Senescence of the Lentinula edodes Fruiting Body After Harvesting

The book discusses the novel scientific approaches for the improvement of the food quality and offers food scientists valuable assistance for the future. The detailed methodologies and their practical applications could serve as a fundamental reference work for the industry and a requisite guide for the research worker, food scientist and food analyst. It will serve as a valuable tool for the analysts improving their knowledge with new scientific data for quality evaluation. Two case study chapters provide data on the improvement of food quality in marine and land organisms in the natural environment.


Introduction
Lentinula edodes, or shiitake mushroom as it is more popularly known, is one of the most economically important edible cultivated mushrooms. However, postharvest spoilage, such as browning of the gills and softening of the fruiting body, results in loss of freshness and consequent loss of food value 1980a, b). Numerous studies addressing the mechanisms of quality loss during postharvest storage have revealed that browning of the L. edodes fruiting body is associated with increased activities of tyrosinase (Tyr; Kanda et al., 1996a, b) and laccase (Lcc; Nagai et al., 2003) after harvest. In addition to being an important fresh food source, shiitake mushrooms also have medicinal value. Lentinan, a β-1,3-glucan used for tumor immunotherapy (Chihara et al., 1969), is purified from fresh shiitake mushrooms. However, lentinan content decreases during postharvest storage (Minato et al., 1999). The reported structure of lentinan is a β-1,3linked-D -glucan with β-1,6 branches (Chihara et al., 1969), and it appears that postharvest degradation of lentinan during mushroom storage is mediated by β-1,3-glucanase (Minato et al., 1999). Four glucanases have been reported in L. edodes fruiting bodies, two exo-β-1,3glucanases, EXG1 (Sakamoto et al., 2005a) and EXG2 (Sakamoto et al., 2005b), and two endoβ-1,3-glucanases, TLG1 (Sakamoto et al., 2006) and GLU1 . An endoβ-1,6-glucanase, Pus30, was also purified from L. edodes fruiting body . Except for EXG1, these glucanases are involved in lentinan degradation after harvesting.
Postharvest changes are considered to be complex, and there is currently little information about the changes in gene transcription following harvest of the L. edodes fruiting bodies. It has been reported that the expression of several genes increases during the postharvest period in Agaricus bisporus (button mushroom, Eastwood, 2001). Eastwood et al., (2001) revealed that argininosuccinate lyase increases after harvest, and the relationship between elevated levels of argininosuccinate lyase and postharvest physiological changes of the A. bisporus fruiting body has been investigated in detail (Wagemaker et al., 2007;Eastwood et al., 2008). Several studies have analyzed changes in gene transcription during development of the fruiting body of L. edodes (Hirano et al., 2004;Miyazaki et al., 2005). More recently, EST analysis (Suizu et al., 2008) and SAGE analysis (Chum et al., 2008(Chum et al., , 2011 were carried out on L. edodes. Changes in gene transcription after harvest of the L. edodes fruiting body were investigated, revealing that many genes were newly expressed after harvest, such as putative chitinases, chitosanase, and a transcription factor . This chapter will discuss about genes involved in fruiting body senescence in mushrooms, especially in L. edodes.

