strongly increased the luminescence (13). However, they did not describe that hispidin was the
luciferin precursor or luciferin for the bioluminescence of the M.
chlorophos fruiting body and that hispidin was detected in the
fruiting bodies of M. chlorophos. In their report, it was shown that
the luminescence spectrum of the in vitro mixture was identical

* Correspondence to: K. Teranishi, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie, 514-8507 Japan. E-mail: teranisi@bio.
mie-u.ac.jp
Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu,
Mie 514-8507, Japan
Abbreviations: CCD, charge coupled device; FAD, flavin adenine dinucleotide
disodium; HPLC, high-performance liquid chromatography; PDA, photodiode
array

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K. Teranishi

Figure 1. Bioluminescence of one fruiting body of M. chlorophos at each growth stage over time. 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. These results are cited from (15).

to the fluorescence emission spectrum of hispidin in methanol, but

the interpretation on identification of these spectra was not
explained. At present, little chemical information is available
concerning the bioluminescence of M. chlorophos. At present, no
information is available concerning the light emission of M.
chlorophos. Here we report identification of possible light emitters
in the M. chlorophos pilei.

Tesque, Inc.) for analysis were used, except for HPLCfluorescence

analysis 1 in section Analysis of fluorescent components. Mass
analysis was done using a ZQ 4000 mass spectrometer (Waters
Corporation, Milford, MA, USA) in an electrospray ionization (ESI)positive or an ESI-negative mode. Bioluminescence intensity was
measured using a Microplate Luminometer Centro (Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany).

Experimental

Fruiting bodies of M. chlorophos and chemicals

Instruments
Fluorescence and bioluminescence spectra were measured using
an FP-6600 spectrofluorometer ( JASCO Corp., Tokyo, Japan) at
20 C. The excitation wavelength (bandwidth, 10 nm) was 450 nm,
and the emission bandwidth was 10 nm. Micrographs of fluorescence with blue excitation and bioluminescence in the dark from
the surface of fresh gills of M. chlorophos were obtained using a microscope (Axiovert 200; Carl Zeiss Co., Ltd, Oberkochen, Germany)
equipped with an objective lens (Plan-Apochromat 20/0.75; Carl
Zeiss Co., Ltd) and a monochrome charge coupled device (CCD)
camera (AxioCam MRm; Carl Zeiss Co., Ltd) at 20 C and approximately 80% relative humidity. Photons of fluorescence and bioluminescence were counted for 0.03 and 60 sex, respectively, in a
1 1 binning mode. Images were acquired using AxioVision 4.8
software (Carl Zeiss Co., Ltd.). Filter set 17 (excitation bandpass,
485/20 nm; beam splitter, ft510 nm; and emission bandpass,
515565 nm; Carl Zeiss Co., Ltd.) was used for fluorescence imaging. Isolation and analysis of components was performed using
an HPLCphotodiode array (PDA)fluorescence system with a
MD-910 detector ( JASCO Corp., Tokyo, Japan) and a FP-1520
fluorescence detector ( JASCO Corp.). A Cosmosil 5C18-PAQ
column (20 250 mm; Nacalai Tesque, Inc., Kyoto, Japan) for isolation and a Cosmosil 5C18-PAQ column (4.6 250 mm; Nacalai

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A mycelium strain of M. chlorophos (Berkeley & M.A. Curtis) Saccardo

was purchased from the National Institute of Technology and Evaluations Biological Resource Center, Japan. Fruiting bodies of M.
chlorophos were cultivated from the mycelia at 20 C and at approximately 90% relative humidity in the dark, as described previously
(14). The pilei were carefully harvested and were immediately used
for various experiments or were stored in liquid nitrogen for extraction of fluorescent components. trans-p-Hydroxycinnamic acid was
purchased from TCI Chemicals (Tokyo, Japan). Other chemicals were
purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan).
Extraction of green-fluorescent components
Extraction method 1. Fresh gills (0.2 g) at three growth stages, i.e.,
unexpanded pileus and weak bioluminescence (stage I), expanded
pileus and active light emission (stage II), and collapsed pileus and
weak bioluminescence (stage III), were obtained from a large pileus of M. chlorophos. The gills were stored in liquid nitrogen and
were homogenized with 0.2 mL of 0.1 M phosphate buffer
(pH = 7.0) using a Vibra-Cell ultrasonic disintegrator (Sonics &
Materials Inc., Newtown, CT, USA) in an ice bath for 5 sec, and were
centrifuged at 12,000 rpm and 0 C for 10 min. After separating the
supernatant, the pellet was extracted four times with 0.2 mL of
0.1 M phosphate buffer (pH = 7.0). Supernatant obtained after each

