Identification of regulatory candidate genes for Lentinula edodes pileus development based on transcriptome analysis

TL;DR

This study identifies key genes involved in the development of the mushroom cap in Lentinula edodes using transcriptome analysis.

Contribution

The study identifies three candidate regulatory genes for pileus development in Lentinula edodes through RNA sequencing and validation.

Findings

283 conserved differentially expressed genes were identified across three developmental stages.

Three candidate regulatory genes (Alpha-amylase, HSP70, Phosphatidylserine decarboxylase) were validated for their role in pileus development.

Stage-specific variations in antioxidant enzyme activities and membrane lipid peroxidation levels were observed.

Abstract

The pileus serves as a primary determinant of market grade and commercial value in Lentinula edodes. To elucidate the molecular mechanisms governing pileus development, we conducted comparative transcriptome analysis via RNA sequencing across 3 distinct developmental stages: Early button stage, Young fruiting body stage, and mature fruiting body stage. Gene expression profiling revealed a substantial number of differentially expressed genes (DEGs) between stages, with 283 conserved DEGs spanning the entire developmental continuum. Systematic mining of these conserved DEGs identified 3 candidate regulatory genes encoding: Alpha-amylase, Heat shock protein 70 (HSP70), Phosphatidylserine decarboxylase. Quantitative PCR validation confirmed the accuracy of both RNA-Seq data and DEG identification. Enzymatic activity assays demonstrated significant stage-specific variations in: Antioxidant enzyme activities, Membrane lipid peroxidation levels. This study provides valuable insights into the molecular framework underlying pileus morphogenesis in Lentinula edodes.

Figures

Characteristics of fruiting body samples subjected to transcriptome sequencing. The white scale bar represents 1 cm.

Volcano plot and Venn diagram of differential gene expression at characteristic stages of pileus development. Differential gene expression in EBS vs YFB a); Differential gene expression in YFB vs MFB b); Differential gene expression in EBS vs MFB c); and the total number of differentially expressed genes in the comparison combination d); Functional enrichment analysis of differentially expressed genes e).

Analysis of differential gene enrichment during the development of Le.M-sd2 strain pileus. GO enrichment of differentially expressed genes in EBS vs YFB a), KEGG enrichment of differentially expressed genes in EBS vs YFB b), GO enrichment of differentially expressed genes in EFB vs MFB c), KEGG enrichment of differentially expressed genes in EFB vs MFB d), GO enrichment of differentially expressed genes in YFB vs MFB e), and KEGG enrichment of YFB vs MFB differential genes f).

Top 10 pathways for differential gene enrichment during pileus opening. The values used for plotting are FPKM values.

Validation of the DEGs in different comparisons using qRT-PCR. The expression level of each gene was normalized by the level of GADPH as reference gene. The Error bars represent SD for 3 biological replicates.

Changes in antioxidant enzyme activities and lipid peroxidation levels during Cap development in Le.M-sd2. a) Superoxide dismutase (SOD) activity, b) catalase (CAT) activity, () peroxidase (POD) activity, d) malondialdehyde (MDA) content. The Error bars represent SD for 3 biological replicates. The letters on the bar chart indicate their level of significance, P < 0.05.

Functions exercised by candidate genes in pileus. The abbreviated content of the figure MGAM stands for maltase-glucoamylase, CTP for phosphatidate cytidylyltransferase, Psd for phosphatidyl-L-serine carboxy-lyase, PA for Phosphatidate, PS stands for Phosphatidylserine, PE stands for Phosphatidylethanolamine, PssA stands for PS synthase, Ub stands for ubiquitin protein, and BUQLN2 stands for ubiquinone protein 2. The light green bottom part is α-Amy in The main function exercised by the pileus opening process is to hydrolyze macromolecular carbohydrates to supply cells in the pileus site for rapid proliferation. The light blue bottom part is the main function exercised by Hsp70, which is mainly involved in endoplasmic reticulum-related degradation, binding to BUQLN2 and stretching unfolded proteins, and then using BUQLN2 as a shuttle for the proteasome to degrade insoluble ubiquitinated aggregates. The light pink bottom part is the main function exercised by the Psd, which converts PS to PE during lipid metabolism, and PE then allows the ATG gene to esterify to form the ATG-PE complex, which participates in the autophagic program of the cell.

