Hydrogen-rich water mediates redox regulation of the antioxidant system, mycelial regeneration and fruiting body development in Hypsizygus marmoreus
Introduction
Hypsizygus marmoreus (Peck) H.E. Bigelow, also known as shimeji or beech mushroom, is a popular edible mushroom with a small, light-brown or white umbrella-shaped cap (Akavia et al., 2006). Cultivated worldwide, H. marmoreus has exceptional medicinal applications, including as an antitumor agent, in addition to its popular/exotic culinary status (Akavia et al., 2009, Matsuzawa et al., 1998). H. marmoreus is an important industrialized mushroom in Asia. According to data from the Chinese Edible Fungi Association, the total production of H. marmoreus in China in 2016 was 360,348.78 tons, ranking third among industrialized mushrooms; this production suggests a rapidly increasing demand for this mushroom (Zhang et al., 2016).
For cultivation, mechanical injury such as scratching is needed to stimulate fruiting body production in mushrooms, such as H. marmoreus. In both plants and fungi, mechanical damage-induced changes are mediated by elaborate signaling networks, including receptors/sensors, calcium (Ca2+) influx, ATP release, kinase cascades, reactive oxygen species (ROS), and oxylipin signaling (Hernández_Oñate et al., 2012). For example, Zhang et al. (2015a) studied expression of different genes at four developmental stages of H. marmoreus and found that ROS had important roles in fruiting body development. Moreover, enhancing laccase activity induced H. marmoreus primordium initiation and increased fruiting body production (Zhang et al., 2016, Zhang et al., 2015a). However, the developmental mechanism of H. marmoreus has remained unclear, and the defects of basic biological studies have hindered further development of the commercial value of H. marmoreus.
ROS are particularly important for maintaining the balance between cell proliferation and differentiation. In fungi, important biological roles of ROS in growth and development have been demonstrated (Brun et al., 2009, Takemoto et al., 2007, Zheng et al., 2015). The link between ROS and differentiation in fungi was first established in Aspergillus nidulans, whereby NADPH oxidase A (NoxA) was shown to be necessary for cleistothecium development (Lara-Ortiz et al., 2003). Furthermore, ROS balance regulates Epichloë festucae hyphal morphogenesis and growth in culture and in plants (Takemoto et al., 2011). During the sclerotia formation of Botrytis cinerea, ROS play important roles, which are the basis for fruiting body development (Kim et al., 2011, Segmueller et al., 2008). Regarding mushrooms, ROS have also been shown to act as important factors in the mycelial growth and fruiting body development of Ganoderma lucidum (Mu et al., 2014, Ren et al., 2016, Zhang et al., 2017). Although ROS production is clearly crucial for signaling, other than a general association with mitogen-activated protein kinase (MAPK) cascades, the pathways involved have yet to be elucidated. Regardless, the scarcity of studies on the ROS system has hindered a deeper understanding of how the redox balance regulates the growth and development of H. marmoreus.
Research on gaseous hydrogen has progressed from focusing on its role as a fuel to its role as matter that is able to regulate biological and pharmacological functions. For instance, hydrogen-rich water (HRW) inhibited glucose- and α, β-dicarbonyl compound-induced ROS production in rat kidney homogenates in vitro (Katakura et al., 2012). HRW re-establishes the ROS homeostasis of radish sprouts by blocking the UV-A-induced increase in hydrogen peroxide (H2O2) and superoxide anion (O2−) accumulation and by enhancing the UV-A-induced increase in superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities (Su et al., 2014). HRW also significantly decreased the ROS content, maintained the biomass and polar growth morphology of mycelium, and decreased secondary metabolism in G. lucidum under HAc-induced oxidative stress (Ren et al., 2016). By regulating ROS levels under oxidative stress, HRW increased the antioxidant activity and mycelial growth of H. marmoreus (Zhang et al., 2015a, Zhang et al., 2015b). However, there is no research to date on the effects of HRW on H. marmoreus fruiting body development.
Despite being one of the most important industrial mushrooms in Asia, H. marmoreus development is a long process. Our major research objective is to identify effective and safe methods to increase fruiting body production of H. marmoreus. As a reducer gas, it is unknown whether H2 has a function in H. marmoreus fruiting body regulation. Thus, the aim of the current study was to examine the effect of HRW on fruiting body development and mycelial regeneration after mechanical damage. We also investigated the possible mechanism by which HRW affects ROS levels, the activities of the key enzymes involved in oxidative metabolism and the expression levels of ROS, Ca2+, MAPK and oxylipin signaling pathway-related genes.
Section snippets
Sample preparation and collection
H. marmoreus samples were obtained from the China General Microbiological Culture Collection Center (Beijing) (No. CGMCC5.01974) and grown at 25 °C on potato dextrose agar (PDA) medium for 14 d. The fungus was transferred once to a new PDA plate for 14 d, and HRW or distilled water was added for 12 h. HRW or the distilled water was removed, and the samples were cut into a grid pattern with a scalpel to detect ROS levels.
For cultivation experiments, primary cultures were grown in potato dextrose
Effects of HRW on the fruiting body development of H. marmoreus
The effects of HRW on H. marmoreus fruiting body development were investigated (Fig. 1). On the 4th d, the mycelial regeneration stage, the mycelia in the HRW groups had regenerated more quickly and were thicker than those in the CK group (Fig. 1A); on the 8th d, the mycelial pigmentation stage, the mycelia in the HRW groups had completed pigmentation, whereas the mycelia in the CK group had not completed pigmentation (Fig. 1A). In addition, the HRW groups also developed earlier than the CK
Discussion
Due to their important commercial value, large basidiomycete fungi have attracted much attention. However, the poor understanding of their biology has limited the further development of the commercial value of these fungi. In 2014, Zhang et al. (2014) sequenced the transcriptomes of the four developmental stages of H. marmoreus. In addition, it was found that laccase has important roles in primordium initiation and that KA might regulate primordium initiation of H. marmoreus by influencing ROS
Conclusions
The results of this study indicated that HRW regulates H. marmoreus on the oxidative stress resistance, mycelial regeneration and fruiting body development after mechanical injury. The mechanical damage induced by scratching caused an apparent increase in ROS production, and HRW increased antioxidant enzyme (SOD, CAT, GR and GPX) activities and antioxidant (AsA and GSH) levels. Furthermore, HRW might activate the MAPK, Ca2+ and oxylipin signaling pathways by regulating the ROS balance, which is
Funding
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31601802 and 31401932), the Youth Talent Development Plan of Shanghai Municipal Agricultural System of China (Grant No. 20160111) and the earmarked fund for Shanghai Modern edible fungi-industry Technology Research System (Grant No. 201709).
Acknowledgments
We thank Jing Zhao for giving some helps in performing the experiments and thank the AJE company for providing language editing help.
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