Phenol oxidases involved in browning of fruiting body after harvesting
The postharvest preservation of L. edodes fruiting bodies causes gill browning, which is commercially undesirable since it causes an unpleasant appearance. In general, melanins involved in gill browning are considered to be synthesized from β-(3,4dihydroxyphenyl)alanine (DOPA), derived from tyrosine. DOPA can be oxidized enzymatically to quinones, which polymerize nonenzymatically to form the melanin pigments. Oxidation of tyrosine is commonly catalyzed by tyrosinasae (Tyr: EC1.14.18.1). The mechanisms of mushroom browning have been investigated extensively in A. bisporus (Burton, 1988;Espín et al., 1999). Browning in this species is mainly due to DOPA and cglutaminyl-3,4-dihydroxy-benzene (GDHB) melanins (Jolivet et al., 1998), and Tyr seems to play the most important role in their synthesis (Turner, 1974). Burton (1988) reported that epidermal tissues of A. bisporus had a greater activity of non-latent Tyr and a greater concentration of phenols than did the fresh. It has been reported that Tyr activity of L. edodes fruiting bodies increases after harvest, and a Tyr has been purified (Kanda et al., 1996a). Laccases (Lccs: EC 1.10.3.2) catalyze the single-electron oxidation of phenols or aromatic amines to form different products via various pathways. Lcc belongs to a group of polyphenol oxidases that contain copper atoms in their catalytic center; thus, they are typically referred to as multicopper oxidases. Lcc catalyzes the single-electron oxidation of phenolic substrates or aromatic amines to form different products via a variety of biochemical pathways (Leonowicz et al., 2001). There are many reports in the literature of the purification and characterization of Lcc isoforms from white-rot fungi, and several Lccencoding genes have been isolated (reviewed by Kumar et al., 2003;Baldrian, 2006). A correlation between melanin synthesis and intracellular Lcc in Cryptococcus neoformans has been reported (Ikeda et al., 2002). Lcc in Aspergillus nidulans is considered to form L-DOPA to synthesize melanins in conidia (Aramayo and Timberlake, 1990). Activity of Lcc increases after harvest of the L. edodes fruiting body, and a Lcc in L. edodes purified from fruiting bodies after harvest can oxidize DOPA (Nagai et al., 2003). Therefore, it is considered that Tyr and Lcc are involved in melanin synthesis after harvest of L. edodes fruiting bodies.

Tyr involvement in gill browning after harvest
Tyr (EC 1.14.18.1) is an ubiquitous enzyme in nature and the key enzyme in the process of melanin biosynthesis (van Gelder et al., 1997). Tyr catalyzes oxidation of phenolic substrates to quinone, which spontaneously polymerizes into dark-colored pigments known as melanin in the presence of nucleophilic moieties. Tyrs are found in a wide range of organisms, including prokaryotic and eukaryotic microorganisms, plants, invertebrates, and mammals. Tyrs are involved in a variety of biological functions, for example skin pigmentation in mammals and browning in plants and mushrooms. In mushrooms, browning after harvest has been investigated, especially in A. bisporus and L. edodes. Browning after harvest is considered a consequence of the Tyr-catalyzed oxidation of phenolic substrates (e.g., DOPA) into quinones leading to the formation of dark pigments of melanins. The enzymatic pigmentation of mushrooms is mediated largely by Tyr (Jolivet et al., 1998).
Several reports revealed that Tyr is related to gill browning after harvest of L. edodes fruiting bodies. It was reported that Tyr activity increases in the gills during postharvest preservation (Kanda et al., 1996a). A Tyr (LeTyr) was purified and characterized as the L. edodes Tyr, and LeTyr can catalyze tyrosine to DOPA (Kanda et al. 1996a(Kanda et al. , 1996b. The gene encoding LeTyr (Letyr) was isolated, and anti-serum was synthesized to LeTyr . Sato et al., (2009) showed that LeTyr increased after harvest, suggesting that LeTyr catalyzes DOPA synthesis for melanin formation after harvest in L. edodes. Letyr is the only Tyr encoding gene in L. edodes ). On the other hand, Tyr is involved in browning of the surface of vegetative mycelia (Sano et al., 2010), suggesting that LeTyr is involved in melanin synthesis in different tissues. Browning of the surface of vegetative mycelia is regulated by light, and the blue light receptor PHRB regulates Letyr (Sano et al., 2009). This suggests that melanin synthesis in vegetative mycelium under light is caused by an increase of LeTyr via PHRB. PHRB is a homolog of WC2 in Neurospora crassa, and expression of the WC2 homolog in L. edodes is low in the L. edodes fruiting body after harvest . This suggests that expression of Letyr in the fruiting body after harvest is regulated by transcription factor(s) other than PHRB.