Tables

Table 1.. Data filtering analysis results.

Sample	Raw Read	Clean read	Q20 Rate (%)	Useful Read (%)	Uniquely Mapped (%)	Mapped to Gene (%)	Mapped To Exon (%)
EBS 1	39,166,944	36,512,114	97.62	93.22	98.68	86.87	97.44
EBS 2	44,799,028	40,970,880	97.70	91.45	98.87	88.19	97.76
EBS 3	36,436,100	33,599,022	97.65	92.21	98.88	88.03	97.69
YFB 1	45,267,800	35,710,590	97.65	91.95	98.93	87.79	97.69
YFB 2	46,063,166	42,312,126	97.48	92.55	98.88	87.25	97.69
YFB 3	37,350,804	33,856,732	97.51	92.73	98.92	88.10	97.71
MFB 1	38,940,230	41,626,558	97.66	91.70	98.94	88.03	97.67
MFB 2	45,405,968	42,634,550	97.72	93.18	98.93	87.76	97.53
MFB 3	36,216,534	34,638,606	97.82	93.48	98.91	87.89	97.63

Full text

Introduction

Edible mushrooms have garnered significant consumer attention due to their substantial nutritional and medicinal value. Nutritionally, they provide distinctive umami profiles (Harada-Padermo et al. 2020; Yalman et al. 2023), contain complete spectra of essential amino acids serving as premium protein sources (El-Ramady et al. 2022; Parikh et al. 2022), and are characterized by low lipid content, minimal caloric density, and abundant vitamins and minerals (Azeem et al. 2022). Medicinally, multiple species—including Cordyceps sinensis, Tricholoma matsutake, and Agaricus blazei—harbor bioactive compounds such as polysaccharides (Zhou et al. 2024), demonstrating therapeutic properties encompassing immunomodulation, anticancer activity, and cholesterol reduction (Tan et al. 2022; Liu et al. 2023; Narayanan et al. 2023).

As a representative agaricomycete fungus of the order Agaricales, Lentinula edodes exhibits fruiting body morphology, particularly pileus characteristics that serves as the primary determinant of commercial grading and economic viability. Fruiting body development in edible fungi is governed by genetic determinants and modulated by environmental factors (Wessels 1993). Elucidating the molecular mechanisms underlying fungal morphogenesis constitutes a critical research frontier in mycology, with profound implications for enhancing yield and quality traits. Recent advances in molecular biology have progressively unveiled the mechanistic framework of fruiting body development. Key revelations include: Identification of a β-1,3-glucanase gene (GH55 family) encoded by 55 exons in Volvariella volvacea; and the Characterization of 11 laccase genes in Flammulina velutipes (Wang et al. 2015). These breakthroughs collectively illuminate the complex genetic and biochemical networks orchestrating fruiting body formation and development in L. edodes.

Nevertheless, despite the pileus constituting the primary edible organ of L. edodes, research efforts over decades have disproportionately emphasized stipe elongation control—aiming to develop short-stipe phenotypes that reduce postharvest losses (Kim et al. 2021; Kishikawa et al. 2022). In stark contrast, pileus development remains largely unexplored, with only isolated investigations into etiological factors of pileus malformation (Yan et al. 2021). To date, no comprehensive characterization of molecular mechanisms or regulatory networks governing normal pileus morphogenesis has been documented.

In this study, comparative transcriptomic analyses were conducted at 3 critical developmental stages of the mushroom cap to identify key genes involved in L. edodes Pileus development. This work deepens our understanding of the evolutionary mechanisms underlying fungal morphogenesis and provides molecular-level solutions to address industrial challenges such as postharvest losses and varietal improvement.

