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Expression of tyrosinase genes associated with fruiting body formation and pigmentation in Grifola frondosa
Mycoscience 60 (2019) 262e269
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Expression of tyrosinase genes associated with fruiting body formation and pigmentation in Grifola frondosa Nobuhisa Kawaguchi a, Mirai Hayashi b, Shota Nakano b, Norihiro Shimomura b, Takeshi Yamaguchi b, Tadanori Aimi b, * a b
Laboratory, Biological Business Department, Ichimasa Kamaboko Co Ltd., 77-1 Junishin, Agano-shi, Niigata, 959-1936, Japan Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori, 680-8553, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 December 2018 Received in revised form 2 April 2019 Accepted 2 April 2019 Available online 11 April 2019
Chemical and physical properties (including ultraviolet spectrum) of a brown pigment isolated from Grifola frondosa fruiting bodies were almost identical to those of fungal melanin. From the full genome sequence of G. frondosa, genes involved in the melanin biosynthesis pathway of this polypore mushroom were identified, including tyrosinases (tyr1 and tyr2). Transcriptional analyses showed dramatic changes only in the expression of tyr2 in the primordial stage. Immuno-electron microscopy using anti-tyrosinase antibodies showed that TYR2 localizes in the cell walls of primordia. Therefore, TYR2 may be closely associated with melanin biosynthesis in this polypore mushroom G. frondosa, and melanin might be produced in the cell wall. © 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Albino Cell wall Immuno-electron microscopy Melanin
1. Introduction The fruiting bodies of mushrooms are very colorful. However, in some typical edible mushrooms, such as Lentinula edodes (shiitake), Pholiota microspora (nameko), Grifola frondosa (maitake), and Hypsizygus tessellatus (bunashimeji), the fruiting bodies of the wild-type strain are black or brown (Hongo, Ueda, & Izawa, 1994, p. 384; Imazeki, Otani, & Hongo, 1988, p. 623). In general, the brown pigment in the pileus of mushrooms is often melanin. Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents, and ionizing radiation (Eisenman & Casadevall, 2012). Fruiting body development is initiated within the tree rootstock and extends to the external environment. The primordia and young fruiting body tissues are then exposed to sunlight, resulting in the production of melanin to protect the intracellular DNA from mutation by UV light. Therefore, it is strongly suggested that pigment production is closely linked to fruiting body development. Grifola frondosa, a white-rot fungus widely distributed in Asia, North America, and Europe, is a pleasant-tasting edible mushroom.
* Corresponding author. E-mail address: [email protected] (T. Aimi).
Irradiation with visible light is required for the initiation of pileus formation in artificially cultivated G. frondosa. When a dark-grown vegetative mycelium is exposed to light, the buff-brown color of the surrounding spawn surface is induced, and subsequently, the primordia form. Therefore, it is possible that the color of mycelia is closely associated with the initiation of primordia formation and could be a trigger for fruiting body formation. However, neither the identity of the brown pigment nor its synthesis pathway in G. frondosa has been reported. A similar phenomenon was observed in Polyporus arcuraius. The brown pigmentation of mycelia in P. arcularius occurred only in a dikaryotic strain grown under visible light before development of primordia and did not occur in a monokaryon strain, even when grown under visible light. Moreover, pigmentation of the dikaryotic mycelia did not occur when grown in the dark. Reverse transcription PCR (RT-PCR) products of tyrosinase 1 gene (tyr1) in dikaryon and monokaryon P. arcularius could not be detected without exposing the strains to visible light, and the amplification products disappeared when the strains were transferred back to the dark (Kanda et al., 2007). These data suggest that exposure to visible light, tyrosinase gene induction, brown pigment production, and primordia formation are closely linked. However, there is no direct evidence that the brown pigment is melanin, and the relationship between fruiting and tyrosinase gene expression in both P. arcularius and G. frondosa is unclear.
https://doi.org/10.1016/j.myc.2019.04.003 1340-3540/© 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
N. Kawaguchi et al. / Mycoscience 60 (2019) 262e269
To elucidate the molecular mechanism of pigment formation in G. frondosa, several genes potentially related to brown color formation were identified from genome sequence databases, such as genes encoding tyrosinases (TYR1 and TYR2). Expression of the two genes was investigated in the different stages of fruiting body development. The primary goal of this study was to enhance understanding of the factors that trigger the initiation of fruiting body formation in “polypore mushrooms.” 2. Materials and methods 2.1. Strains and culture conditions The wild-type dikaryotic strain IM-BM11 and albino dikaryotic strain IM-WM1 were obtained from Ichimasa Kamaboko Co., Ltd (Agano-shi, Niigata, Japan). The albino monokaryotic strain IMWM1-25 was obtained by basidiospore isolation of the fruiting bodies of IM-WM1. The wild-type monokaryotic strain IM-BM11P21 was derived from regenerated protoplasts of wild-type dikaryotic strain IM-BM11. Grifola frondosa dikaryotic strains were cultivated on a sawdust substrate, consisting of sawdust and rice bran at a volumetric ratio of 10:1. The moisture content of the substrate was adjusted to 65%. All ingredients were combined and mixed, and 500 g of the prepared substrate was bagged into high-density polyethylene bags (SE-25ES; Sakato Sangyo Co., Ltd., Minakami-machi, Tone-gun, Gunma, Japan) sealed with a heat sealer, sterilized by autoclaving (121 C, 60 min), cooled, and then inoculated with mycelia. The substrate was incubated at 25 C in the dark for 40 d to allow the substrate to become fully colonized by the mycelia (this process was designated “spawn running”). After spawn running, the fully colonized substrate was irradiated with 200 lux visible light for 1 wk at 25 C (stage 1). The substrate was then incubated at 15 C under 200 lux visible light until fruiting body maturation. The polyethylene bags were opened when the mycelium had turned brown (stage 2), and the moisture was maintained at 90% to initiate primordia formation. RNA was isolated from mycelia in the sawdust substrate at stage 1 (mycelial stage) and stage 2 (colored mycelial stage). We also isolated RNA from primordia and fruiting body tissues at different stages. 