Lcc involvement in gill browning after harvest
White-rot fungi produce several isoforms of extracellular lignin degrading enzymes, including lignin peroxidase, manganese peroxidase, and Lcc. These lignin degrading enzymes are considered as secreted enzymes, but several Lccs are intracellular enzymes.
Two Lccs (Lcc1 and Lcc2) have been purified from L. edodes (Nagai et al., 2002 and, an Lcc-encoding gene has been cloned and characterized, and six Lcc encoding genes (lcc1-lcc6; lcc1: AB035409; lcc2: AB035410; lcc3: AB046713; Lcc4: AB446445; Lcc5: AB543788; Lcc6: AB543787) have been deposited in the DNA data bank (DDBJ) (Zhao and Kwan, 1999;Sakamoto et al., 2008Sakamoto et al., , 2009Yano et al., 2010). Lcc1 is secreted from vegetative mycelia in culture (Nagai et al., 2002) and is encoded by lcc1 (Sakamoto et al., 2008). Lcc2 is expressed in the brown gills of fruiting bodies after harvesting (Nagai et al., 2003). The putative amino acid sequence of lcc4 includes identical amino acid sequences to the N-terminal amino acid sequence of an enzymatically digested peptide of Lcc2 . We expressed the lcc4 gene heterologously in Aspergillus oryzae, and observed Lcc activity of the recombinant enzyme (Yano et al. 2009). These data suggest that lcc4 encodes Lcc2, as designated by Nagai et al. (2003), and the gene lcc2 (Accession No. AB035410) does not. Therefore, the Lcc2 purified by Nagai et al. (2003) is designated Lcc4 in this paper. Expression of lcc1 is high in vegetative mycelia (Sakamoto et al., 2008), but lcc4 is expressed only in the fruiting body and increases after harvest . Furthermore, Lcc1 cannot oxidize DOPA (Nagai et al., 2002), but Lcc4 can (Nagai et al., 2003;Yano et al., 2009). These observations collectively suggest that Lcc4 is involved in melanin synthesis after harvesting by catalyzing L-DOPA to DOPA quinone. Gill browning after harvesting of L. edodes fruiting bodies is considered to be caused by melanin synthesis due to cooperation of LeTyr and Lcc4 and their increased levels after harvest.

Cell wall degrading enzymes involved in fruiting body autolysis
Fruiting body softening occurs due to cell wall degradation. The cell wall of L. edodes is constructed of several polysaccharides, such as β-1,3-glucan, β-1,6-glucan, chitin, and chitosan (Shida et al. 1981). Thus, β-1,3-glucanase, β-1,6-glucanase, chitinase, chitosanase are involved in cell wall degradation after harvesting. Cell wall degrading enzymes found in the L. edodes fruiting body after harvest will be introduced. One of the cell wall components, β-1,3-1,6-glucan, called lentinan, is used for antitumor therapy. Lentinan degradation after harvesting is caused by an increase in β-1,3-glucanase activity after harvesting. Thus, controlling β-1,3-glucanase expression is very important to keep the lentinan content in the L. edodes fruiting body after harvest. There are few reports on cell wall degrading enzymes, such as endoglucanase and exoglucanase encoding in basidiomycetous fungi, such as in A. bisporus. However, there were fewer reports on glucanases related to senescence of mushrooms when we started our research on senescence in L. edodes. There were several studies on chitinase activity in several mushrooms, but there was no evidence of a relationship between chitinases and mushroom senescence at that time. Cell wall degrading enzymes in L. edodes fruiting bodies after are discussed in this section. www.intechopen.com
It has been reported that some of the enzymes in the GH16 family have endo--1,3glucanase activity. One GH16 family gene, mlg1, was found in fruiting bodies after harvest . Furthermore, several other GH16 family genes, mlg2 (DJ432070), ghf16.1 (DJ432068), and ghf16.2 (DJ432069) were isolated from L. edodes fruiting bodies. However, the enzymatic activities of the proteins encoded by these genes are not known; therefore, there is no clear evidence for a relationship between GH16 family enzymes and autolysis of the L. edodes fruiting body so far.

endo-β-1,6-glucanase
Some mycoparasitic fungi such as Trichoderma species produce an extracellular β-1,6glucanase, member of GH30, for attack and degradation of host cell walls during their mycoparasitic action (De la Cruz and Llobell, 1999;Djonović et al., 2006;Montero et al., 2005). However, little information is known about the physiological function and role of the fungal β-1,6-glucanases. Sakamoto et al. (2009) found one GH30 protein, ghf30 is upregulated after harvest. Furthermore, LePus30A, an endo-type -1,6-glucan hydrogenase, and classified as a member of GH family 30, was purified from L. edodes . LePus30A was the first purified basidiomycetous protein characterized as a GH 30 member . LePus30A has high levels of similarity to proteins from basidiomycetous species such as L. bicolor, S. commune and C. cinerea, suggesting that -1,6-glucanases widely conserved in basidiomycetous fungi. The transcript level of lepus30a in fruiting bodies undergoing postharvest preservation for 2-4 days was also significantly higher than at other stages of the life cycle. This result supported an important role for LePus30A in the degradation of the cell wall's complex structure during fruiting body senescence after harvest. LePus30 has no activity toward lentinan, but the enzyme showed activity for the cell wall glucans from L. edodes fruiting bodies . In addition, LePus30A degrades cell wall glucans producing glucose and -1,6-linked oligoglucosides. Therefore, LePus30A is mainly contribute to a degradation of the -1,6-glucan rich content, during fruiting body senescence. The expression of lepus30a was also observed in mycelium and young fruiting body. This result implicates that the -1,6glucanase also contributes in hyphal growth and branching, or development (Djonović et al. 2006;Moy et al. 2002).

