Transcription of laccase and cellulase genes of Lentinula edodes was examined during growth and development under different temperature and moisture levels on a sawdust-based substrate. RNA was extracted from samples of mycelium and fruit bodies at various stages of development and gene expression was determined by competitive RT-PCR. The level of laccase transcripts was maximal during the mycelial growth stage, and then declined rapidly at the fruiting stage. In contrast, the cellulase transcript level peaked at the veil–break stage during fruit body development. Gene expressions for laccase and cellulase were triggered by microclimatic changes, specifically lower temperature and osmotic pressure.
Lentinula edodes (Berk.) Pegler (shiitake) is one of the traditionally important cultivated edible mushrooms in the world. In 1997, worldwide production of shiitake reached 1 564 400 ton, or about 25.4% of the total mushroom supply . Altering various factors, e.g., genotype, culture substrates (natural logs and sawdust-based substrate), nutrient supplements and environmental conditions [2–5] have improved cultivation efficiency of L. edodes. These improvements have contributed to a consistent market supply of high quality mushrooms. In particular, use of sawdust-based cultivation as a replacement for natural logs has contributed to expanding production and consumption of L. edodes. Sawdust based substrates usually consist of hardwood sawdust, rice bran, wheat bran, ground corncobs and other agricultural waste materials. Our work has led to a wider acceptance of sawdust-based substrates by commercial growers [6–9].
The secreted enzymes of L. edodes include both hydrolases and oxidases. The activities of two of these enzymes, laccase (EC 188.8.131.52) and cellulase (endoglucanase, EC 184.108.40.206), are strongly regulated during fruit body development [10,11]. We have already examined the transcriptional regulation of laccase and cellulase genes in L. edodes. Here, the effect of temperature and substrate moisture content was tested in regard to the mRNA expression for laccase and cellulase associated with mycelial growth and fruit body development.
2Materials and methods
The strain used in this study was L. edodes (KS-58: stock culture of Kyushu University) originally from a commercial source. The strain was subcultured on potato dextrose agar at 4°C at 6 mo intervals. Spawn was prepared by incubating a mycelial culture for 14 days at 23°C on Quercus acutissima sawdust-based substrate. For mushroom production substrate, ingredients (sawdust, rice bran, wheat bran and corncob meal) were mixed in the ratio of 7:1:1:1 and water was added to raise final moisture content to 60%. The mixture was packed into 1600-ml capacity polypropylene bottles and sterilized at 1.2 kg cm−2 for 30 min. Cooled substrate then was spawned and incubated at 22°C for 90 days.
At the end of the incubation period, temperatures were lowered (chilling) from 22°C to 15°C by transferring the colonized substrate to a separate incubator. Relative humidity (RH) was maintained at 90% and 3000 lux illumination was provided for 12 h daily. Fruit body formation was initiated 7 to 10 days after transfer to the lower temperature.
Seven growth development phases were specified for sampling and were characterized as follows: day 90, colonization (C); day 94, mycelial aggregate formation (A); day 98, pin formation (P); day 100, button (B); day 102, veil–break (V); day 104, senescence (S); day 140, second flush (2F). At day 130, substrates were soaked in water (18°C) for 24 h. The water soaking treatment induced fruit body initiation for the second flush. Water potential in the cultures was determined as described previously . A Wescor psychrometer HR-33T equipped sample chamber CF-52 was used in all experiments.
Ergosterol content was measured to judge culture maturity, using previously reported procedures . Saponification was carried out with methanol, ethanol and KOH. Ergosterol was measured by high-performance liquid chromatography (HPLC) using a reversed-phase system consisting of a μ-Bondapak-C18 column (Waters Associates Inc.) and 100% HPLC-grade methanol.
The samples of sawdust-based substrate were homogenized in acetate-acetic buffer (pH 4.2), and the resulting supernatants were purified using an ammonium sulfate precipitation method. Crude enzyme was obtained after dialysis with a Visking tube. Quantification of laccase and cellulase activities in culture supernatants was carried out as described previously .
Laccase activity was determined using p-phenylenediamine as a substrate. Cellulase was assayed by determining the amounts of reducing sugar liberated from carboxymethyl cellulose (CMC). One unit of laccase activity was defined as a change in absorbance of 0.001 per min at 525 nm. One unit of cellulase activity was defined as the amount of enzyme required to produce 1 μmol of glucose per min.
RNA isolation and competitive RT-PCR analysis were carried out as described previously . Total RNA was isolated following procedures by Raguz et al.  using 5-g samples of colonized and fruiting sawdust-based substrates. At least three subsamples from each substrate were combined and used for each RNA isolation. The frozen samples were blended to a fine powder with CO2 (dry ice) in an electric coffee grinder. RNA was extracted using triisopropylnaphthalene sulfonate and phenol/cresol followed by ethanol precipitation.