2.2. DNA preparation and genome sequencing Genomic DNA was extracted according to the method described by Dellaporta, Wood, and Hicks (1983). The complete nucleotide sequence of the genomic DNA of albino monokaryotic strain WM125 was determined using Illumina HiSeq 2000 paired-end technology (Hokkaido System Science Co., Ltd., Sapporo, Hokkaido, Japan). This sequencing run yielded 34,502,348 high-quality filtered reads with 101-bp paired-end sequencing. The genomic sequence was assembled using Velvet Assembler version 1.1.02 (hash length, 75 bp). The final assembly contained 7354 contigs of a total length of 35,343,167 bp, with an n50 length of 94,048 bp. 2.3. RNA preparation and cDNA synthesis RNA was extracted using a MagExtractor kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. cDNA was synthesized with total RNA as the template using ReverTraAce qPCR RT Master Mix with a gDNA Remover kit (Toyobo). Amplification of cDNA fragments and 30 -rapid amplification of cDNA ends (RACE) were performed using a Takara RNA PCR (AMV) version 3.0 kit (Takara Bio, Kusatsu, Shiga, Japan) and 50 -RACE with a 50 -Full RACE Core Set (Takara Bio). PCR was carried out according to the kit manufacturer’s instructions using the oligonucleotide
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primers listed in Table 1. The amplified fragments were subcloned into a pMD20 T-vector (Takara Bio) and sequenced Table 2. 2.4. Identification of genes associated with melanin synthesis within the genome of G. frondosa In order to design nucleotide primers for quantitative RT-PCR, nucleotide sequences of tyrosinase, tyrosinase-related protein, and polyketide synthase (PKS) genes of G. frondosa were identified from the genome sequence data of strain IM-WM1-25. Sequences exhibiting homology with protein sequences of L. edodes tyrosinase (BAB71736.1) were searched using the tblastn program (Altschul, Gish, Miller, Myers, & Lipman, 1990). Two tyrosinase genes were identified and designated tyr1 and tyr2. Positions of the ATG start codons and introns in tyr1 and tyr2 were determined by 30 - and 50 RACE PCR and RT-PCR. All of the introns began with the nucleotides GT and ended with AG. The genomic DNA sequences of tyr1, tyr2, and act1 of IM-BM11-P21 were submitted to DDBJ under accession numbers LC380840, LC380841 and LC380842, respectively. 2.5. Real-time PCR assay We used the actin gene (DDBJ No. LC380842) as the housekeeping gene. Primers were designed according to their cDNA sequences using GENETYX 10.0 software (Genetyx, Tokyo, Japan). All primers were tested to ensure amplification of single bands with no primer-dimers. Plasmid extraction was performed according to the modified method reported by Birnboim (1983). Four 10-fold dilutions of the plasmid were performed to construct standard curves. Real-time PCR was conducted using KOD SYBR qPCR Mix (Toyobo) and a PikoReal 96 Real-time PCR System (Thermo Fisher Scientific, Tokyo, Japan). Each reaction was run twice. The cycling parameters were 98 C for 120-s pre-denaturation, and then 40 cycles of 98 C for 10 s, 65 C for 10 s, and 68 C for 30 s. Melting curves were determined according to the manufacturer’s instructions. After real-time PCR, samples were also run on a 1.5% agarose gel to confirm the specificity of amplification. The data were analyzed according to the manufacturer’s instructions. 2.6. Immuno-electron microscopy Tissues of the vegetative mycelial and primordial stages of the wild-type strain IM-BM-11 and albino strain IM-WM-1 were fixed in 4% paraformaldehyde for 5 h at 25 C. The samples were then washed with distilled water four times for 15 min, dehydrated in an ethanol series (70, 80, 90, 95, and 100%), and infiltrated in a 4:1 mixture of LR-White resin in 100% ethanol for 2 h at 25 C. Next, the samples were infiltrated in 100% LR-White and polymerized for 6 d at 25 C under ultraviolet (UV) light. Thin sections (70e100 nm) were cut with a diamond knife using an MT-7000 ultramicrotome (RMC, Tucson, USA), placed on nickel grids, and processed for colloidal gold immunolabeling as follows. The sections were immersed in 10% H2O2 for 40 min at 25 C. After washes in distilled water, the sections were treated with Tris-HCl buffer (TBS, pH 7.2) containing skim milk (0.25 g/5 mL TBS) with 0.05% Tween 20 (Wako, Osaka, Japan) for 60 min at 25 C in a humidified chamber to block nonspecific background staining. The sections were then incubated at 5 C overnight with a primary antibody elicited by immunization against a synthesized partial peptide of TYR1 ([H]CTDPKLKTKYGDAAKRFR-[OH]) and TYR2 ([H]-CRNPELRARYGRAAQRFR-[OH]), diluted 1:1000 in TBS containing skim milk and Tween 20. Negative control treatments were also prepared without any primary antibody. After washing ten times in TBS, the sections were incubated at 5 C overnight with goat anti-rabbit IgG (wholemolecule alkaline phosphatase antibody produced in goats, Sigma)
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Table 1 Primers used in this study. Primer
Sequence
Use
TYR1RT3-F1 TYR1RT5-P TYR1RT5-S1 TYR1RT5-A1 TYR1RT5-S2 TYR1RT5-A2 TYR2RT3-F1 TYR2RT5-P TYR2RT5-S1 TYR2RT5-A1 TYR2RT5-S2 TYR2RT5-A2 TYR1Re-F1 TYR1Re-R1 TYR2Re-F2 TYR2Re-R1 ACTRe-F1 ACTRe-R1
50 -GGTGCGGGGCATATGGGTAA-30 5’-(P)CTCGTATAGCACTTG-30 50 -CACATGGCACCGCCCTTATGTGGC-30 50 -TGACGCCATTCCAAGGGGTATATG-30 50 -CCCTTATGTGGCCCTCATAGAGCA-30 50 -GCCATTCCAAGGGGTATATGGCAA-30 50 -AGGCGGGCATATGGCTACTG-30 5’-(P)CAGATCACTTGCTCC-30 50 -GCTATTGTACTCACGGAACTGTC-30 50 -AGTCCCCAATATTGACATCATTCC-30 50 -CTCACGGAACTGTCCTCTTC-30 50 -TTCCAAGGAGTGTATGGCAA-30 50 -TGCACATCGCTGCTTCGTACACC-30 50 -GCTTGGCGCACTGATGACGCTAAC-30 50 -CATGCTAATGCACATAGCCAGCCT-30 50 -CCATACTCCTGTGCGTGGAGA-30 50 -GAGAAGATCTGGCATCACACGTTC-30 50 -ACGACAGGACGGCTTGGATG-30
30 -RACE of TYR1 50 -RACE of TYR1 (50 -end of this oligonucleotide was phosphorylated) 50 -RACE of TYR1
30 -RACE of TYR2 50 -RACE of TYR2 (50 -end of this oligonucleotide was phosphorylated) 50 -RACE of TYR2
Used for real-time PCR of TYR1 Used for real-time PCR of TYR2 Used for real-time PCR of Actin1
Table 2 Characteristics of melanin pigment isolated from Grifola frondosa. Test
Result of G. frondosa
Result of Tuber melanosporum (Harki et al., 1997).