Chitinase
As chitin is one of the fungal cell wall components, chitinase and chitin synthase are very important for morphogenesis of fungi. There are several reports on chitinase activities in basidiomycetous mushrooms (for example, Kamada et al. 1981), but few on the relationship between chitinase and fruiting body senescence. Higher chitinase activity is observed in fruiting bodies following harvest compared to the activity just after harvest (Fig. 5). Genes encoding chitin degrading enzymes were identified in fruiting bodies after harvest (chi1, Fig. 4. Scheme of degradation of -1,3-1,6-glucan after harvest of the L. edodes fruiting body www.intechopen.com chi2; Sakamoto et al., 2009). The putative amino acid sequences of chi1 and chi2 contain a motif found in the GH18 family (Fig. 6). These genes do not have significant similarity to known chitinase encoding genes, but have significant similarities to hypothetical genes in basidiomycetous genomes such as S. commune, Serpula lacrymans and Postia placenta.
A putative chitinase encoding gene has also been found in an EST sequence from L. edodes (Suizu et al. 2008); the cloned full-length gene, chi3, has a GH18 domain and chitin binding domains (Fig. 6). Expression of chi3 also increased after harvest (Fig. 7). These observations suggest that increased expression of chitinases has an important role in fruiting body Fig. 5. chtinase activity after harvest measured by using 4-MUF-chitin (Hood 1991). The X axis indicates 0: fresh fruiting body; 1: fruiting body at day 1 after harvest ; 2 fruiting body at day 2 after harvest; 3 fruiting body at day 3 after harvest; 4: fruiting body at day 4 after harvest. The Y axis indicates unit/g protein. www.intechopen.com senescence after harvest. Other genes encoding putative enzymes related to chitin modification were cloned from fruiting bodies after harvest , including chitin deacetylase (chd1) and chitosanase (cho1). The putative amino acid sequence of chd1 has a polysaccharide deacetylase domain in the middle and a serine-rich region at the C-terminus that are present in the chitin deacetylase from C. neoformans (Levitz et al., 2001;Fig 6). The DNA sequence of the chitosanase from L. edodes, cho1, has high similarity with that of A. oryzae, but there are no significantly similar sequences in the basidiomycetous genome databases available so far. Chitosan is also a cell wall component of L. edodes (Pochanavanich et al., 2002). It was reported that chitin deacetylase (chd1) and chitosanase (cho1) were upregulated after harvesting of the L. edodes fruiting body . The observation that the chi1, chi2 and cho1 genes do not have significant similarity to other basidiomycetous genes suggests that L. edodes has a unique chitin and chitosan metabolism system. These data indicate that the cell wall of the L. edodes fruiting body is possibly degraded as a result of increased glucanase and chitinase activity following harvest.

Regulation of senescence related genes
As shown above, many genes involved in fruiting body senescence increased after harvest of the L. edodes fruiting body, including those for phenol oxidases, involved in gill browning, and cell wall degrading enzymes involved in autolysis after harvest. Expression of many other genes increased after harvest of the L. edodes fruiting body ). The functions of these genes in senescence of the fruiting body are still unclear, but certainly gene expression is drastically changed after harvest. Transcription factors or chromatin remodelling could cause these drastic changes in gene expression. A gene encoding a putative transcription factor, exp1, was isolated from L. edodes fruiting bodies after harvest. The putative amino acid sequence of exp1 displays high similarity with the sequence for exp1 from C. cinerea (Accession No. AB363984; Muraguchi et al., 2008), and contains two HMG boxes in the C-terminus. Proteins that have HMG boxes have functions in transcription factor or chromatin remodeling. In this section, genes other than phenol oxidases and cell wall degradation enzymes, but expressed after harvest will be discussed.