Materials and methods

Sample source and selected methods

The experimental strain L. edodes Le.M-sd2, domesticated from wild isolates by our research group, is preserved at the Institute of Edible Fungi, Guizhou University. This strain has been viability-verified and deposited at the China Centre for Type Culture Collection (CCTCC) under accession number M20221091. For activation, the strain was cultured on potato dextrose agar (PDA) plates at 25 °C under light-deprived conditions for 7 d. Subsequently, it was transferred to liquid PDA medium (agar-free) and incubated at 25 °C with orbital shaking (130 rpm) for 15 d. The activated mycelia were then inoculated into sterilized cultivation bags (polypropylene; substrate composition: 78% sawdust, 20% wheat bran, 1% glucose, 1% gypsum; 60% moisture content; 1 kg wet weight). Primary mycelial growth proceeded at 25 °C for 28 d, followed by transfer to an IoT-enabled climatic chamber (25 °C, 85% RH) for fruiting body induction. Pileus developmental progression was documented from the primordium stage. One-quarter of the pileus tissue samples were collected using a scalpel at 3 critical stages (Fig. 1): Early button Stage (0 d; EBS), Young fruiting body stage (2 d; YFB), Mature fruiting body stage (4 d; MFB). All samples were promptly cryopreserved in liquid nitrogen and stored at −80 °C for RNA extraction.

Characteristics of fruiting body samples subjected to transcriptome sequencing. The white scale bar represents 1 cm.

RNA extraction and sequencing

Total RNA was extracted from 0.5 g of pileus tissues of L. edodes strain Le.M-sd2 at 3 developmental stages using TRIzol Reagent (Vazyme, Nanjing, China). The concentrations, integrity, and purity of the RNA were assessed using 1% agarose gel electrophoresis, using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA libraries were constructed using NEBNext Ultra RNA Library Prep Kits for Illumina (NEB) according to the manufacturer's instructions. The sequencing library was then sequenced on NovaSeg 6000 platform (Illumina) Shanghai Personal Biotechnology Cp. Ltd.

Transcriptome analysis

Raw data from each sample were processed through quality filtering using fastp (v0.22.0), which removed adapter-contaminated sequences at 3′-termini and reads with average quality scores below Q20. All subsequent analyses utilized these high-quality processed reads. Filtered sequences were aligned to the L. edodes reference genome (strain G408PP-4; Assembly GCA_002003045.1) available at Ensembl Fungi: http://fungi.ensembl.org/Lentinula_edodes_gca_002003045/Info/Index?db=core. HTSeq (v0.9.1) quantified read counts mapped to each gene as raw expression values. For cross-gene and cross-sample expression comparability, normalization was performed using fragments per kilobase (FPKM of transcript per million mapped fragments). For paired-end sequencing, FPKM counts only fragments where both reads align to the same transcript. During functional annotation of differentially expressed genes (DEGs) through GO enrichment, we conducted stratified analysis across 3 developmental comparisons (EBS vs YFB, YFB vs MFB, EBS vs MFB) for: Biological Processes (BP), Cellular Components (CC), Molecular Functions (MF). The top 10 significantly enriched terms per ontological category were selected for visualization. Concurrently, KEGG annotations were categorized into 5 functional hierarchies: Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems. Bubble plots were generated to display the 20 most significantly enriched pathways (FDR-adjusted P < 0.05).

Real-time qPCR analysis

Total RNA was extracted from fruiting body samples using TRIzol Reagent (Vazyme, Nanjing, China). First-strand cDNA synthesis was performed with 1 μg of total RNA per sample using M-MLV (H⁻) reverse transcriptase (Vazyme, Nanjing, China) according to the manufacturer's protocol. Quantitative real-time PCR (qRT-PCR) reactions were conducted in 20 μL volumes containing: 10 μL of 2× Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 1 μL cDNA template, 1 μL gene-specific primers (10 μM). Amplification was carried out on a CFX96 Touch RT-qPCR System (Bio-Rad, Hercules, CA, USA) under the following thermal profile: Initial denaturation: 94 °C for 3 min (1 cycle), Amplification: 40 cycles of: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 3. Final extension: 72 °C for 1 min (1 cycle). Primer sequences are detailed in Supplementary Table 1.