Distilled water 1M NaOH 2M HCl Organic solvents Reaction with oxidizing agent (H2O2) Reaction for polyphenols (FeCl3 test)
Insoluble Soluble Precipitated readily Insoluble Decolorized Brown precipitate
Insoluble Soluble Precipitated readily Insoluble Decolorized Brown precipitate
antibody conjugated with 10-nm colloidal gold as a secondary antibody, diluted 1:10 in TBS containing skim milk and Tween 20. The sections were washed five times in TBS and then in distilled water five times. After labeling, sections were stained with uranyl acetate followed by lead citrate. The localization of colloidal gold was observed using a transmission electron microscope (JEM-1400 Plus; JEOL, Tokyo, Japan) at 80 kV. Next, a total of 100 mycelial tissue samples of wild-type strain IM-BM-11 and albino strain IMWM-1 for each vegetative mycelial and primordial stage were analyzed as a single group, and the ratio of label observed in both stages was calculated as follows: ratio of label in observed tissues (%) ¼ (label observed in mycelial tissues/observed mycelial tissues) 100. 2.7. Extraction and purification of melanin The 4-g cap was cut off the fruiting body (IM-BM11 and IMWM1), boiled for 10 min in 8 mL of 50 mM Tris-HCl buffer (pH 7.2), and centrifuged for 15 min (4000g). The supernatant was adjusted to pH 10 with 1 M NaOH and subsequently acidified to pH 3 with 6 M HCl, and then incubated at 4 C overnight to precipitate melanin. The precipitate was washed with distilled water and dried before use in further studies. The precipitate from wild-type IMBM11 and albino IM-WM1 was dissolved in 1 M NaOH and spotted onto silica thin-layer chromatography plates, and then developed in 2-propanol:water (5:5 [v/v]). A band appearing only in the IMBM11 sample was collected, and its physical and chemical characteristics were analyzed in detail. 2.8. Chemical analysis of the pigment Chemical analysis of the pigment was carried out according to the modified method reported by Thomas (1955). Solubility of the
brown pigment was individually assessed in distilled-deionized water, 2 N HCl, 1 N NaOH, ethanol, acetone, chloroform, benzene, and phenol. Reactions with oxidizing agents such as 30% hydrogen peroxide (H2O2) were also analyzed. The pigment also precipitated in 1% FeCl3. 2.9. Spectroscopic analyses of the pigment The pigment obtained from the fungus was dissolved in 1 M NaOH, and its UV spectrum (200e700 nm) was recorded using a spectrophotometer (Hitachi, Japan) with 1 M NaOH as the reference blank. 3. Results 3.1. Identification of the component responsible for the brown color of G. frondosa In order to identify the pigment imparting color to the fruiting body, pigment was extracted from both the wild-type strain IMBM11 and albino strain IM-WM1. Brown pigment was detected in IM-BM11, but not in IM-WM1 (Fig. 1A and B). The chemical and physical properties of the brown pigment extracted from IM-BM11 were then investigated (see Table 2). In the IM-BM11 extract, a brown spot appeared on the silica gel thin-layer chromatograph (TLC) with Rf value of 0.7875 (Fig. 1C). The chemical properties of the brown pigment are summarized in Table 1. The pigment was insoluble in pure water, acid, ethanol, benzene, chloroform and acetone, but was soluble in alkaline solution. The dissolved pigment was decolorized by treatment with oxidizing and reducing reagents such as H2O2. The pigment tested positive for polyphenols with FeCl3 and produced a flocculent brown precipitate. The brown pigment extracted from G. frondosa exhibited all of the physical and
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chemical properties common to natural melanin, as previously identified in Tuber melanosporum (Harki, Talou, & Dargent, 1997). The nature of the pigment was further confirmed based on its spectral properties. The UV spectrum exhibited a profile typical of melanin. The pigment absorbed strongly in the UV region, but progressively less so as the wavelength increased (Bell & Wheeler, 1986). The absorption spectrum showed characteristic peaks in the UV region spanning 200e300 nm, but not in the visible region (400e600 nm). The absorption spectrum of an alkaline solution of melanin from T. melanosporum also showed no maximum and minimum absorption in the visible range (Harki et al., 1997). An overall characteristic absorption peak was observed at 260 nm (Fig. 1D). This peak is associated with numerous complex conjugated structures in the melanin molecule (Cockell & Knowland, 1999). In the case of melanin, an almost linear decrease in absorption with increasing wavelength is observed. Collectively, these results strongly suggest that the brown pigment extracted from G. frondosa is melanin. 3.2. Expression of genes associated with melanin synthesis during fruiting body formation
Fig. 1. A: Pigment extraction from Grifola frondosa wild-type strain IM-BM11 and albino strain IM-WM1 fruiting bodies. Melanin was extracted using an alkaline method. B: Thin-layer chromatography of melanin extracted from G. frondosa fruiting bodies. Lane 1, wild-type strain IM-BM11; dark brown pigment. Lane 2, albino strain IM-WM1; no pigment production. C: Ultraviolet and visible spectra of melanin extracted from wild-type strain IM-BM11 G. frondosa fruiting bodies.
The expression of tyr1 and tyr2 in wild-type strain BM11 was analyzed. Transcription of tyr1 and tyr2 at various stages of the developmental cycle on sawdust medium is shown in Fig. 2A and B, respectively. At least four stages have been identified in the developmental cycle of G. frondosa in our laboratory: vegetative mycelial growth and colonization of the growth substrate, light-induced brown mycelial formation, primordial initiation, and fruiting body development. The formation of a brown color on the surface of mature mycelia normally precedes the appearance of brown-colored primordia, which has been depicted as a key step in the morphogenesis of G. frondosa fruiting bodies. Therefore, brown color formation is closely associated with the initiation of primordial
Fig. 2. Transcription levels of melanin biosynthesiseassociated genes at different developmental stages in Grifola frondosa wild-type strain IM-BM11 grown on sawdust medium. A: TYR1. B: TYR2.
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Fig. 3. Immuno-electron microscopic localization of TYR1 and TYR2 in Grifola frondosa wild-type strain IM-BM11 and albino strain IM-WM1. Tissues were isolated from vegetative mycelia and primordia cultivated with sawdust substrate. Sections treated with anti-TYR1 primary antibody: vegetative stage of wild-type (A) and albino (B) strains, and primordial stage of wild-type (C) and albino (D) strains. Sections treated with anti-TYR2 primary antibody: vegetative stage of wild-type (E) and albino (F) strains, and primordial stage of wildtype (G) and albino (H) strains. Arrows indicate gold-labeled particles. CM, cell membrane; CP, cytoplasm; CW, cell wall. Bars: AeC, E, F 200 nm; D, G, H 500 nm.
formation, and it is expected that one or more tyrosinases catalyze melanin-formation reactions, similar to tyrosinase expression in P. arcularius (Kanda et al., 2007). From the analysis of gene expression profiles, it became clear that the expression of tyr1 is not correlated with brown coloration, as this gene exhibited the highest expression in mycelia and decreased dramatically in the colored mycelia, primordia, fresh fruiting bodies, and post-harvest fruiting body tissues. The level of tyr2 transcription increased rapidly in primordia and peaked in young fruiting bodies before decreasing dramatically in mature
fruiting bodies. The gradual increase in the level of tyr2 transcription in young fruiting bodies suggests that this gene is associated with melanin formation. 3.3. Subcellular localization of tyrosinases in G. frondosa The subcellular localization of tyrosinases in G. frondosa was determined using immunoeelectron microscopy with anti-TYR1 and -TYR2 primary antibodies and a goat anti-rabbit IgG antibody conjugated with 10-nm colloidal gold as the secondary antibody
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(Fig. 3). No colloidal gold particles were observed when the primary antibody was omitted. When anti-TYR1 was used as the primary antibody, colloidal gold particles were observed in tissues of the vegetative mycelial and primordial stages of both wild-type strain IM-BM11 and albino strain IM-WM1 (Fig. 3AeD). The labeling patterns in the vegetative stage of wild-type strain IM-BM11 and albino strain IM-WM1 were similar, showing an accumulation of colloidal gold particles in the cytoplasm (Fig. 3A and B). In the primordial stage, the labeling pattern was also similar between wild-type strain IM-BM11 and albino strain IM-WM1, with colloidal gold particles localized mainly within the cell membrane and wall (Fig. 3C and D). The mycelia of both the wild-type and albino strains were not colored. However, TYR 1 and TYR 2 were detected in the cell
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cytoplasm of mycelia and primordia in both the wild-type and albino strains. On the other hand, TYR2 was observed only in the cell wall of colored primordia of the wild-type strain. TYR1 was not observed in the cell wall of either strain, while TYR2 was not detected in the cell wall of non-colored primordia in the albino strain (Fig. 3). These results indicated that subcellular localization of TYR2 might be required for the formation of colored primordia and fruiting bodies. When anti-TYR2 was used as the primary antibody, colloidal gold particles were also observed in both stages of the wild-type and albino strains (Fig. 3EeH), and the labeling pattern was the same as that observed with anti-TYR1 antibodies. The ratio of labelpositive to -negative tissues in the vegetative stage was the same in the wild-type and albino strains for both TYR1 and TYR2 (Fig. 4B).
Fig. 4. Ratio of cells observed with gold-labeled particles in mycelial tissues treated with TYR1 or TYR2 from wild-type strain IM-BM11 and albino strain IM-WM1 in the vegetative mycelial stage (A) and primordial stage (B). Control, no primary antibody. The ratio was calculated as follows: ratio of label observed in tissues (%) ¼ (labeled mycelial tissues/ observed mycelial tissues) 100.