Unknown genes increased after harvesting
Expression of numerous genes increases after harvest of L. edodes fruiting bodies, but their functions in senescence are still unknown. For example, genes encoding riboflavin forming enzyme (baw28) and malate dehydrogenase (mdh) were isolated from L. edodes fruiting bodies. The putative amino acid sequence of baw28 gene displays 53% identity to the C. albicans riboflavin forming enzyme and 33% identity to the L. edodes riboflavin forming enzyme (Accession No. AB116639) that is expressed specifically in the fruiting body (Hirano et al., 2004). The putative amino acid sequence of baw28 has a barwin-like endoglucanase domain in the N-terminus, and a serine-rich region in the C-terminus. The mdh gene identified in this study does not contain any significant motifs, but displays 34% identity to Aspergillus fumigatus malate dehydrogenase. Expression of the genes increased after harvest (Fig. 7); therefore, the genes presumably have some biological function in senescence, but their function is unclear.
Many genes upregulated after harvest in the L. edodes fruiting body have been reported but do not have significant similarities to other genes Fig. 8). On the other www.intechopen.com hand, genome sequence information in basidiomycetous fungi is increasing, so extensive new information has been found from unknown genes. One of them, a gene that has similarity to glucoamylase (Fig. 8 amy). Expression of amy1 is weak in vegetative mycelia and young fruiting bodies, but significantly increases after harvest. The ghf79 has similarity to GH79 family which includes enzymes glucronidase. The gene lup33 has high similarity to hypothetical proteins found in basidiomycetous genomes. The function of the hypothetical proteins is unclear, but expression of the gene specifically increases in the L. edodes fruiting body after harvest. On the other hand, there are several genes that have no similarity to any other genes, including hypothetical proteins in the basidiomycetous genomes available so far. These genes are L. edodes specific genes. Expression of these L. edodes unknown protein encoding genes, lup23, lup410, lup66, and lup48, increases significantly after harvest (Sakamoto et al., 2009, Fig. 8), but is weak in vegetative mycelia and young fruiting bodies. The function of these genes is still unknown, but these genes would have a specific role in senescence of the L. edodes fruiting body after harvest.

putative senescence related gene transcription regulating factor exp1
Putative transcription factors, exp1 which is up-regulated after harvest are isolated from L. edodest. As the mRNA level of exp1 was higher three days after harvest than on the day of harvest, exp1 is likely to be involved in fruiting body senescence after harvesting. The L. edodes exp1 gene, a homolog of exp1 isolated from C. cinerea (Muraguchi et al., 2008), contains two HMG boxes in the C-terminus. The HMG1/2 class proteins have been considered architectural components of chromatin that have a general role in the regulation of chromosomal functions (Thomas et al., 2001). Several proteins that have an HMG box are considered to be transcription factors, or have been shown to interact with transcription factors (Wissmüller, 2006). C. cinerea opens the cap of its fruiting body by lysing lamellae in the cap during spore diffusion, and the C. cinerea exp1 mutant cannot open its cap (Muraguchi et al., 2008). This suggests that exp1 in C. cinerea controls cap autolysis during spore diffusion by regulating the genes that encode cell wall lysing enzymes. For example, expression of tlg1, which is a homolog of one of the cell wall degrading enzymes for senescence in L. edodes, is suppressed Fig. 8. Expression pattern of unknown genes found in the L. edodes fruiting body. The Y axis represents the ratio of mRNA levels of each gene to that of gpd. The X axis indicates 1: mycelium from liquid culture. 2: young fruiting bodies under 1 cm. 3: young fruiting bodies, 1-2 cm. 4: stipe of young fruiting bodies, 2-3 cm. 5: pileus of young fruiting bodies, 2-3 cm. 6: stipe of young fruiting bodies, 3-5 cm. 7: pileus of young fruiting bodies, 3-5 cm. 8: gill of mature fruiting body; 9: gill of fruiting body at 1 day after harvest; 10: gill of fruiting body at 2 days after harvest; 11: gill of fruiting body at 3 days after harvest; 12: gill of fruiting body at 4 days after harvest www.intechopen.com in the C. cinerea exp1 mutant (Fig. 9). Cell wall lysis after spore diffusion is observed in L. edodes, and expression of the cell wall degrading enzymes EXG2, TLG1 and GLU1 increases after spore diffusion (Sakamoto et al., 2005b(Sakamoto et al., , 2006 Fig. 3). Expression of these cell wall degrading enzymes also increased after harvest, suggesting that the systems for cell wall lysis in the fruiting body after harvesting and after spore diffusion are similar in L. edodes. These results suggest that exp1 in L. edodes might control senescence of the fruiting body by regulating genes that are expressed after harvesting, such as genes that encode cell wall degrading enzymes. Fig. 9. expression pattern of the tlg1 in exp1. The Y axis represents the ratio of mRNA levels of the tlg1 to that of gpd.

