Pleurotus giganteus is a high-temperature wood-rotting edible mushroom. The optimal temperature for P. giganteus fruiting body production is around 25–32°C. Most commercially cultivated mushrooms are low-temperature or middle-temperature mushrooms, therefore, not suitable for non-industrial cultivation in summer. As a high-temperature mushroom, P. giganteus could cultivate in the greenhouse in summer, when most mushrooms are not suitable for cultivation. Therefore, it was widely welcomed by individual farmers. P. giganteus favored the consumers for its culinary tastes and health benefits (1). Pleurotus giganteus is rich in carbohydrates, and total protein (2), has unsaturated fatty acids (such as oleic acid, linoleic acid, and eicosadienoic acid), and lower saturated fat. Freeze-dried P. giganteus fruiting bodies showed hepatoprotective effects against thioacetamide-induced liver injury in rats (3). Phan et al. reported that the phenolics in P. giganteus showed antioxidant activity in vitro and the mushroom extracts presented neuritogenic activity (2). The lipid extracts from ethyl acetate fraction significantly inhibited the growth of Candida species, which could be further explored as antifungal agents against Candida species (4). In addition, P. giganteus also show anti-inflammatory activity, antidiabetic, antiproliferative, antidengue, and genoprotection activities (1), suggesting that P. giganteus has a health benefit for consumers.

The P. giganteus fruiting body shape of the harvest maturity is like a goblet and funnel with a long stipe and round flat pileus. The pileus has soft, meaty, and tender textures, which is suitable for cooking like other mushrooms. However, the stipe of the harvest-mature fruiting body of P. giganteus has an inedible hard shell, which has to be shaved off before cooking. Therefore, it is necessary to analyze genes and protein expression during the development of the P. giganteus the fruiting body. This information could help us better understand the molecular mechanism underlying nutrients synthesis and fruiting body texture changes (1, 5).

The growth and development of P. giganteus could be divided into three main phases: phase I (vegetable growth of mycelia in cultivation bag, including physiological maturation stage), phase II (transition phase or primordial inducing phase), and phase III (fruiting body development phase, from primordial to mature fruiting body). In phase I, P. giganteus secreted lignocellulolytic enzymes into the solid media. Polymers of solid substrates were depolymerized by these enzymes into small molecules, which were absorbed by P. giganteus mycelia. After the mycelium occupies the whole cultivation bag, P. giganteus undergo a further cultivation period to reach physiological maturation (6). In phase II, the cultivation environment was dramatically changed. The soil was added to the top of the open cultivation bag (7), the water that was lost during phase I was replenished, temperature stimulation was provided, and light exposure was necessary. Changed environmental factors stimulate the formation of hyphal knots and primordia. In phase III, primordial differentiated into pileus and stipe, which finally form a mature fruiting body. In the present study, phase III of P. giganteus fruiting body development was selected for further analysis.

The fruiting body development of many edible mushrooms has been investigated by transcriptomic or proteomic analysis, including Dictyophora indusiata (8, 9), Agrocybe aegerita (10), Lentinula edodes (11, 12), Hypsizygus marmoreus (13, 14), Flammulina filiformis ((15, 16)), Stropharia rugosoannulata (17, 18), Pleurotus tuoliensis (19), Pleurotus eryngii (20, 21), Botrytis cinerea (22), Ganoderma lucidum (23), et al. In a previous study, we found that the protein expression patterns between the cap and stipe of mature Stropharia rugosoannulata show a lower correlation (with Pearson correlation coefficients between 0.50 and 0.58) with each other. Proteins related to carbon metabolism, energy production, and stress-response-related biological processes had higher abundances in stipe tissue compared with those in the cap, while proteins associated with fatty acid synthesis and mRNA splicing showed higher expression levels in the cap (18). Our previous research about H. marmoreus indicated that proteins involved in biomass increase, cell proliferation, signal response, and differentiation might play key roles in fruiting body development (13). Omics research of the fruiting development could help us understand the molecular mechanism underlying this important biological process and mine key genes responsible for development. However, the differential expression analysis of P. giganteus development was not reported to date.

Recently, a high-quality genome of P. giganteus was sequenced and assembled by our lab (7). The genome size of P. giganteus is ~ 40.00 Mb in 27 contigs. Comparative genomic and phylogenetic analysis revealed that P. giganteus belongs to Pleurotus species with a closer relationship to Pleurotus tuber-regium and Pleurotus citrinopileatus, which also have a funnel shape fruiting body. CAZymes analysis confirmed the robustness of the lignocellulose degradation capacity of P. giganteus. However, the development information about this mushroom especially at a molecular level is still scanty. In the present study, comparative transcriptome and proteome analysis of the P. giganteus fruiting body in four different developing stages were performed to obtain a comprehensive and systematic understanding of the morphological development process and nutritional changes of the fruiting body underlying molecular mechanisms.