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By contrast, the ratio in the primordial stage differed between the wild-type and albino strains (Fig. 4B). The ratios of TYR1-to TYR2positive tissues in the wild-type strain were both 100%, although the TYR1-and TYR2-positive ratios in the albino strain were 64 and 11%, respectively. A significant accumulation of colloidal gold particles was observed in wild-type strain IM-BM11. These results indicate that TYR1 and TYR2 are localized primarily in the cytoplasm of mycelia, whereas these proteins are localized in the cell membrane and wall of primordia. TYR2 was expressed at high levels in primordia and localized in the cell wall, suggesting that melanin production occurs in the cell wall of primordia and is catalyzed by TYR2. TYR1 expression was detected in both stages of the wild-type and albino strains. However, the expression of TYR2 in the primordial stage of the albino strain was significantly lower than that of the wild-type strain, as shown in Figs. 3 and 4. Therefore, it is possible that the lack of coloration in the albino strain can be attributed to TYR 2 deficiency.
4. Discussion In this study, we demonstrated that the brown pigment in G. frondosa fruiting bodies is likely melanin. Two pathways of melanin synthesis have been reported in fungi (Eisenman & Casadevall, 2012). The first pathway (pathway 1, or the 8dihydroxynaphthalene [DHN] pathway) is catalyzed by PKS (Schumann & Hertweck, 2006). Gene expression analyses of pks in G. frondosa over the whole life cycle revealed that the DHN pathway is dispensable for melanin synthesis in this mushroom. The second melanin synthesis pathway is known as the L-3, or 4-dihyroxyphenylalanine (L-DOPA) pathway. There are two possible starting molecules in this pathway: L-DOPA and tyrosine. When L-DOPA is the starting molecule, it is oxidized by laccase. If tyrosine is the starting molecule, the reaction is catalyzed by tyrosinase (Land, Ramsden, & Riley, 2004; Langfelder, Streibel, Jahn, Haase, & Brakhage, 2003; Riley, 1997). The tyrosinase genes (tyr1 and tyr2) were identified in the G. frondosa draft genome. Only tyr2 levels were dramatically increased in the primordial stage, indicating that the polypore mushroom G. frondosa produces melanin via the L-DOPA melanin biosynthetic pathway. Thus, tyrosinase is an essential enzyme for melanin production in this mushroom. In Agaricales, the color of the fruiting body depends on polyphenol oxidases such as tyrosinase and laccase. The mechanisms of mushroom browning have been investigated extensively in Agaricus bisporus. Browning in this species is mainly due to melanin (Jolivet, Arpin, Wichers, & Pellon, 1998), and tyrosinase seems to be the principal enzyme in its synthesis. In L. edodes, tyrosinase activity increases in the fruiting body during postharvest storage, resulting in an increase in gill browning (Kanda et al., 2007). The intracellular laccase Lcc2 from L. edodes also plays an important role in melanin synthesis in the fruiting body. The albino strain is capable of producing fruiting bodies; thus, this indicates that melanin production is not essential for fruiting body development. However, tyr2 expression occurred simultaneously with primordial formation in the wild-type strain. Therefore, it is possible that regulation of tyr2 expression might be associated with primordial formation. The molecular mass and maturation (processing of the carboxyl terminus) of TYR1 and TYR2 were examined using SDS polyacrylamide gel electrophoresis followed by western blotting with an anti-TYR2 antibody. However, we did not detect a positive band (data not shown), indicating that TYR2 is tightly bound to or inserted within the cell wall. We could not extract the protein from the cell wall in the soluble fraction, although we tested many
different detergents for the extraction. The relationship between tyrosinase activity and pigmentation is experimentally unclear. We therefore performed immunoelectron microscopy to identify the precise location(s) of the tyrosinase protein within G. frondosa. TYR2 was found predominantly in the G. frondosa cell wall in the primordial stage. Interestingly, limited anti-TYR1 antibodies were localized in the cell wall and membrane of the primordia, perhaps due to variations in TYR1 expression during the different stages of the G. frondosa life cycle. Other possible explanations could include TYR2 synthesized melanin at this stage. Cell wall localization of TYR2 would place melanin in a position most likely to protect the organism from adverse environmental conditions, such as ambient UV light and drying. Melanin could also contribute to fungal survival by promoting cell wall integrity. To confirm the relationship between melanin biosynthesis and TYR2, an albino dikaryotic strain of G. frondosa will be used as the parental strain to isolate a monokaryotic strain carrying mutant tyr2, with subsequent backcrossing analysis. Possible tyr2 mutated phenotypes include alteration of subcellular localization, lost/diminished enzyme activity, and so on, which would account for the presence of TYR2 in the primordial stage of the albino strain. In summary, this investigation demonstrated that G. frondosa synthesizes melanin, which is associated with the cell wall and cytoplasm to protect the fungus from environmental insults. The present study is the first to examine the melanin biosynthesis pathway in polypore mushrooms at the molecular level, and the results should facilitate targeted breeding and selection strategies for developing mushroom strains that are less susceptible to discoloration. Acknowledgments This work was partially supported by a Grant-in-Aid for Scientific Research (C) 18K05763 by the Japan Society for the Promotion of Science (JSPS). References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403e410. http:// cshprotocols.cshlp.org/cgi/pmidlookup?view¼long&pmid¼21357135. Bell, A. A., & Wheeler, M. H. (1986). Biosynthesis and functions of fungal melanins. Annual Review of Phytopathology, 24, 411e451. https://doi.org/10.1146/ annurev.py.24.090186.002211. Birnboim, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods in Enzymology, 100, 243e255. Cockell, C. S., & Knowland, J. (1999). Ultraviolet radiation screening compounds. Biological Reviews, 74, 311e345. https://doi.org/10.1017/S0006323199005356. Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA mini preparation: Version II. Plant Molecular Biology Reporter, 1, 19e21. https://link.springer.com/ content/pdf/10.1007/BF02712670.pdf. Eisenman, H. C., & Casadevall, A. (2012). Synthesis and assembly of fungal melanin. Applied Microbiology and Biotechnology, 93, 931e940. https://link.springer.com/ article/10.1007%2Fs00253-011-3777-2. Harki, E., Talou, T., & Dargent, R. (1997). Purification, characterization and analysis of melanin extracted from Tuber melanosporum Vitt. Food Chemistry, 58, 69e73. https://doi.org/10.1016/S0308-8146(96)00215-4. Hongo, T., Ueda, T., & Izawa, M. (1994). Mushrooms. Tokyo: Yama-kei Publishers Co., Ltd. Imazeki, R., Otani, Y., & Hongo, T. (1988). Nihon no kinoko [fungi of Japan. Tokyo, Japan: Yama-Kei Publishers Co., Ltd. Jolivet, S., Arpin, N., Wichers, H. J., & Pellon, G. (1998). Agaricus bisporus browning: A review. Mycological Research, 102, 1459e1483. https://doi.org/10.1017/ S0953756298006248. Kanda, S., Aimi, T., Masumoto, S., Nakano, K., Kitamoto, Y., & Morinaga, T. (2007). Photoregulated tyrosinase gene in Polyporus arcularius. Mycoscience, 48, 34e41. https://link.springer.com/article/10.1007/s10267-006-0327-3. Land, E. J., Ramsden, C. A., & Riley, P. A. (2004). Quinone chemistry and melanogenesis. Methods in Enzymology, 378, 88e109. https://doi.org/10.1016/S00766879(04)78005-2.
N. Kawaguchi et al. / Mycoscience 60 (2019) 262e269 Langfelder, K., Streibel, M., Jahn, B., Haase, G., & Brakhage, A. A. (2003). Biosynthesis of fungal melanins and their importance for human pathogenic fungi. Fungal Genetics and Biology, 38, 143e158. https://doi.org/10.1016/S1087-1845(02) 00526-1. Riley, P. A. (1997). Melanin. The International Journal of Biochemistry & Cell Biology, 29, 1235e1239. https://doi.org/10.1016/S1357-2725(97)00013-7.
