Comparative Proteomic Analysis within the Developmental Stages of the Mushroom White Hypsizygus marmoreus

(1) Background: The white Hypsizygus marmoreus is a popular edible mushroom in East Asia markets. Research on the systematic investigation of the protein expression changes in the cultivation process of this mushroom are few. (2) Methods: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of six groups of samples collected in different growth stages. A total of 3468 proteins were identified. The UpSetR plot analysis, Pearson correlation coefficient (PCC) analysis, and principal component (PC) analysis were performed to reveal the correlation among the six groups of samples. The differentially expressed proteins (DEPs) were organised by One-way ANOVA test and divided into four clusters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to divide the DEPs into different metabolic processes and pathways in each cluster. (3) Results: The DEPs in cluster 1 are of the highest abundance in the mycelium and are mainly involved in protein biosynthesis, biosynthesis of cofactors, lipid metabolism, spliceosome, cell cycle regulation, and MAPK signaling pathway. The DEPs in cluster 2 are enriched in the stem and are mainly associated with protein biosynthesis, biosynthesis of cofactors, carbon, and energy metabolism. The DEPs in cluster 3 are highly expressed in the primordia and unmatured fruiting bodies and are related to amino acids metabolism, carbon and carbohydrate metabolism, protein biosynthesis and processing, biosynthesis of cofactors, cell cycle regulation, MAPK signaling pathway, ubiquitin-mediated proteolysis, and proteasome. The DEPs in cluster 4 are of the highest abundance in the cap and are mainly associated with spliceosome, endocytosis, nucleocytoplasmic transport, protein processing, oxidative phosphorylation, biosynthesis of cofactors, amino acids metabolism, and lipid metabolism. (4) Conclusions: This research reports the proteome analysis of different developmental stages during the cultivation of the commercially relevant edible fungi the white H. marmoreus. In the mycelium stage, most of the DEPs are associated with cell proliferation, signal response, and mycelium growth. In the primordia and unmatured fruiting bodies stage, the DEPs are mainly involved in biomass increase, cell proliferation, signal response, and differentiation. In the mature fruiting body stage, the DEPs in the stem are largely associated with cell elongation and increase in biomass, and most of the DEPs in the cap are mainly related to pileus expansion. Several carbohydrate-active enzymes, transcription factors, heat shock proteins, and some DEPs involved in MAPK and cAMP signaling pathways were determined. These proteins might play vital roles in metabolic processes and activities. This research can add value to the understanding of mechanisms concerning mushroom development during commercial production.

Genetic structure and evolutionary diversity of mating-type (MAT) loci in Hypsizygus marmoreus

The mating compatibility in fungi is generally governed by genes located within a single or two unlinked mating type (MAT) loci. Hypsizygus marmoreus is an edible mushroom in the order Agaricales with a tetrapolar system, which contains two unlinked MAT loci-homeodomain (HD) transcription factor genes and pheromone/pheromone receptor genes (P/R). In this study, we analyzed the genetic structure and diversity of MAT loci in tetrapolar system of H. marmoreus through sequencing of 54 heterokaryon and 8 homokaryon strains. Although within the HD loci, the gene order was conserved, the gene contents were variable, and the HD loci haplotypes were further classified into four types. By analyzing the structure, phylogeny, and the HD transmissibility based on the progeny of these four HD mating-type loci types, we found that they were heritable and tightly linked at the HD loci. The P/R loci genes were found to comprise three pheromone receptors, three pheromones, and two pheromone receptor-like genes. Intra- and inter-specific phylogenetic analyses of pheromone receptors revealed that the STE3 genes were divided into three groups, and we thus theorize that they diverged before speciation. Comparative analysis of the MAT regions among 73 Basidiomycete species indicated that the diversity of HD and P/R loci in Agaricales and Boletales may contribute to mating compatibility. The number of HD genes were not correlated with the tetrapolar or bipolar systems. In H. marmoreus, the expression levels of these genes at HD and P/R loci of compatible strains were found higher than in those of homonuclear/homokaryotic strains, indicating that these mating genes acted as switches for mating processes. Further collinear analysis of HD loci in interspecific species found that HD loci contains conserved recombination hotspots showing major rearrangements in Coprinopsis cinerea and Schizophyllum commune, suggesting different mechanisms for evolution of physically linked MAT loci in these groups. It seems likely that gene rearrangements are common in Agaricales fungi around HD loci. Together, our study provides insights into the genomic basis of mating compatibility in H. marmoreus.

The Utilisation of Pholiota nameko, Hypsizygus marmoreus, and Hericium erinaceus Spent Mushroom Substrates in Pleurotus ostreatus Cultivation

The feasibility of utilising spent mushroom substrates (SMSs) as a growing medium component for Pleurotus ostreatus cultivation was investigated. P. ostreatus was cultivated on traditional wheat straw (control) and wheat straw substrate supplemented with SMSs from Pholiota nameko (N-SMS), Hypsizygus marmoreus (M-SMS), and Hericium erinaceus (E-SMS) in varying supplementation rates (10%, 20%, and 30%). The yield, biological efficiency (BE), dry matter, and protein content of P. ostreatus fruiting bodies grown on ten substrates were evaluated. Significant differences in yield, BE, protein content, and dry matter of P. ostreatus were found among the studied substrates. The highest yield was recorded in 20% E-SMS (254.33 g), 20% N-SMS (253.43 g), and 10% E-SMS (251.67 g). The biological efficiency ranged from 66.48% (30% M-SMS) to 72.67% (20% E-SMS) and followed a similar trend to yield. The highest protein content was recorded in 30% M-SMS (29.93 g∙100 g dry weight−1). The highest dry matter of P. ostreatus was noticed in 30% of M-SMS (23.74 g) and 10% of M-SMS (23.06 g). Therefore, the spent mushroom substrates of Ph. nameko, H. marmoreus, and H. erinaceus could be used as a potential, low-cost sustainable alternative (10–30%) and as a renewable component of traditional growing media for P. ostreatus cultivation.

Combined Effects of Amino Acids in Garlic and Buna-Shimeji (Hypsizygus marmoreus) on Suppression of CCl4-Induced Hepatic Injury in Rats

The combination of the garlic-derived amino acid, S-allyl-l-cysteine sulfoxide (ACSO), and ornithine or arginine on CCl4-induced hepatic injury was examined. After investigating the effectiveness of the mixture of ACSO and ornithine or arginine in preventing hepatic injury in vivo, an extract rich in ACSO and ornithine was prepared by converting arginine in garlic to ornithine by arginase from Hypsizygus marmoreus (buna-shimeji), after screening the productivity of ornithine among 12 kinds of mushrooms. Co-administration of ACSO with ornithine or arginine suppressed the increase in aspartate transaminase, alanine transaminase, and thiobarbituric acid reactive substance, and the decrease in glutathione S-transferase and cytochrome p450 2E1 activities after CCl4 injection more effectively than a single administration of ACSO. All extracts prepared from garlic and buna-shimeji with low and high contents of ACSO and arginine or ornithine significantly suppressed CCl4-induced hepatic injury in rats. Considering that ACSO is tasteless, odourless, and enhances taste, and ornithine has a flat or sweet taste and masks bitterness, the extract rich in ACSO and ornithine from garlic and buna-shimeji could be considered a potential antioxidant food material that can be added to many kinds of food to prevent hepatic injury.