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Bioluminescence of Mycena chlorophos

extraction was stored at 80 C until used for HPLCfluorescence
analysis. Concentrations of green-fluorescent components were
measured using the supernatant obtained after each extraction.
This extraction process was performed for five pilei.
Actively luminescent gills (30 g) were
obtained from the large pilei of M. chlorophos, stored in liquid
nitrogen, and homogenized with 30 mL water using an ultrasonic
disintegrator in an ice bath for 30 sec. The homogenate was centrifuged at 12,000 rpm and 0 C for 20 min. After separating the
supernatant, the pellet was homogenized in 30 mL water using
an ultrasonic disintegrator in an ice bath for 30 sec and was centrifuged at 12,000 rpm and 0 C for 10 min. The supernatants were
combined and were mixed with 180 mL methanol. The suspension
was centrifuged at 12,000 rpm and 0 C for 10 min, and the supernatant was separated and evaporated under reduced pressure to
60 mL. The resultant solution was washed with 15 mL CH2Cl2,
and water extract was centrifuged at 12,000 rpm and 0 C for
5 min. The supernatant was evaporated under reduced pressure
to 15 mL and was stored at 80 C until used for isolating fluorescent components and HPLCPDAmass analysis.
Extraction method 2.

Isolation of fluorescent components

The extract solution obtained using extraction method 2 was centrifuged at 12000 rpm and 5 C for 10 min. The supernatant was
separated and filtered through a 0.45-m membrane filter. The
filtrate (3 mL) was purified using the HPLCPDAfluorescence system. The mobile phase was a mixture of (A) aqueous 2 mM phosphate buffer (pH = 7.0) and (B) methanol. A linear gradient was
achieved starting with 0% of B and reaching 100% of B in 60 min
at room temperature and at a flow rate of 5 mL/min. Fluorescent
components were eluted between 20 and 25 min and between
39 and 41 min. HPLC was also performed for 12 mL of the extract
solution. The fractions eluted between 39 and 41 min were evaporated under reduced pressure to 0.6 mL and were used for measuring fluorescence spectra. The fractions eluted between 20 and
25 min were evaporated under reduced pressure to 4 mL, and
the resulting solution was filtered through a 0.45-m membrane
filter. The filtrate was purified using an HPLCPDAfluorescence
system. The mobile phase was a mixture of (A) aqueous 10 mM
phosphate buffer (pH 7.0) and (B) methanol. A linear gradient
was achieved starting with 15% of B and reaching 40% of B in
60 min at room temperature and at a flow rate of 5 mL/min.
Fluorescent components were eluted between 21 and 22 min
and between 22 and 23 min. The eluted fractions were evaporated
under reduced pressure to 0.1 mL and were used to measure the
fluorescence spectra.

using extraction method 1, an HPLCfluorescence system was

used at room temperature. The following two mobile systems
were used at a flow rate of 0.8 mL/min. Mobile system 1: mixture
of (A) aqueous 10 mM phosphate buffer (pH = 7.0) and (B)
methanol; linear gradient was achieved starting with 15% of B
and reaching 40% of B in 30 min. Mobile system 2: mixture of (A)
aqueous 0.1% (v/v) trifluoroacetic acid solution and (B) 0.1% (v/v)
trifluoroacetic acid methanol solution; linear gradient was
achieved starting with 20% B and reaching 45% B in 30 min.
HPLCPDAmass analysis. To identify the fluorescent components in the extract solution obtained using extraction method 2,
we used an HPLCPDAmass system. The mobile system 2 mentioned above was used as the mobile phase. Injection sample
was prepared as follows: 2 mL extract solution obtained using
extraction method 2 was filtered through a 0.45-m membrane filter, and the filtrate was concentrated under reduced pressure to
0.1 mL. The resultant solution (0.02 mL) was injected into the HPLC
column. Commercially available riboflavin 5-monophosphate sodium salt (FMN-Na, 1 mM), flavin adenine dinucleotide disodium
salt (FAD-Na2, 1 mM), and riboflavin solution (1 mM) in 0.1 M phosphate buffer (pH = 7.0) were used as standard compounds.
Fluorescent components were identified using UVvis absorption
spectra obtained by PDA detection and mass analysis.

Concentrations of fluorescent components in the extract solutions prepared using extraction method 1 were quantified using HPLCfluorescence
analysis 2 with mobile system 1. Calibration curves of FMN, FAD,
and riboflavin were used as standards. Their concentrations in
the gills were expressed as mean standard deviation (SD) of five
independent measurements and as nmol of fluorescent component equivalents per 1 g of fresh wet gills.