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Schumann, J., & Hertweck, C. (2006). Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. Journal of Biotechnology, 124, 690e703. https://doi.org/10.1016/j.jbiotec.2006.03.046. Thomas, M. (1955). Melanins. In K. Patch, & M. V. Tracey (Eds.), Modern methods of plant analysis (Vol. 4, pp. 661e675). Berlin: Springer - Verlag.
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Expression of tyrosinase genes associated with fruiting body formation and pigmentation in Grifola frondosa Nobuhisa Kawaguchi a, Mirai Hayashi b, Shota Nakano b, Norihiro Shimomura b, Takeshi Yamaguchi b, Tadanori Aimi b, * a b
Laboratory, Biological Business Department, Ichimasa Kamaboko Co Ltd., 77-1 Junishin, Agano-shi, Niigata, 959-1936, Japan Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori, 680-8553, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 December 2018 Received in revised form 2 April 2019 Accepted 2 April 2019 Available online 11 April 2019
Chemical and physical properties (including ultraviolet spectrum) of a brown pigment isolated from Grifola frondosa fruiting bodies were almost identical to those of fungal melanin. From the full genome sequence of G. frondosa, genes involved in the melanin biosynthesis pathway of this polypore mushroom were identified, including tyrosinases (tyr1 and tyr2). Transcriptional analyses showed dramatic changes only in the expression of tyr2 in the primordial stage. Immuno-electron microscopy using anti-tyrosinase antibodies showed that TYR2 localizes in the cell walls of primordia. Therefore, TYR2 may be closely associated with melanin biosynthesis in this polypore mushroom G. frondosa, and melanin might be produced in the cell wall. © 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Albino Cell wall Immuno-electron microscopy Melanin
1. Introduction The fruiting bodies of mushrooms are very colorful. However, in some typical edible mushrooms, such as Lentinula edodes (shiitake), Pholiota microspora (nameko), Grifola frondosa (maitake), and Hypsizygus tessellatus (bunashimeji), the fruiting bodies of the wild-type strain are black or brown (Hongo, Ueda, & Izawa, 1994, p. 384; Imazeki, Otani, & Hongo, 1988, p. 623). In general, the brown pigment in the pileus of mushrooms is often melanin. Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents, and ionizing radiation (Eisenman & Casadevall, 2012). Fruiting body development is initiated within the tree rootstock and extends to the external environment. The primordia and young fruiting body tissues are then exposed to sunlight, resulting in the production of melanin to protect the intracellular DNA from mutation by UV light. Therefore, it is strongly suggested that pigment production is closely linked to fruiting body development. Grifola frondosa, a white-rot fungus widely distributed in Asia, North America, and Europe, is a pleasant-tasting edible mushroom.
* Corresponding author. E-mail address: [email protected] (T. Aimi).
Irradiation with visible light is required for the initiation of pileus formation in artificially cultivated G. frondosa. When a dark-grown vegetative mycelium is exposed to light, the buff-brown color of the surrounding spawn surface is induced, and subsequently, the primordia form. Therefore, it is possible that the color of mycelia is closely associated with the initiation of primordia formation and could be a trigger for fruiting body formation. However, neither the identity of the brown pigment nor its synthesis pathway in G. frondosa has been reported. A similar phenomenon was observed in Polyporus arcuraius. The brown pigmentation of mycelia in P. arcularius occurred only in a dikaryotic strain grown under visible light before development of primordia and did not occur in a monokaryon strain, even when grown under visible light. Moreover, pigmentation of the dikaryotic mycelia did not occur when grown in the dark. Reverse transcription PCR (RT-PCR) products of tyrosinase 1 gene (tyr1) in dikaryon and monokaryon P. arcularius could not be detected without exposing the strains to visible light, and the amplification products disappeared when the strains were transferred back to the dark (Kanda et al., 2007). These data suggest that exposure to visible light, tyrosinase gene induction, brown pigment production, and primordia formation are closely linked. However, there is no direct evidence that the brown pigment is melanin, and the relationship between fruiting and tyrosinase gene expression in both P. arcularius and G. frondosa is unclear.
https://doi.org/10.1016/j.myc.2019.04.003 1340-3540/© 2019 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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To elucidate the molecular mechanism of pigment formation in G. frondosa, several genes potentially related to brown color formation were identified from genome sequence databases, such as genes encoding tyrosinases (TYR1 and TYR2). Expression of the two genes was investigated in the different stages of fruiting body development. The primary goal of this study was to enhance understanding of the factors that trigger the initiation of fruiting body formation in “polypore mushrooms.” 2. Materials and methods 2.1. Strains and culture conditions The wild-type dikaryotic strain IM-BM11 and albino dikaryotic strain IM-WM1 were obtained from Ichimasa Kamaboko Co., Ltd (Agano-shi, Niigata, Japan). The albino monokaryotic strain IMWM1-25 was obtained by basidiospore isolation of the fruiting bodies of IM-WM1. The wild-type monokaryotic strain IM-BM11P21 was derived from regenerated protoplasts of wild-type dikaryotic strain IM-BM11. Grifola frondosa dikaryotic strains were cultivated on a sawdust substrate, consisting of sawdust and rice bran at a volumetric ratio of 10:1. The moisture content of the substrate was adjusted to 65%. All ingredients were combined and mixed, and 500 g of the prepared substrate was bagged into high-density polyethylene bags (SE-25ES; Sakato Sangyo Co., Ltd., Minakami-machi, Tone-gun, Gunma, Japan) sealed with a heat sealer, sterilized by autoclaving (121 C, 60 min), cooled, and then inoculated with mycelia. The substrate was incubated at 25 C in the dark for 40 d to allow the substrate to become fully colonized by the mycelia (this process was designated “spawn running”). After spawn running, the fully colonized substrate was irradiated with 200 lux visible light for 1 wk at 25 C (stage 1). The substrate was then incubated at 15 C under 200 lux visible light until fruiting body maturation. The polyethylene bags were opened when the mycelium had turned brown (stage 2), and the moisture was maintained at 90% to initiate primordia formation. RNA was isolated from mycelia in the sawdust substrate at stage 1 (mycelial stage) and stage 2 (colored mycelial stage). We also isolated RNA from primordia and fruiting body tissues at different stages. 