The conditions for reverse transcription, amplification, and electrophoresis were similar to that of Smith . Primers used were 5′-GATCAGAGCTCCTATGACTG-3′ and 5′-AGTTGAGGGTGATGTGCTTAT-3′ for laccase, and 5′-TCAAGCTCCTCCTCCCAC-3′ and 5′-GGGCAGATCGTAGACAAC-3′ for cellulase. Total RNA (2 μg) was used as the template to generate first-strand cDNA. Competitive PCRs were performed with 0.1 to 100 pg of competitor genomic DNA template. Amplification was performed with the same conditions as described previously . Gels were stained with ethidium bromide (0.5 μg l−1) visualized with UV transilluminator and photographed using Polaroid 667 film .
3Results and discussion
3.1Mycelial growth and fruit body development
Ergosterol content was monitored as an indicator of culture maturity [19–22]. The content of ergosterol increased gradually prior to fruit body initiation and then peaked at the pinning stage (Fig. 1).
As expected, ergosterol content in the sawdust-based substrate accumulated through the pinning stage and then began to decline during fruit body development and maturation. This pattern indicates normal growth and development of L. edodes on the sawdust-based substrate .
3.2Activities of laccase and cellulase
Laccase activity was high during colonization, and then declined rapidly during fruit body development (Fig. 2). While laccase activity decreased to a low level at the aggregate stage, cellulase activity rose sharply. Cellulase activity peaked at the veil break stage of fruit body development. A decrease in laccase activity was associated with primordium formation while cellulase activity increased during the same time period.
Changes in laccase and cellulase have been reported during Agaricus bisporus[23–25] and L. edodes[10–12] development; the former enzyme showing high levels in vegetative mycelium and rapidly declining at the start of fruiting. Decline of laccase activity was necessary for fruit body initiation. The laccase and cellulase activities found in the sawdust-based substrates of L. edodes exposed to low temperature and high moisture conditions could be related to the rapid response for fruiting of these enzymes in the vegetative mycelium.
3.3Transcripts of mRNA of laccase and cellulase
Transcriptional regulation of laccase and cellulase mRNA was closely correlated with changes in enzyme activity (Fig. 3). The level of laccase transcripts was greatest in mycelium from colonized substrate prior to fruiting, and then declined to low levels during fruiting. For the cellulase gene, low levels of mRNAs were detected from mycelium in colonized substrate while transcript levels rose to a maximum at the veil break stage and then declined.
Temperature reduction triggered shifts in transcriptional regulation of laccase and cellulase (Fig. 4). Laccase mRNAs rapidly decreased to a low level while cellulase mRNA gradually increased through the fruiting stage.
Water potential of the substrate was estimated at various stages of mycelial and fruit body development. Water potential of the soaked substrate was approximately −0.5 MPa immediately after soaking and then gradually decreased as fruit body development continued (Fig. 5). This trend followed a typical pattern observed by previous research for fruiting substrates . Substrate rehydration also affected transcriptional regulation in a manner similar to the chilling treatment (Fig. 6).
Physiological stress can affect the initiation of fruit bodies [27,28]. Temperature reduction and substrate rehydration is a commercially used treatment for inducing fruit body production of L. edodes. This study shows the effect of environmental stimuli on laccase and cellulase gene expression in a commercially valuable mushroom. Researchers could team with commercial producers to evaluate the effects of altering the environment on mushroom production and yield by monitoring transcript levels of either cellulase or laccase. In Japan and the USA, shiitake are produced on supplemented sawdust using different cultural practices. In Japan, for example, shiitake are produced on substrate contained in bottles or bags, while in the USA, production is from substrate contained only in bags. In addition, many growers in Japan use a technique whereby the entire spawn run (approximately 60–90 days) occurs in the bottle or bag. In the USA, many growers use a much shorter spawn run period (18–23 days) in the bag followed by a browning period (28 days) outside the bag. Thus, enzyme profiles may be different for each system and total enzyme levels may vary from system to system and cycle to cycle. It would be of interest to determine if overall transcript levels were higher for a particular segment(s) of the cropping cycle and if the transcript levels could be correlated to first or second break mushroom yields.
In conclusion, the results of this study on L. edodes indicate that gene expression for laccase and cellulase correlates directly to the measurable level of extracellular enzyme activity and biosynthesis of enzyme production. The potential for commercial application of these data lies in gaining additional information on gene expression during various crop cycle periods and cropping systems. Comparison of transcript levels from various parts of the cropping cycle in various systems may allow growers to combine the most efficient components from each system.
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (C 12660153).