Quantification of fluorescent components.

Measurement of the bioluminescence activity of the living

gills
The gill bioluminescence activity of the living gills was measured
by the following method. Phosphate buffer (10 mM, pH = 7.0;
3 L) and gill sections (2 mm 2 mm) of M. chlorophos at stage I
were placed in a black 96-well plate (96/V-PP, Eppendorf AG,
Hamburg, Germany). The bioluminescence intensity was measured every 1 min for approximately 20 min at 25 C to confirm
the steadiness of bioluminescence intensity. Then, 10 L of
0.3 mM trans-p-hydroxycinnamic acid in 10 mM phosphate buffer
(pH = 7.0) was added to the wells, and bioluminescence intensity
was measured every 1 min for 20 min at 25 C. At each time point,
the intensities were accumulated for 1 sec. The bioluminescence
intensities are provided as mean of independent measurements
(n = 4).

Analysis of fluorescent components

HPLCfluorescence analysis 1.

Results and discussion

HPLCfluorescence analysis 2. To identify and quantify the fluorescent components in the extract solutions (0.02 mL) obtained

Although the whole pileus of M. chlorophos was actively bioluminescent when visually observed (Fig. 1; stage II), the bioluminescence region was localized in the microscopic level (Fig. 2b) as
previously reported (15). The green-fluorescent region was
localized in the bioluminescent region (Fig. 2c). The shape and
maximum wavelength of bioluminescence spectra of the gills
were almost the same as those of fluorescence spectra on the
surface of the gills (Fig. 3). These results indicated that the greenfluorescent components were abundant and served as light
emitters for bioluminescence in the gills. Next, we aimed to isolate

To preliminarily detect the fluorescent components in the extract solutions (0.02 mL) obtained using
extraction method 1, we used an HPLCfluorescence system with a
Cosmosil Protein-R column (4.6 250 mm; Nacalai Tesque, Inc.).
The mobile phase was a mixture of (A) aqueous 10 mM phosphate
buffer (pH = 7.0) and (B) methanol. A linear gradient was achieved
starting with 0% of B and reaching 100% of B in 30 min and subsequently 100% of B in 10 min at room temperature and at a flow
rate of 0.8 mL/min.

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K. Teranishi

Figure 2. Micrographs of the surface of fresh gills of actively bioluminescent M.

chlorophos. (a) Under white light, (b) bioluminescence in the dark, and (c) green
fluorescence after B-excitation. Imaging conditions are described in the Experimental
section.

Figure 3. Fluorescence and bioluminescence spectra of fresh gills of actively bioluminescent M. chlorophos. Measurement conditions are described in the Experimental
section.

and chemically identify the possible light emitters involved in the

bioluminescence of M. chlorophos.
Fluorescent components were extracted from actively bioluminescent gills using extraction method 1. The extracted fluorescent
components were analyzed by performing HPLCfluorescence
analysis 1 with a Protein-R column for analyzing proteins and
low-molecular-weight compounds (see Experimental section).
The analysis showed three peaks for fluorescent components
(Fig. 4a). These peaks were effectively separated by performing
HPLCfluorescence analysis 2 using Cosmosil 5C18-PAQ column
for low-molecular-weight compounds and mobile system 1

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Figure 4. HPLCfluorescence chromatograms of the extract of fresh gills of actively

bioluminescent M. chlorophos, FMN, FAD, and riboflavin. (a) HPLCfluorescence analysis 1 of the extract; (b) HPLCfluorescence analysis 2 of the extract using mobile system 1; (c) HPLCfluorescence analysis 2 of FMN, FAD, and riboflavin using mobile
system 1; (d) HPLCfluorescence analysis 2 of the extract using mobile system 2; (e)
HPLCfluorescence analysis 2 of FMN, FAD, and riboflavin using mobile system 2.
The extract was prepared using extraction method 1. The chromatograms shown
are representative. HPLC conditions are described in the Experimental section.