2.2. DNA preparation and genome sequencing Genomic DNA was extracted according to the method described by Dellaporta, Wood, and Hicks (1983). The complete nucleotide sequence of the genomic DNA of albino monokaryotic strain WM125 was determined using Illumina HiSeq 2000 paired-end technology (Hokkaido System Science Co., Ltd., Sapporo, Hokkaido, Japan). This sequencing run yielded 34,502,348 high-quality filtered reads with 101-bp paired-end sequencing. The genomic sequence was assembled using Velvet Assembler version 1.1.02 (hash length, 75 bp). The final assembly contained 7354 contigs of a total length of 35,343,167 bp, with an n50 length of 94,048 bp. 2.3. RNA preparation and cDNA synthesis RNA was extracted using a MagExtractor kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. cDNA was synthesized with total RNA as the template using ReverTraAce qPCR RT Master Mix with a gDNA Remover kit (Toyobo). Amplification of cDNA fragments and 30 -rapid amplification of cDNA ends (RACE) were performed using a Takara RNA PCR (AMV) version 3.0 kit (Takara Bio, Kusatsu, Shiga, Japan) and 50 -RACE with a 50 -Full RACE Core Set (Takara Bio). PCR was carried out according to the kit manufacturer’s instructions using the oligonucleotide
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primers listed in Table 1. The amplified fragments were subcloned into a pMD20 T-vector (Takara Bio) and sequenced Table 2. 2.4. Identification of genes associated with melanin synthesis within the genome of G. frondosa In order to design nucleotide primers for quantitative RT-PCR, nucleotide sequences of tyrosinase, tyrosinase-related protein, and polyketide synthase (PKS) genes of G. frondosa were identified from the genome sequence data of strain IM-WM1-25. Sequences exhibiting homology with protein sequences of L. edodes tyrosinase (BAB71736.1) were searched using the tblastn program (Altschul, Gish, Miller, Myers, & Lipman, 1990). Two tyrosinase genes were identified and designated tyr1 and tyr2. Positions of the ATG start codons and introns in tyr1 and tyr2 were determined by 30 - and 50 RACE PCR and RT-PCR. All of the introns began with the nucleotides GT and ended with AG. The genomic DNA sequences of tyr1, tyr2, and act1 of IM-BM11-P21 were submitted to DDBJ under accession numbers LC380840, LC380841 and LC380842, respectively. 2.5. Real-time PCR assay We used the actin gene (DDBJ No. LC380842) as the housekeeping gene. Primers were designed according to their cDNA sequences using GENETYX 10.0 software (Genetyx, Tokyo, Japan). All primers were tested to ensure amplification of single bands with no primer-dimers. Plasmid extraction was performed according to the modified method reported by Birnboim (1983). Four 10-fold dilutions of the plasmid were performed to construct standard curves. Real-time PCR was conducted using KOD SYBR qPCR Mix (Toyobo) and a PikoReal 96 Real-time PCR System (Thermo Fisher Scientific, Tokyo, Japan). Each reaction was run twice. The cycling parameters were 98 C for 120-s pre-denaturation, and then 40 cycles of 98 C for 10 s, 65 C for 10 s, and 68 C for 30 s. Melting curves were determined according to the manufacturer’s instructions. After real-time PCR, samples were also run on a 1.5% agarose gel to confirm the specificity of amplification. The data were analyzed according to the manufacturer’s instructions. 2.6. Immuno-electron microscopy Tissues of the vegetative mycelial and primordial stages of the wild-type strain IM-BM-11 and albino strain IM-WM-1 were fixed in 4% paraformaldehyde for 5 h at 25 C. The samples were then washed with distilled water four times for 15 min, dehydrated in an ethanol series (70, 80, 90, 95, and 100%), and infiltrated in a 4:1 mixture of LR-White resin in 100% ethanol for 2 h at 25 C. Next, the samples were infiltrated in 100% LR-White and polymerized for 6 d at 25 C under ultraviolet (UV) light. Thin sections (70e100 nm) were cut with a diamond knife using an MT-7000 ultramicrotome (RMC, Tucson, USA), placed on nickel grids, and processed for colloidal gold immunolabeling as follows. The sections were immersed in 10% H2O2 for 40 min at 25 C. After washes in distilled water, the sections were treated with Tris-HCl buffer (TBS, pH 7.2) containing skim milk (0.25 g/5 mL TBS) with 0.05% Tween 20 (Wako, Osaka, Japan) for 60 min at 25 C in a humidified chamber to block nonspecific background staining. The sections were then incubated at 5 C overnight with a primary antibody elicited by immunization against a synthesized partial peptide of TYR1 ([H]CTDPKLKTKYGDAAKRFR-[OH]) and TYR2 ([H]-CRNPELRARYGRAAQRFR-[OH]), diluted 1:1000 in TBS containing skim milk and Tween 20. Negative control treatments were also prepared without any primary antibody. After washing ten times in TBS, the sections were incubated at 5 C overnight with goat anti-rabbit IgG (wholemolecule alkaline phosphatase antibody produced in goats, Sigma)
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Table 1 Primers used in this study. Primer
Sequence
Use
TYR1RT3-F1 TYR1RT5-P TYR1RT5-S1 TYR1RT5-A1 TYR1RT5-S2 TYR1RT5-A2 TYR2RT3-F1 TYR2RT5-P TYR2RT5-S1 TYR2RT5-A1 TYR2RT5-S2 TYR2RT5-A2 TYR1Re-F1 TYR1Re-R1 TYR2Re-F2 TYR2Re-R1 ACTRe-F1 ACTRe-R1
50 -GGTGCGGGGCATATGGGTAA-30 5’-(P)CTCGTATAGCACTTG-30 50 -CACATGGCACCGCCCTTATGTGGC-30 50 -TGACGCCATTCCAAGGGGTATATG-30 50 -CCCTTATGTGGCCCTCATAGAGCA-30 50 -GCCATTCCAAGGGGTATATGGCAA-30 50 -AGGCGGGCATATGGCTACTG-30 5’-(P)CAGATCACTTGCTCC-30 50 -GCTATTGTACTCACGGAACTGTC-30 50 -AGTCCCCAATATTGACATCATTCC-30 50 -CTCACGGAACTGTCCTCTTC-30 50 -TTCCAAGGAGTGTATGGCAA-30 50 -TGCACATCGCTGCTTCGTACACC-30 50 -GCTTGGCGCACTGATGACGCTAAC-30 50 -CATGCTAATGCACATAGCCAGCCT-30 50 -CCATACTCCTGTGCGTGGAGA-30 50 -GAGAAGATCTGGCATCACACGTTC-30 50 -ACGACAGGACGGCTTGGATG-30
30 -RACE of TYR1 50 -RACE of TYR1 (50 -end of this oligonucleotide was phosphorylated) 50 -RACE of TYR1
30 -RACE of TYR2 50 -RACE of TYR2 (50 -end of this oligonucleotide was phosphorylated) 50 -RACE of TYR2
Used for real-time PCR of TYR1 Used for real-time PCR of TYR2 Used for real-time PCR of Actin1
Table 2 Characteristics of melanin pigment isolated from Grifola frondosa. Test
Result of G. frondosa
Result of Tuber melanosporum (Harki et al., 1997).