Assays of antioxidant enzyme activities during pileus development in Lentinula edodes

Fresh pileus tissues from 3 developmental stages of L. edodes were assayed for key antioxidant enzymes using commercial kits (Grace Biotechnology, Suzhou, China): Superoxide Dismutase (SOD; Kit No. G0101F), Catalase (CAT; Kit No. G0105F), Peroxidase (POD; Kit No. G0107F) following manufacturer's protocols. Concurrently, malondialdehyde (MDA) content was quantified via the thiobarbituric acid (TBA) method.

Statistical analysis

Adobe Photoshop 2020 was used to plot physiological phenotype images, and the heatmaps were performed using TBtools. IBM SPSS Statistics 22 was used to analyze changes in parameters between treatments. GraphPad Prism 8.0.2 was used to plot histograms and line graphs of potential and well-known errors. A few comparisons between redress were performed using the LSD (last-significant difference) method (P < 0.05).

Results

Raw data and quality assessment

The raw sequencing data for L. edodes strain Le.M-sd2 have been deposited at the National Center for Biotechnology Information (NCBI) under BioSample accession PRJNA1392787: https://www.ncbi.nlm.nih.gov/sra/PRJNA1392787. Following data filtering and trimming, 341,862,378 high-quality clean reads were generated. Low-quality reads were removed from each sample's raw data, with resultant metrics detailed in Table 1. All samples exhibited Q20 scores >97%, confirming base-call quality met analytical standards. Post-filtering useful reads exceeded 90% across all 9 samples, indicating robust sequencing reproducibility. Critical alignment metrics demonstrated: Uniquely mapped reads: >98%, Reads mapped to gene regions: >85%, Exon-mapped reads: >97%. These results demonstrate close phylogenetic proximity between strain Le.M-sd2 and the reference genome (G408PP-4), along with comprehensive genomic annotation integrity.

Differential gene analysis

This study delineates differential gene expression between 3 pivotal stages of L. edodes pileus development: Early button stage (EBS), Young fruiting body stage (YFB), and Mature fruiting body stage (MFB). Genes with an absolute log2 Fold Change greater than 1 and an adjusted P-value (FDR) less than 0.05 were considered differentially expressed. Our transcriptomic analysis revealed dynamic gene expression patterns across key developmental transitions. Specifically, we identified 894 differentially expressed genes (DEGs) between the early button stage (EBS) and young fruiting body (YFB) stage, marking the primordium-to-transition phase, with 557 upregulated and 337 downregulated genes (Fig. 2a, Supplementary Fig. 1). The comparison between YFB and mature fruiting body (MFB) stages showed 927 DEGs (693 upregulated, 234 downregulated), reflecting transcriptional reprograming during maturation (Fig. 2b, Supplementary Fig. 1). The broadest expression changes occurred from EBS to MFB, encompassing 2,156 DEGs (1,265 upregulated, 891 downregulated), which underscores the extensive genetic restructuring throughout development (Fig. 2c, Supplementary Fig. 1). A Venn analysis further identified a core set of 283 DEGs that were conserved across all 3 transitions, highlighting genes potentially fundamental to fruiting body development (Fig. 2d). These persistently regulated genes likely represent core genetic determinants orchestrating pileus morphogenesis throughout fruiting body development.

Volcano plot and Venn diagram of differential gene expression at characteristic stages of pileus development. Differential gene expression in EBS vs YFB a); Differential gene expression in YFB vs MFB b); Differential gene expression in EBS vs MFB c); and the total number of differentially expressed genes in the comparison combination d); Functional enrichment analysis of differentially expressed genes e).

Analysis of the 283 conserved differentially expressed genes (DEGs) (Fig. 2e) revealed 230 upregulated genes enriched in critical pathways: Arachidonic acid metabolism, glycerophospholipid metabolism, starch and sucrose metabolism, FoxO signaling pathway, ascorbate and aldarate metabolism, ubiquinone and terpenoid-quinone biosynthesis, PI3K-Akt signaling pathway, Meiosis, cAMP signaling pathway. These pathways coordinate fundamental biological processes including cell cycle progression, proliferation, apoptosis, antioxidant response, and fruiting body morphogenesis. Conversely, the 53 downregulated genes participated in: N-Glycan biosynthesis, mRNA surveillance pathway, Nucleocytoplasmic transport implicating roles in hyphal cell wall biogenesis and nuclear-cytoplasmic trafficking of regulatory molecules. Collectively, this transcriptional reprogramming demonstrates upregulation of genes governing senescence, autophagy, carbohydrate metabolism, redox homeostasis, and signal transduction, concomitant with downregulation of cell wall biogenesis and homeostatic maintenance genes during pileus development.