(Fig. 4b). The retention times of peaks A, B, and C (indicated in

Fig. 4b) were the same as those of FMN, FAD, and riboflavin, respectively (Fig. 4c). HPLCfluorescence analysis 2 of a mixture of
extract solution, FMN, FAD, and riboflavin showed that peaks A,
B, and C were identical to the peaks of FMN, FAD, and riboflavin,
respectively. In addition, HPLCfluorescence analysis 2 of the extract solution with the mobile system 2 (see Experimental section)
provided three peaks for fluorescent components (Fig. 4d). The

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Bioluminescence of Mycena chlorophos

elution times of peaks A, B, and C were identical to those of FMN,
FAD, and riboflavin, respectively (Fig. 4e), suggesting that peaks A,
B, and C corresponded to FMN, FAD, and riboflavin, respectively.
To reliably identify the fluorescent components corresponding
to peaks A, B, and C in the chromatograms in Fig. 4, UVvis absorption and mass spectra of these components were measured.
Because extraction method 1 produced only small amounts of
these components, high concentrations of these components required for measuring their spectra were obtained using extraction
method 2 (see Experimental section). UVvis absorption and mass
spectra were obtained by performing HPLCPDA-mass analysis
(see Experimental section) using FMN, FAD, and riboflavin as the
standards (Fig. S1, Supporting information). The shapes of the
UVvis absorption spectra of peaks A, B, and C obtained using
HPLCPDA analysis were identical to those of FMN, FAD, and
riboflavin (Fig. 5). Mass spectra showed that peaks A, B, and C
had molecular weights of m/z 457 (M + 1)+, m/z 786 (M + 1)+, and
m/z 377 (M + 1)+, respectively (Fig. S2, Supporting information).
These results indicated that the peaks A, B, and C corresponded
to FMN, FAD, and riboflavin, respectively. However, the unknown

fluorescent compound detected in M. chlorophos fruiting bodies

by Hayashi et al. (12) and lampteroflavin detected in L. japonicus
fruiting bodies by Isobe et al. (9) were not detected in the present
study.
Fluorescence spectra of the fluorescent components corresponding to peaks A, B, and C in the chromatograms shown in
Fig. 4 were measured using fluorescent compounds isolated from
the extract. The shapes and maximum wavelengths of fluorescence spectra were the same as those of FMN, FAD, and riboflavin
(Fig. 6). These data supported that findings that the fluorescence
components were FMN, FAD, and riboflavin. Moreover, the fluorescence spectra were identical to the bioluminescence spectra of the
gills shown in Fig. 3. Therefore, we concluded that the light emitters in M. chlorophos gills were FMN, FAD, and/or riboflavin.
FMN, FAD, and riboflavin in each extract prepared using extraction method 1 were quantified by performing HPLCfluorescence
analysis 2 with mobile system 1 (see Experimental section). Highest
concentration of FMN, FAD, and riboflavin were effectively extracted during the fifth extraction (Fig. S3 Supporting information).
The resulting residues after the fifth extraction showed no green

Figure 5. UVvis absorption spectra of peaks A, B, and C; FMN; FAD; and riboflavin.
The spectra were obtained by performing HPLCPDA analysis. HPLC conditions are
described in the Experimental section.

Figure 6. Fluorescence spectra of the isolated fluorescent compounds A, B, and C

corresponding to peaks A, B, and C in Fig. 4 and FMN, FAD, and riboflavin. Measurement method is described in the Experimental section.

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K. Teranishi
fluorescence, indicating that almost of the green-fluorescent
components were efficiently extracted. Concentrations of FMN,
FAD, and riboflavin in the five extracts of each gill sample were
measured, and total concentrations were expressed as nmol of
fluorescent component equivalents per 1 g of fresh wet gills.
Figure 7 shows the concentrations of FMN, FAD, and riboflavin in
the gills during the three growth stages. Although concentrations
of FMN, FAD, and riboflavin were abundant in the gills during all
the three growth stages, their concentrations were higher at stage
II than those during other growth stages. When the pileus
collapsed after active bioluminescence, the concentrations of
these fluorescent components decreased.
In recent times, we found that a component, transp-hydroxycinnamic acid, in the gills of a bioluminescent fungus
M. chlorophos increased the bioluminescence activity of the gills
at stage I, whereby the original bioluminescence capacity was approximately 1% compared with that of the gills at stage II (16).
However, even when a sufficient amount of transp-hydroxycinnamic acid was added to the gills, the resulting
bioluminescence capacity was approximately 12% of that at
stage II (16). This result suggests that the bioluminescence activity must be regulated by trans-p-hydroxycinnamic acid and other
components. In the present study, we hypothesized that one of
the other components was a green-fluorescent flavin component
serving as the light emitter. We sometimes encountered a

situation in which the gills at stage I exhibited bioluminescence

activity that was not significantly increased even by the addition
of trans-p-hydroxycinnamic acid. Using a microscope, we observed
the intensity and localization of green fluorescence of the gills at
stage I, in which bioluminescence activity was increased by the
addition of trans-p-hydroxycinnamic acid, as shown in the graph
in Fig. 8 line A and the micrographs (Fig. 9a-1, a-2). We observed
other instances in which the bioluminescence intensity was not
increased despite this addition, as shown in the graph Fig. 8 line
B and the micrographs (Fig. 9b-1, b-2. The gills of the former had
a large amount of green-fluorescent basidia cells (Fig. , 9a-2),
whereas those in the latter had little green-fluorescent basidia cells
(Fig. 9b-2). These findings indicated that the amount of greenfluorescent component regulated the bioluminescence activity in
the presence of trans-p-hydroxycinnamic acid. In particular, it