Distilled water 1M NaOH 2M HCl Organic solvents Reaction with oxidizing agent (H2O2) Reaction for polyphenols (FeCl3 test)
Insoluble Soluble Precipitated readily Insoluble Decolorized Brown precipitate
Insoluble Soluble Precipitated readily Insoluble Decolorized Brown precipitate
antibody conjugated with 10-nm colloidal gold as a secondary antibody, diluted 1:10 in TBS containing skim milk and Tween 20. The sections were washed five times in TBS and then in distilled water five times. After labeling, sections were stained with uranyl acetate followed by lead citrate. The localization of colloidal gold was observed using a transmission electron microscope (JEM-1400 Plus; JEOL, Tokyo, Japan) at 80 kV. Next, a total of 100 mycelial tissue samples of wild-type strain IM-BM-11 and albino strain IMWM-1 for each vegetative mycelial and primordial stage were analyzed as a single group, and the ratio of label observed in both stages was calculated as follows: ratio of label in observed tissues (%) ¼ (label observed in mycelial tissues/observed mycelial tissues) 100. 2.7. Extraction and purification of melanin The 4-g cap was cut off the fruiting body (IM-BM11 and IMWM1), boiled for 10 min in 8 mL of 50 mM Tris-HCl buffer (pH 7.2), and centrifuged for 15 min (4000g). The supernatant was adjusted to pH 10 with 1 M NaOH and subsequently acidified to pH 3 with 6 M HCl, and then incubated at 4 C overnight to precipitate melanin. The precipitate was washed with distilled water and dried before use in further studies. The precipitate from wild-type IMBM11 and albino IM-WM1 was dissolved in 1 M NaOH and spotted onto silica thin-layer chromatography plates, and then developed in 2-propanol:water (5:5 [v/v]). A band appearing only in the IMBM11 sample was collected, and its physical and chemical characteristics were analyzed in detail. 2.8. Chemical analysis of the pigment Chemical analysis of the pigment was carried out according to the modified method reported by Thomas (1955). Solubility of the
brown pigment was individually assessed in distilled-deionized water, 2 N HCl, 1 N NaOH, ethanol, acetone, chloroform, benzene, and phenol. Reactions with oxidizing agents such as 30% hydrogen peroxide (H2O2) were also analyzed. The pigment also precipitated in 1% FeCl3. 2.9. Spectroscopic analyses of the pigment The pigment obtained from the fungus was dissolved in 1 M NaOH, and its UV spectrum (200e700 nm) was recorded using a spectrophotometer (Hitachi, Japan) with 1 M NaOH as the reference blank. 3. Results 3.1. Identification of the component responsible for the brown color of G. frondosa In order to identify the pigment imparting color to the fruiting body, pigment was extracted from both the wild-type strain IMBM11 and albino strain IM-WM1. Brown pigment was detected in IM-BM11, but not in IM-WM1 (Fig. 1A and B). The chemical and physical properties of the brown pigment extracted from IM-BM11 were then investigated (see Table 2). In the IM-BM11 extract, a brown spot appeared on the silica gel thin-layer chromatograph (TLC) with Rf value of 0.7875 (Fig. 1C). The chemical properties of the brown pigment are summarized in Table 1. The pigment was insoluble in pure water, acid, ethanol, benzene, chloroform and acetone, but was soluble in alkaline solution. The dissolved pigment was decolorized by treatment with oxidizing and reducing reagents such as H2O2. The pigment tested positive for polyphenols with FeCl3 and produced a flocculent brown precipitate. The brown pigment extracted from G. frondosa exhibited all of the physical and
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chemical properties common to natural melanin, as previously identified in Tuber melanosporum (Harki, Talou, & Dargent, 1997). The nature of the pigment was further confirmed based on its spectral properties. The UV spectrum exhibited a profile typical of melanin. The pigment absorbed strongly in the UV region, but progressively less so as the wavelength increased (Bell & Wheeler, 1986). The absorption spectrum showed characteristic peaks in the UV region spanning 200e300 nm, but not in the visible region (400e600 nm). The absorption spectrum of an alkaline solution of melanin from T. melanosporum also showed no maximum and minimum absorption in the visible range (Harki et al., 1997). An overall characteristic absorption peak was observed at 260 nm (Fig. 1D). This peak is associated with numerous complex conjugated structures in the melanin molecule (Cockell & Knowland, 1999). In the case of melanin, an almost linear decrease in absorption with increasing wavelength is observed. Collectively, these results strongly suggest that the brown pigment extracted from G. frondosa is melanin. 3.2. Expression of genes associated with melanin synthesis during fruiting body formation
Fig. 1. A: Pigment extraction from Grifola frondosa wild-type strain IM-BM11 and albino strain IM-WM1 fruiting bodies. Melanin was extracted using an alkaline method. B: Thin-layer chromatography of melanin extracted from G. frondosa fruiting bodies. Lane 1, wild-type strain IM-BM11; dark brown pigment. Lane 2, albino strain IM-WM1; no pigment production. C: Ultraviolet and visible spectra of melanin extracted from wild-type strain IM-BM11 G. frondosa fruiting bodies.
The expression of tyr1 and tyr2 in wild-type strain BM11 was analyzed. Transcription of tyr1 and tyr2 at various stages of the developmental cycle on sawdust medium is shown in Fig. 2A and B, respectively. At least four stages have been identified in the developmental cycle of G. frondosa in our laboratory: vegetative mycelial growth and colonization of the growth substrate, light-induced brown mycelial formation, primordial initiation, and fruiting body development. The formation of a brown color on the surface of mature mycelia normally precedes the appearance of brown-colored primordia, which has been depicted as a key step in the morphogenesis of G. frondosa fruiting bodies. Therefore, brown color formation is closely associated with the initiation of primordial
Fig. 2. Transcription levels of melanin biosynthesiseassociated genes at different developmental stages in Grifola frondosa wild-type strain IM-BM11 grown on sawdust medium. A: TYR1. B: TYR2.
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Fig. 3. Immuno-electron microscopic localization of TYR1 and TYR2 in Grifola frondosa wild-type strain IM-BM11 and albino strain IM-WM1. Tissues were isolated from vegetative mycelia and primordia cultivated with sawdust substrate. Sections treated with anti-TYR1 primary antibody: vegetative stage of wild-type (A) and albino (B) strains, and primordial stage of wild-type (C) and albino (D) strains. Sections treated with anti-TYR2 primary antibody: vegetative stage of wild-type (E) and albino (F) strains, and primordial stage of wildtype (G) and albino (H) strains. Arrows indicate gold-labeled particles. CM, cell membrane; CP, cytoplasm; CW, cell wall. Bars: AeC, E, F 200 nm; D, G, H 500 nm.
formation, and it is expected that one or more tyrosinases catalyze melanin-formation reactions, similar to tyrosinase expression in P. arcularius (Kanda et al., 2007). From the analysis of gene expression profiles, it became clear that the expression of tyr1 is not correlated with brown coloration, as this gene exhibited the highest expression in mycelia and decreased dramatically in the colored mycelia, primordia, fresh fruiting bodies, and post-harvest fruiting body tissues. The level of tyr2 transcription increased rapidly in primordia and peaked in young fruiting bodies before decreasing dramatically in mature
fruiting bodies. The gradual increase in the level of tyr2 transcription in young fruiting bodies suggests that this gene is associated with melanin formation. 3.3. Subcellular localization of tyrosinases in G. frondosa The subcellular localization of tyrosinases in G. frondosa was determined using immunoeelectron microscopy with anti-TYR1 and -TYR2 primary antibodies and a goat anti-rabbit IgG antibody conjugated with 10-nm colloidal gold as the secondary antibody
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(Fig. 3). No colloidal gold particles were observed when the primary antibody was omitted. When anti-TYR1 was used as the primary antibody, colloidal gold particles were observed in tissues of the vegetative mycelial and primordial stages of both wild-type strain IM-BM11 and albino strain IM-WM1 (Fig. 3AeD). The labeling patterns in the vegetative stage of wild-type strain IM-BM11 and albino strain IM-WM1 were similar, showing an accumulation of colloidal gold particles in the cytoplasm (Fig. 3A and B). In the primordial stage, the labeling pattern was also similar between wild-type strain IM-BM11 and albino strain IM-WM1, with colloidal gold particles localized mainly within the cell membrane and wall (Fig. 3C and D). The mycelia of both the wild-type and albino strains were not colored. However, TYR 1 and TYR 2 were detected in the cell
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cytoplasm of mycelia and primordia in both the wild-type and albino strains. On the other hand, TYR2 was observed only in the cell wall of colored primordia of the wild-type strain. TYR1 was not observed in the cell wall of either strain, while TYR2 was not detected in the cell wall of non-colored primordia in the albino strain (Fig. 3). These results indicated that subcellular localization of TYR2 might be required for the formation of colored primordia and fruiting bodies. When anti-TYR2 was used as the primary antibody, colloidal gold particles were also observed in both stages of the wild-type and albino strains (Fig. 3EeH), and the labeling pattern was the same as that observed with anti-TYR1 antibodies. The ratio of labelpositive to -negative tissues in the vegetative stage was the same in the wild-type and albino strains for both TYR1 and TYR2 (Fig. 4B).