Genes downregulated after harvesting
It has been reported that a large number of genes downregulated after harvest are likely involved in normal fruiting body formation and are needed to maintain freshness of the fruiting body ). Translation, transcription and protein metabolism related genes, in addition to those involved in spore formation, cytoskeleton, and cell cycle are downregulated after harvest, and many more transcription factors are also downregulated after harvest ). Interestingly, genes identified among those upregulated after harvest are completely different from those in the downregulated genes. This suggests that gene transcription is drastically altered after harvesting of the fruiting body.

Transcription, translation, and protein metabolism related genes
Many transcription, translation and protein metabolism related genes are down-regulated after harvesting. Several RNA related genes downregulated after harvest, such as the Pumilio family RNA binding protein, RNA binding protein 5-like protein, ATP-dependent helicase and pre-mRNA splicing factor ). These RNA related proteins are involved in transcription and translation (de Moore et al., 2005;Rogers et al., 2002); therefore, expression of many genes might be changed after harvesting as a result of suppression of these transcription and translation related genes. Heat shock proteins (i.e., Hsp70 and Hsp90), chaperonin and calnexin, which are involved in the proper translation and folding of proteins to control protein quality (Saibil, 2008;Spiess et al., 2004;Williams, 2006), were also down-regulated. This suggests that the protein quality control system in the www.intechopen.com L. edodes fruiting body is less effective following harvest. Hsp70 and Hsp90 were especially highly expressed in young fruiting bodies (Fig. 10); therefore, these proteins have an important function in normal fruiting body development. Several proteases and peptidases, such as aspartic protease (pro1, pep1), metallopeptidase MepB and gamma-glutamyltranspeptidase were downregulated after harvest , and proteasome-related protein encoding genes such as proteasome 26S, proteasome 26S subunit alpha type 6 and proteasome 26S ATPase subunit 3 were also downregulated after harvest ). These Fig. 10. Putative transcription factors found in the L. edodes fruiting body. The Y axis represents the ratio of mRNA levels of each gene to that of gpd. The X axis indicates 1: mycelium from liquid culture. 2: young fruiting bodies under 1 cm. 3: young fruiting bodies, 1-2 cm. 4: stipe of young fruiting bodies, 2-3 cm. 5: pileus of young fruiting bodies, 2-3 cm. 6: stipe of young fruiting bodies, 3-5 cm. 7: pileus of young fruiting bodies, 3-5 cm.
www.intechopen.com proteins comprise a proteasome complex that is involved in protein degradation (Wolf and Hilt, 2004). Decreased protease and proteasome activity would extend the life of proteins expressed after harvest. These data suggest that transcription, translation and posttranslational protein metabolism are drastically altered in fruiting bodies after harvest.

Mitosis and meiosis related genes
Many mitosis and meiosis related genes are down-regulated after harvesting. Several meiosis related proteins, such as a meiotic recombination related protein encoding gene, dmc1, show altered expression in harvested fruiting bodies . DMC1 is a recombinase that is required for DNA pairing during recombination. Two DMC1encoding genes were previously identified in C. cinerea, namely Rad51 (Stassen et al., 1997) and LIM15 (Namekawa et al., 2005). Whereas Rad51 is involved in both mitosis and meiosis, LIM15 is specifically involved in meiosis, being expressed during meiosis and disappearing immediately after meiosis (Nara et al., 1999). The putative amino acid sequence encoded by dmc1 has significant similarity to LIM15/DMC1 in C. cinerea. The dmc1 gene was found to be transcribed abundantly in the gills of fresh fruiting bodies and was downregulated after harvesting . These data suggest that dmc1 is specifically involved in meiosis, and is downregulated after spore formation. Expression of the cell division related genes septin and cdc48 reportedly decreased after harvest . Both of these genes have important functions during cell division (Lindsey and Momany, 2006;Cheeseman and Desai, 2004). Septins are GTPases that form filaments in fungi and animals and are involved in membrane trafficking, coordinating nuclear divisions, and organizing the cytoskeleton (Lindsey and Momany, 2006). cdc48 has a variety of cellular functions; for instance, it is implicated in the membrane fusions that occur after mitosis to reassemble the endoplasmic reticulum and the Golgi apparatus (Cheeseman and Desai, 2004). Genes encoding the cytoskeleton related proteins actin and beta-tubulin were also downregulated after harvest . These genes are essential for progression of cytokinesis (Nanninga, 2001). This suggests that the frequency of cell division decreases after harvest.