Figure 7. Concentrations of FMN, FAD, and riboflavin in the gills of M. chlorophos

during the three growth stages. The concentrations of these components in the gills
are expressed as mean standard deviation (SD) of five independent measurements.
The measurement procedure is described in the Experimental section.

Figure 8. Effect of trans-p-hydroxycinnamic acid addition on the bioluminescence

intensity of gills at stage I. Line A: a gill in which bioluminescence activity was increased by the addition of 0.3 mM trans-p-hydroxycinnamic acid. Line B: a gill in which
bioluminescence activity was not increased despite this addition. The measurement
method for the bioluminescence intensity is described in the Experimental section.

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Figure 9. Green fluorescence from the fresh gills surface of M. chlorophos fruiting bodies
at stage I. The bioluminescence activity of the gills in the (a) micrograph was increased by
the addition of trans-p-hydroxycinnamic acid, as shown in Fig. 8 (line A), and the bioluminescence activity of the gills in the (b) micrograph was not increased despite this addition,
as shown in Fig. 8 (line B). (a-1) The gill surface under white light. (a-2) The gill surface in
the dark after blue excitation. (b-1) The gill surface under white light. (b-2) The gill surface
in the dark after blue excitation. Imaging conditions are described in the Experimental
section. These gills are representative of some gills. Bioluminescence imaging of these gills
could not be performed because of their insufficient bioluminescence intensity.

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Bioluminescence of Mycena chlorophos

indicated that, along with the growth of the fruiting body, the
green-fluorescent components, riboflavin, FMN, and/or FAD, as
light emitters, should be included in the bioluminescence system
in the basidia cells and that trans-p-hydroxycinnamic acid should
be largely biosynthesized to contribute to the bioluminescence
reaction.
Isobe et al. concluded that the light emitter in the bioluminescence of L. japonicus was riboflavin because it was the major
green-fluorescent compound extracted from the fungus and because its fluorescence spectrum was similar to its bioluminescence
spectrum (17). In further studies, however, they reported that the
true light emitter was not riboflavin but was lampteroflavin (9). In
their experiments, most riboflavin was produced as an artifact
during extraction at 3 C for 5 days and lampteroflavin was obtained by extraction at 20 C for 10 h. In the present study, riboflavin, FMN, and FAD were exclusively detected as green-fluorescent
components during the first extraction process at 0 C for approximately 11 min (see the Experimental section). In addition, these
fluorescent components did not decompose because artifacts
were not formed in the homogenate of fresh gills after extraction
at 0 C for 1 h. These results indicated that our experimental procedures were appropriate and led us to conclude that the light emitters involved in the bioluminescence of M. chlorophos gill were
riboflavin, FMN, and/or FAD, whereas we could not determine
which of these fluorescent components emitted light from the
bioluminescent region in the gills. It is possible that a flavin chromophore accepts energy produced by an energy source and
subsequently emits light, similar to that observed with aequorin
and green-fluorescent protein in luminous jellyfish Aequorea (18).
If this assumption is correct, it is likely that the flavin molecule is
very close to the energy source in the bioluminescence system.
Although flavin components were detected in their free forms in
the present study, these components may not exist in their free
forms in the living tissue of luminous gills (such as flavincontaining protein) to effectively accept energy. The fluorescent
components identified in this study could be used to gain more
insights into the bioluminescence system and to study molecular
mechanisms underlying the bioluminescence of M. chlorophos.

Conclusions
The present study identified riboflavin, FMN, and/or FAD as the
possible light emitters in the bioluminescence system in M.
chlorophos gills based on the results of microscopic, HPLCfluorescencePDAmass detection, bioluminescence spectroscopic, and
fluorescence spectroscopic analyses. Along with the growth of
fruiting body, the green-fluorescent component(s) should be included in the bioluminescence system. However, the mechanisms
underlying energy excitation of light emitters to generate light are
still unknown. We are now aiming to identify energy source(s) and
reaction(s) involved in the energy generation for determining
molecular mechanisms underlying the bioluminescence of M.
chlorophos.

Luminescence 2016

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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