Fig. 4. Ratio of cells observed with gold-labeled particles in mycelial tissues treated with TYR1 or TYR2 from wild-type strain IM-BM11 and albino strain IM-WM1 in the vegetative mycelial stage (A) and primordial stage (B). Control, no primary antibody. The ratio was calculated as follows: ratio of label observed in tissues (%) ¼ (labeled mycelial tissues/ observed mycelial tissues) 100.
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By contrast, the ratio in the primordial stage differed between the wild-type and albino strains (Fig. 4B). The ratios of TYR1-to TYR2positive tissues in the wild-type strain were both 100%, although the TYR1-and TYR2-positive ratios in the albino strain were 64 and 11%, respectively. A significant accumulation of colloidal gold particles was observed in wild-type strain IM-BM11. These results indicate that TYR1 and TYR2 are localized primarily in the cytoplasm of mycelia, whereas these proteins are localized in the cell membrane and wall of primordia. TYR2 was expressed at high levels in primordia and localized in the cell wall, suggesting that melanin production occurs in the cell wall of primordia and is catalyzed by TYR2. TYR1 expression was detected in both stages of the wild-type and albino strains. However, the expression of TYR2 in the primordial stage of the albino strain was significantly lower than that of the wild-type strain, as shown in Figs. 3 and 4. Therefore, it is possible that the lack of coloration in the albino strain can be attributed to TYR 2 deficiency.
4. Discussion In this study, we demonstrated that the brown pigment in G. frondosa fruiting bodies is likely melanin. Two pathways of melanin synthesis have been reported in fungi (Eisenman & Casadevall, 2012). The first pathway (pathway 1, or the 8dihydroxynaphthalene [DHN] pathway) is catalyzed by PKS (Schumann & Hertweck, 2006). Gene expression analyses of pks in G. frondosa over the whole life cycle revealed that the DHN pathway is dispensable for melanin synthesis in this mushroom. The second melanin synthesis pathway is known as the L-3, or 4-dihyroxyphenylalanine (L-DOPA) pathway. There are two possible starting molecules in this pathway: L-DOPA and tyrosine. When L-DOPA is the starting molecule, it is oxidized by laccase. If tyrosine is the starting molecule, the reaction is catalyzed by tyrosinase (Land, Ramsden, & Riley, 2004; Langfelder, Streibel, Jahn, Haase, & Brakhage, 2003; Riley, 1997). The tyrosinase genes (tyr1 and tyr2) were identified in the G. frondosa draft genome. Only tyr2 levels were dramatically increased in the primordial stage, indicating that the polypore mushroom G. frondosa produces melanin via the L-DOPA melanin biosynthetic pathway. Thus, tyrosinase is an essential enzyme for melanin production in this mushroom. In Agaricales, the color of the fruiting body depends on polyphenol oxidases such as tyrosinase and laccase. The mechanisms of mushroom browning have been investigated extensively in Agaricus bisporus. Browning in this species is mainly due to melanin (Jolivet, Arpin, Wichers, & Pellon, 1998), and tyrosinase seems to be the principal enzyme in its synthesis. In L. edodes, tyrosinase activity increases in the fruiting body during postharvest storage, resulting in an increase in gill browning (Kanda et al., 2007). The intracellular laccase Lcc2 from L. edodes also plays an important role in melanin synthesis in the fruiting body. The albino strain is capable of producing fruiting bodies; thus, this indicates that melanin production is not essential for fruiting body development. However, tyr2 expression occurred simultaneously with primordial formation in the wild-type strain. Therefore, it is possible that regulation of tyr2 expression might be associated with primordial formation. The molecular mass and maturation (processing of the carboxyl terminus) of TYR1 and TYR2 were examined using SDS polyacrylamide gel electrophoresis followed by western blotting with an anti-TYR2 antibody. However, we did not detect a positive band (data not shown), indicating that TYR2 is tightly bound to or inserted within the cell wall. We could not extract the protein from the cell wall in the soluble fraction, although we tested many
different detergents for the extraction. The relationship between tyrosinase activity and pigmentation is experimentally unclear. We therefore performed immunoelectron microscopy to identify the precise location(s) of the tyrosinase protein within G. frondosa. TYR2 was found predominantly in the G. frondosa cell wall in the primordial stage. Interestingly, limited anti-TYR1 antibodies were localized in the cell wall and membrane of the primordia, perhaps due to variations in TYR1 expression during the different stages of the G. frondosa life cycle. Other possible explanations could include TYR2 synthesized melanin at this stage. Cell wall localization of TYR2 would place melanin in a position most likely to protect the organism from adverse environmental conditions, such as ambient UV light and drying. Melanin could also contribute to fungal survival by promoting cell wall integrity. To confirm the relationship between melanin biosynthesis and TYR2, an albino dikaryotic strain of G. frondosa will be used as the parental strain to isolate a monokaryotic strain carrying mutant tyr2, with subsequent backcrossing analysis. Possible tyr2 mutated phenotypes include alteration of subcellular localization, lost/diminished enzyme activity, and so on, which would account for the presence of TYR2 in the primordial stage of the albino strain. In summary, this investigation demonstrated that G. frondosa synthesizes melanin, which is associated with the cell wall and cytoplasm to protect the fungus from environmental insults. The present study is the first to examine the melanin biosynthesis pathway in polypore mushrooms at the molecular level, and the results should facilitate targeted breeding and selection strategies for developing mushroom strains that are less susceptible to discoloration. Acknowledgments This work was partially supported by a Grant-in-Aid for Scientific Research (C) 18K05763 by the Japan Society for the Promotion of Science (JSPS). References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403e410. http:// cshprotocols.cshlp.org/cgi/pmidlookup?view¼long&pmid¼21357135. Bell, A. A., & Wheeler, M. H. (1986). Biosynthesis and functions of fungal melanins. Annual Review of Phytopathology, 24, 411e451. https://doi.org/10.1146/ annurev.py.24.090186.002211. Birnboim, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods in Enzymology, 100, 243e255. Cockell, C. S., & Knowland, J. (1999). Ultraviolet radiation screening compounds. Biological Reviews, 74, 311e345. https://doi.org/10.1017/S0006323199005356. Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA mini preparation: Version II. Plant Molecular Biology Reporter, 1, 19e21. https://link.springer.com/ content/pdf/10.1007/BF02712670.pdf. Eisenman, H. C., & Casadevall, A. (2012). Synthesis and assembly of fungal melanin. Applied Microbiology and Biotechnology, 93, 931e940. https://link.springer.com/ article/10.1007%2Fs00253-011-3777-2. Harki, E., Talou, T., & Dargent, R. (1997). Purification, characterization and analysis of melanin extracted from Tuber melanosporum Vitt. Food Chemistry, 58, 69e73. https://doi.org/10.1016/S0308-8146(96)00215-4. Hongo, T., Ueda, T., & Izawa, M. (1994). Mushrooms. Tokyo: Yama-kei Publishers Co., Ltd. Imazeki, R., Otani, Y., & Hongo, T. (1988). Nihon no kinoko [fungi of Japan. Tokyo, Japan: Yama-Kei Publishers Co., Ltd. Jolivet, S., Arpin, N., Wichers, H. J., & Pellon, G. (1998). Agaricus bisporus browning: A review. Mycological Research, 102, 1459e1483. https://doi.org/10.1017/ S0953756298006248. Kanda, S., Aimi, T., Masumoto, S., Nakano, K., Kitamoto, Y., & Morinaga, T. (2007). Photoregulated tyrosinase gene in Polyporus arcularius. Mycoscience, 48, 34e41. https://link.springer.com/article/10.1007/s10267-006-0327-3. Land, E. J., Ramsden, C. A., & Riley, P. A. (2004). Quinone chemistry and melanogenesis. Methods in Enzymology, 378, 88e109. https://doi.org/10.1016/S00766879(04)78005-2.
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