Mushroom fruiting bodies are highly complex multicellular structures in fungi, which protect, produce, and disperse sexual spores (24, 25). Fruiting bodies are the main edible parts of mushrooms. For most mushrooms, the ripening time from primordial to harvested (commercial-mature) fruiting body is only 7–10 days, which is only a small part of the overall production cycle. However, this period has a great influence on the commercial quality of the mushrooms. Therefore, mushroom fruiting body development is one of the core research field of mushrooms. The development of mushroom fruiting bodies has extensively been studied at the omics and physiological level (26–38). Our current knowledge of the biological process and molecular mechanism of fruiting body development in P. giganteus is limited. In this study, comparative transcriptomic and proteomic analyses of P. giganteus fruiting body development were performed for the first time.

If we assume that the number of DEGs between two groups represents the differentiated degree between the two samples, the results indicated that the degree of differentiation between the primordium stage and the elongation stage was low while the degrees of fruiting body differentiation between elongation stage, harvest stage, and maturity stage was high, especially in the stem tissue. The morphological change is inconsistent with the number of DEGs. The pileus and stem tissues differentiated from the elongation stage and the gills were first developed in the harvest stage. Continuous development of the fruiting body could be observed from the harvest stage to the maturity stage. The color and shape of the fruiting body were changed and harder shells and flakes were observed in the stem at this stage. The differential expression of genes could reflect these traits. Liu et al. reported that transcriptomic analysis of Flammulina filiformis showed that more DEGs were observed between pileus tissue than stem tissue (15). Different from P. giganteus, the stem of F. filiformis is edible and does not have a dark color and hard shell. The results indicated the metabolic processes in the P. giganteus stem are more complex than those in F. filiformis.

Compared with the PR group, genes encoding Cytochrome P450 monooxygenase and hydrophobin proteins were significantly enriched in up-regulated genes in the EL group. The expression of Cytochrome P450 and hydrophobin proteins during fruiting body development is conserved in fungal (15, 25). Liu et al. (15) reported that genes encoding hydrophobins were significantly up-regulated in the primordium stage compared with vegetative mycelium. Hydrophobins were self-assembled into a rodlet layer on the cell surface, conferring hydrophobic surfaces to hyphae that hinder soaking of fruiting bodies with water. Hsu et al. (39) reported Cytochrome P450 genes were strongly expressed during the formation of fruiting bodies. The mutation of a cytochrome P450 gene in Coprinus cinereus affected the pattern formation in the development of fruit body primordia (40). The expression of several cytochrome P450 genes was significantly changed during the development of the Hypsizygus marmoreus fruiting body (14). All these results indicated that these Cytochrome P450 and hydrophobin genes play key roles in the initial developmental stage in P. giganteus fruiting body development.

Carbon metabolism is the central metabolism during fruiting body development, especially in the pileus. The volume of pileus changed the most during the development. As we know that the main element in fungal mycelia is carbon. Indeed, the expression of carbon metabolism-related genes becomes more active with the development of the P. giganteus fruiting body (Figure 4). The expression of carbon metabolism genes is related to the increasing volume of the fruiting body.

It is worth noting that several inconsistent data were observed in the present study, including the low correlation between gene expression in the transcriptional level and translational level and the negative correlation between protein concentration and nitrogen metabolic. Poor correlation between transcriptomic and proteomic data revealed that the gene expression at transcriptional and translational levels is quite different. This kind of poor correlation was common and also observed in other research (41, 42). The concentration of proteins was influenced by many factors, including mRNA expression level, post-transcriptional regulation, translation regulation, and post-translation modification. In addition, mRNA and protein transportation between different cells will also influence the expression levels of the same gene. Different sample processing procedures, especially for proteomic analysis, could also influence the correlation between transcriptome and proteome data. However, the correlations between different protein extraction and peptide digestion methods are more than 0.9 (43). Therefore, some reasons beyond sample processing must have played a key role in the poor correlation.

Unexpectedly, from the analysis of gene and protein expressions between pileus and stem in the harvest stage (Figure 5), we found that 70 (17.5% of all 400) up-regulated DEGs in the HAP group were secreted proteins, while only 13 (4.3% of all 300) up-regulated DEPs in PHAP group were secreted proteins. This contradiction between high proportion of secreted proteins in DEGs and low proportion of secreted proteins in DEPs could be explained by transportation of mRNA.