Putative transcription factors identified by reverse subtraction
Several putative transcription factors are downregulated after harvest of the L. edodes fruiting body. One putative transcription factor gene, hlh1, which contains a basic helixloop-helix motif involved in DNA binding (Fig. 11), was also cloned . Basic helix-loop-helix proteins are a group of eukaryotic transcription factors that exert a determinative influence on a variety of developmental pathways (Littlewood and Evan, 1995). The putative amino acid sequence of hlh1 contains a nuclear localization signal and displays high similarity to a hypothetical protein from C. cinerea. The hlh is specifically expressed in young fruiting bodies (Fig. 10). Expression of ftf1, ftf2, ftf3, which have a fungal specific transcription factor domain (Fig. 11), was observed in L. edodes fruiting bodies . The putative amino acid sequences of these genes have a similar structure to the priB gene product, which is involved in fruiting body development in L. edodes (Miyazaki et al., 2004). The priB gene is transcribed specifically in the L. edodes fruiting body, and regulates fruiting body-specific genes (Kaneko et al., 2001;Miyazaki et al., 2004).

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The ftf1 ftf2, and ftf3 are highly transcribed in fresh fruiting bodies and their mRNA levels decreased after harvesting ( Fig. 10; Sakamoto et al. 2009), suggesting that the genes regulates genes involved in fruiting body development, but does not regulate genes involved in fruiting body senescence. Fig. 11. Putative transcription factors found in the L. edodes fruiting body The putative protein product of hsf1 has no significant similarity to other known proteins, but has significant similarities to hypothetical proteins in several basidiomycetous fungi, such as C. cinerea and Serpula lacryman. The putative amino acid sequence of hsf1 has a helixturn-helix DNA binding domain for binding to a heat shock element (Fig. 11) that is conserved in the upstream region of heat shock proteins (Pirkkala et al., 2001). Its expression pattern was different from heat shock proteins (Fig. 10); therefore, hsf might not directly regulate the expression of heat shock proteins. The putative transcription factor, zin1 and zin2, were also highly transcribed in fresh fruiting bodies, with a decline in expression after harvesting ( Fig. 10; Sakamoto et al. 2009). The zin1 and zin2 gene products contain a zinc finger motif (Fig. 11). The putative amino acid sequence of zin2 has a MYND-type zinc finger motif as well as significant similarity with the zf-MYND domain containing protein in L. bicolor that is upregulated by infection with ectomycorrhiza (Martin et al., 2008), but there is no significant similar sequence to the zin2 gene sequences in other basidiomycetous genomes available so far. In contrast, there are other basidiomycetous proteins that have a MYND domain; for example, Fuz1 from U. maydis contains an MTND domain and is www.intechopen.com involved in cell morphogenesis (Chew et al., 2008). These results suggest that zin2 is involved in fruiting body development. zip is also specifically expressed in fruiting bodies but not in vegetative mycelia (Fig. 10). The putative amino acid sequence of zip1 has a leucine zipper DNA sequence, which is found in several transcription factors, and also has an Aft osmotic stress domain (Fig. 11). The zip does not have any significant similarity to known transcription factors but has significant similarities to hypothetical proteins in several basidiomycetous fungi, such as C. cinerea and S. lacrymans.
A homolog of the blue light receptor white collar 2 (WC2) in N. crassa was also found in L. edodes fruiting bodies. In N. crassa, WC2 acts as a component of the blue light receptor by interacting with white collar protein 1 (WC1). WC2 in L. edodes was cloned and characterized; the protein has been designated PHRB; Sano et al., 2009). Genes similar to WC1 have also been reported in L. edodes (PHRA; Sano et al., 2008) and in C. cinerea (dst1; Terashima et al., 2005). The C. cinerea dst1 mutant cannot form a mature cap under normal light/dark conditions, and the shape of the fruiting body of the dst1 mutant is very similar to that formed by the wild-type mushroom when grown in complete darkness (Terashima et al., 2005). Since WC2 and PHRA in L. edodes interact with each other, they could act as a blue light receptor in L. edodes that regulates fruiting body development in the light (Sano et al., 2010). Gene disruptionof the WC2 homolog in C. cinerea results in a similar phenotype to that of dst1 (Nakazawa et al. 2011).
These genes might not be directly involved in senescence of the L. edodes fruiting body after harvest, but these putative transcription factor genes presumably would be involved in normal fruiting body morphology. A decrease in expression of the putative transcription factor genes would influence morphology after harvest.