Functional enrichment analysis of differentially expressed gene

Comparative analysis of EBS vs YFB stages identified enrichment across 941 GO terms, with categorical distribution as follows: Biological Processes (BP) 56%, Cellular Components (CC) 7%, Molecular Functions (MF) 37%. Within the top 10 enriched GO terms (Fig. 3a): BP exhibited predominant enrichment for metabolic processes, CC showed significant enrichment for membrane-associated terms: integral component of membrane, intrinsic component of membrane, membrane with additional fungal-specific terms including fungal-type cell wall. MF was primarily enriched for redox and enzymatic activities: catalytic activity, oxidoreductase activity. Concurrently, DEGs participated in 82 KEGG pathways categorized as: Metabolism: 71%, Genetic Information Processing: 20%, Environmental Information Processing: 2%, Cellular Processes: 6%, Organismal Systems: 1%. Analysis of the top 20 enriched pathways (Fig. 3b) revealed active engagement in: Glycolysis/Gluconeogenesis, Pyruvate metabolism, Starch and sucrose metabolism, Tryptophan metabolism, Arginine and proline metabolism. indicating prioritized nutrient utilization and storage during this developmental transition.

Analysis of differential gene enrichment during the development of Le.M-sd2 strain pileus. GO enrichment of differentially expressed genes in EBS vs YFB a), KEGG enrichment of differentially expressed genes in EBS vs YFB b), GO enrichment of differentially expressed genes in EFB vs MFB c), KEGG enrichment of differentially expressed genes in EFB vs MFB d), GO enrichment of differentially expressed genes in YFB vs MFB e), and KEGG enrichment of YFB vs MFB differential genes f).

Comparative analysis of EBS vs MFB stages revealed enrichment across 1,752 GO terms, distributed as: biological processes (BP) 54%, cellular components (CC) 11%, molecular functions (MF) 35%. Among the top 10 enriched GO terms (Fig. 3c): BP demonstrated pronounced enrichment for organic metabolism terms: organic substance metabolic process, primary metabolic process, macromolecule biosynthetic process. CC exhibited strongest enrichment in cellular anatomical entity, with additional terms including ribosome and mitochondrial membrane, MF primarily featured structural constituent of ribosome, hydrolase activity, and coenzyme binding DEGs participated in 103 KEGG pathways categorized as: metabolism: 70%, genetic information processing: 19%, environmental information processing: 3%, cellular processes: 7%, organismal systems: 1%. Analysis of the top 20 enriched pathways (Fig. 3d) identified ribosomal biogenesis as the most differentially regulated process between these stages. This reflects transcriptomic reprogramming during primordium-to-maturity transition rather than pileus-specific expansion. However, ribosomal genes likely modulate expansion morphogenesis secondarily. Notably, methane metabolism and pantothenate and CoA biosynthesis pathways also showed significant enrichment.

The YFB to MFB transition represents a critical phase in pileus expansion, with 791 enriched GO terms distributed as: biological processes (BP) 50%, cellular components (CC) 8%, molecular functions (MF) 42%. Among the top 10 enriched GO terms (Fig. 3e): BP primarily encompassed membrane transport and metabolic regulation: transmembrane transport, lipid metabolic process, carbohydrate metabolic process, polysaccharide metabolic process, glycogen catabolic process. CC featured integral component of membrane, intrinsic component of membrane, and mitochondrial nucleoid, MF was dominated by redox catalysis terms: hydrolase activity, catalytic activity. This developmental stage phenotypically corresponds to partial veil rupture and active pileus expansion. DEGs participated in 75 KEGG pathways categorized as: metabolism: 69%, genetic information processing: 20%, environmental information processing: 3%, cellular processes: 7%, organismal systems: 1%. Analysis of the top 20 enriched pathways (Fig. 3f) identified dominant regulation of: starch and sucrose metabolism, glycerophospholipid metabolism, longevity regulating pathway—multiple species, pentose and glucuronate interconversions, Protein processing in endoplasmic reticulum, indicating coordinated metabolic restructuring during pileus morphogenesis.