CAZymes play key roles in substrate utilization in edible mushrooms which provided carbon and nitrogen sources for mycelia growth and fruiting body development. Besides, CAZymes also play important roles in fruiting body development as reported in omics studies (11, 44, 45). We analyzed all the 489 predicted CAZymes in the P. giganteus genome, 176 (36.0%) CAZymes genes were among the DEGs described in Figures 1 and 5 (Supplementary Table S10). CAZymes analysis between different groups indicated that most lignin modification enzymes (LMEs) in AA1 and AA2 families down-regulated with the development of the fruiting body. However, most of the CAZymes related to cellulose and hemicellulose degradation (from AA9, GH11, GH5, GHJ7, GH9, GH51, GH22) were up-regulated with the development of the fruiting body. As shown in Figure 7A, cellulose and hemicellulose degradation genes from GHs family were mainly upregulated in PR stage and maturation stage (MDBPgig11_11494, MDBPgig11_05949, MDBPgig11_02040, MDBPgig11_13109, MDBPgig11_04488, MDBPgig11_04779). Lytic polysaccharide monooxygenases (LPMOs) from AA9 families are responsible for the degradation of cellulose, most of the LPMOs were also highly expressed in PR and maturation stages (Figure 7B), which is in consistent with that of DEGs from GHs families.

Considering the poor correlation between transcriptome and proteome data, the unusually high proportion of secreted DEGs in fruiting body samples, and the CAZymes analysis results, we assumed that there is a long-distance reverse transportation mechanism of these mRNAs from the fruiting body to substrate mycelia as in plant (46). Up-regulation of (hemi)cellulose CAZymes and down-regulation of lignin modification enzymes during fruiting body development has also been reported in Agaricus bisporus and Ganoderma lucidum, which revealed a two-step lignocellulose degradation strategy in these mushrooms (47, 48). Both studies detect the transcriptome of mycelia in solid media and the gene expression of these genes in mycelia is consistent with the abundance of the secreted proteins. However, none of these studies explained the regulation mechanism of the expression of these CAZymes during development. Similar results were also reported in other mushrooms, that the expression of carbohydrate hydrolases was highly expressed in the fruiting body of several mushrooms (25). As described by Krizsán et al. that the targets of these CAZymes families in fruiting bodies are currently unknown. Considering the correlation between the secreted proteins and the gene expression in the fruiting bodies, the results lead us to the hypothesis that the need for nutrients in the fruiting body could induce the expression of corresponding genes. mRNA of lignocellulose degradation genes could transport from the fruiting body to substrate mycelia and induce the secreted protein synthesis and secretion (Figure 8).

The negative correlation between protein concentration and nitrogen/amino acid metabolism also supports the above speculation to a certain extent. As we observed in proteomic analysis, DEPs related to the arginine biosynthetic process, peptidyl-prolyl cis-trans isomerase activity, and cysteine biosynthetic process from serine GO terms were up-regulated in the stem. In addition, the DEGs related to the nitrogen compound metabolic process GO term were significantly enriched in the HAP group. Compared with the EL group, nitrogen compound metabolic processes were enriched in up-regulated DEGs in the HAS group but not in the HAP group. All the results indicated that the stem is the center for nitrogen metabolism and amino acid synthesis. The synthesized amino acids might be transported to pileus to synthesize protein or transported to substrate mycelia with mRNA to synthesize secreted proteins.

There must be other factors besides transportation that influenced the expression patterns of genes and proteins in the fruiting body. The expression of the Roc1 regulator gene, which regulates the expression of cellulose degradation genes in Schizophyllum commune, homologous in P. giganteus was not induced at any developmental stages (49). Therefore, the expression of these CAZymes might be induced by other factors, which need further investigation.

Integrated transcriptomic and proteomic analysis revealed that Cytochrome P450 and hydrophobin genes play key roles in the initial developmental stage in P. giganteus fruiting body development. While in the mature fruiting bodies carbon metabolism is the central metabolism during fruiting body development, especially in the pileus. To obtain the source of carbon, the substrate utilization genes may be inducibly expressed and the mRNA is transported to vegetative mycelia to synthesize secreted proteins. The nitrogen metabolism and amino acid synthesis process were more active in the stem and the products might transport to pileus together with the reducing sugar from the substrates. As a result, pileus has higher protein and reducing sugar concentrations. To further investigate the observations, there is a need to better understand the regulation mechanism of these differentially expressed genes and proteins.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

HiY, QL and HoY: conceptualization. CS: data curation. NJ and HoY: formal analysis. HiY: funding acquisition. MY and DZ: investigation. MY, XC, and LZ: methodology. CS: supervision. CS and MZ: validation. HoY: writing—original draft. JL and HiY: writing—review and editing. All authors contributed to the article and approved the submitted version.

This research was funded by Shanghai Science and Technology Commission Action of Scientific and Technological Creation (23N61900300), China Agriculture Research System (CARS20), SAAS Program for Excellent Research Team ([2022]001), and Shanghai Science and Technology Commission The Belt and Road Project (20310750500).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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