Future perspectives
As shown above, numerous genes are related to senescence of the L. edodes fruiting body after harvest. However, the mechanism of senescence of the L. edodes fruiting body after harvest is not fully understood. To understand the mechanism involved, an understanding of total gene expression changes after harvest is needed. Transcriptome analysis such as microarray and SAGE analysis is useful to understand total gene expression changes. In Lentinula edodes, there are several reports of transcriptome analysis, such as an EST study (Suizu et al., 2009) and SAGE analysis (Chem 2011). For SAGE analysis, genomic sequence data are needed to increase accuracy. Public genomic sequence data of L. edodes are not yet available so far, but genomic sequencing will be easier than before using a next-generation genome sequencer, such as the Illumina GA IIx. Furthermore, SAGE analysis is optimized for next-generation sequencing to obtain a larger amount of sequence tagged data (Matsumura et al. 2003). Therefore, gene expression profile changes after harvesting of the L. edodes fruiting body will be revealed in more detail by using a combination of genome sequence analysis and Super-SAGE (Matsumura et al. 2003) with a next-generation sequencer. In C. cinerea, autolysis after spore dispersion is similar to that of L. edodes, and public genomic sequence data of C. cinerea are available, so it is possible to compare gene expression profiles during autolysis in C. cinerea and L. edodes. Such research will provide a basic understanding of mushroom senescence.
To prove the function of genes, gene disruption or gene silencing studies will be needed. Gene disruption systems have been constructed in several mushrooms such as C. cinerea (Nakazawa et al. 2011). A homologous gene recombination system has also been constructed (Irie et al. 2003), but a gene disruption system by homologous recombination has not yet been constructed in L. edodes. However, gene silencing by RNAi has succeeded in L. edodes . Therefore, genes upregulated after harvest will be knocked down by RNAi, which will reveal the functions of the genes, such as exp1 and exg2, in senescence. This research will provide useful data for breeding strains with fruiting bodies that remain fresh for a longer period after harvest.

Conclusion
After harvesting of the L. edodes fruiting body, drastic gene expression changes occur. In particular, expression of phenol oxidases (Tyrs and Lccs) and cell wall enzymes (glucanases and chitinases) increase after harvest. This suggests that a gene regulation system for senescence exists in L. edodes. exp1, which is a putative transcription factor or chromatin remodeling related protein, is one of the candidates for regulation of the drastic gene expression changes involved in senescence. The process would include programmed cell death, but there are few studies on the relationship between programmed cell death and mushroom morphology. Therefore, studies on senescence in mushrooms will be important not only for applied agricultural science, but also for basic science.

Acknowledgment
The authors thank Dr. Kamada, Okayama University and Dr. Muraguchi, Akita prefectural University for giving us the exp1 mutant and related information in C. cinerea. The authors also thank to previous members of Microorganism lab. in Iwate biotechnology Research Center.  The book discusses the novel scientific approaches for the improvement of the food quality and offers food scientists valuable assistance for the future. The detailed methodologies and their practical applications could serve as a fundamental reference work for the industry and a requisite guide for the research worker, food scientist and food analyst. It will serve as a valuable tool for the analysts improving their knowledge with new scientific data for quality evaluation. Two case study chapters provide data on the improvement of food quality in marine and land organisms in the natural environment.