Screening of critical regulatory genes governing pileus development

Phenotypic divergence associated with cap maturation primarily manifests during the developmental transition from the young fruiting body (YFB) to the mature fruiting body (MFB) stage. Analysis of the Top 10 KEGG pathways enriched for differentially expressed genes (DEGs) between these 2 stages (Fig. 4) revealed that the pathways exhibiting the most significant enrichment of DEGs were starch and sucrose metabolism, glycerophospholipid metabolism, and protein processing in endoplasmic reticulum. Visualization via heatmaps based on FPKM values identified 1 gene within each of these 3 pathways demonstrating markedly differential expression patterns during cap development, with pronounced upregulation specifically at the cap-opening stage. These genes are: LENED_004582 (encoding Alpha-amylase, α-Amy), LENED_010478 (encoding heat shock protein 70, HSP70), and LENED_011132 (encoding phosphatidylserine decarboxylase, Psd). Consequently, these 3 genes were identified as candidate regulators governing cap development.

Top 10 pathways for differential gene enrichment during pileus opening. The values used for plotting are FPKM values.

Validation of RNA-Seq data by qRT-PCR

To validate the gene expression profiles obtained from RNA-Seq, quantitative real-time PCR (qRT-PCR) was employed to assess the relative expression levels of differentially expressed genes (DEGs). In addition to the 3 previously screened candidate genes—LENED_004582(α-Amy), LENED_010478 (Hsp70), and LENED_011132 (Psd)—6 genes were randomly selected from the DEG pool for mRNA expression level validation. The results demonstrated that the expression trends of these genes across the 3 developmental stages of the fruiting body were largely consistent with the RNA-seq data (Fig. 5), confirming the reliability of the transcriptomic dataset.

Validation of the DEGs in different comparisons using qRT-PCR. The expression level of each gene was normalized by the level of GADPH as reference gene. The Error bars represent SD for 3 biological replicates.

Characteristics of pileus development and changes in enzyme activity

We cultivated and documented the developmental progression of Le.M-sd2 fruiting bodies from primordia to mature sporocarps (Supplementary Fig. 2). The results revealed that approximately 72 h after the initiation of primordium differentiation (when distinct cap and stipe structures became discernible), the fruiting bodies reached the operatively defined cap-mature stage characterized by cap expansion. Furthermore, during cap development, the superoxide dismutase (SOD) activity at the mature fruiting body (MFB) stage reached 131.33 ± 2.49 U/g, showing statistically significant differences compared to the other 2 developmental stages (Fig. 6a). In contrast, peroxidase (POD) activity at this stage was the lowest (13.00 ± 2.05 U/g, Fig. 6c), also exhibiting significant differences from the other 2 periods (P < 0.05). However, no statistically significant differences (P > 0.05) were observed in catalase (CAT) activity (Fig. 6b) or malondialdehyde (MDA) content (Fig. 6d) among the 3 key stages of cap development.

Changes in antioxidant enzyme activities and lipid peroxidation levels during Cap development in Le.M-sd2. a) Superoxide dismutase (SOD) activity, b) catalase (CAT) activity, () peroxidase (POD) activity, d) malondialdehyde (MDA) content. The Error bars represent SD for 3 biological replicates. The letters on the bar chart indicate their level of significance, P < 0.05.

Discussion

Functional annotation during pileus development

Fruiting body ontogeny is orchestrated by the sequential execution of interconnected developmental subroutines. Upon inoculation onto growth substrates, mycelia secrete diverse hydrolytic enzymes including cellulases, ligninases, and proteases—to depolymerize complex organic matter, thereby providing carbon sources and nutrients essential for hyphal proliferation (Widiastuti et al. 2008; Barh et al. 2022; Hao et al. 2019). Concurrently, hyphae undergo radial extension and network formation through coordinated cell division and elongation (Callejas-Negrete et al. 2025). Following complete substrate colonization, vegetative growth transitions to the maturation phase. External stimuli (e.g. light, temperature) induce primordial initiation, during which mycelia intensively mobilize nutrients from decomposed substrates to support hyphal knot formation and subsequent differentiation (Nagy et al. 2023). This phase exhibits marked cellular hyper-proliferation, establishing the critical biomass foundation for morphogenesis. Tissue differentiation-governed by transcriptional regulation and intercellular signaling-drives structural specialization into discrete pileus, lamellae, and stipe tissues, each adapted to specific physiological functions (Hao et al. 2019). Post-initiation, primordia enter the cortication phase characterized by accelerated growth and cuticular melanization initiating at the apical dome. The melanized region develops into the incurving pileus, whose diameter subsequently exceeds that of the stipe, marking the morphogenic phase where characteristic agaricoid architecture emerges. Rapid pileus expansion coincides with volatile organic compound biosynthesis and umami metabolite accumulation. Universal veil rupture ultimately exposes the hymenophore, signifying reproductive maturity. Following sporulation, programed senescence ensues (Li et al. 2023), activating oxidative stress response pathways.

Functional analysis of differentially expressed genes (Fig. 2e) revealed that upregulated genes were primarily involved in arachidonic acid metabolism, glycerophospholipid metabolism, multiple signaling pathways (FoxO, PI3K-Akt, cAMP), and ascorbate metabolism. By regulating cell cycle progression, proliferation, differentiation, apoptosis, and oxidative stress response, these genes actively drive rapid morphogenesis of the pileus (He et al. 2023; Xu et al. 2023). In contrast, downregulated genes were mainly associated with fundamental cellular processes including N-glycan biosynthesis, mRNA surveillance, and nucleocytoplasmic transport. Their suppressed expression suggests implementation of a strategic resource reallocation mechanism in the fungus, whereby energy-intensive housekeeping functions are streamlined to prioritize resources for critical processes essential for pileus expansion-including rapid cell division, signal transduction, and structural biosynthesis (Zhang et al. 2020; Cui et al. 2025). Future investigations should transition from correlative observations to causal validation. By employing an interdisciplinary approach integrating functional genomics, cell biology, metabolomics, and biochemical assays, we aim to construct a molecular blueprint of cap development. This blueprint will delineate the function of individual genetic components, quantify the logic of strategic resource reallocation, and ultimately reveal the adaptive significance of this highly efficient morphogenetic strategy in fungi.

Transcriptomic analysis reveals distinct functional enrichment patterns across developmental transitions: EBS vs YFB: GO term enrichment predominantly involves somatic membrane dynamics and enzymatic cell wall biogenesis. Hyphal-derived carbohydrates are utilized for chitin synthesis and septation, driving quantitative cellular expansion that manifests phenotypically as fruiting body enlargement. This represents a phase of biomass accumulation. EBS vs MFB: Enriched terms center on ribosomal biogenesis and mitochondrial organization, indicating proteomic restructuring and metabolic reprogramming. These shifts correspond phenotypically to natural senescence and basidiospore dispersal mechanisms, consistent with apoptotic processes. YFB vs MFB (critical cap-expansion transition): GO analysis highlights enhanced carbohydrate catabolism and mitochondrial hyperactivity. Concurrent upregulation of oxidoreductases—potentially facilitated by iron-porphyrin cofactors (Vercellino and Sazanov 2022)—characterizes this phase. Peroxidases (ubiquitous oxidoreductases utilizing H₂O₂ as electron acceptor) exhibit tissue-specific functions including ethylene biosynthesis (Chagué 2010). We therefore propose that redox-mediated ethylene signaling induces localized apoptosis during this stage, facilitating universal veil rupture and subsequent cap expansion.

KEGG pathway enrichment analysis of DEGs revealed stage-specific metabolic signatures: EBS vs YFB: Significant enrichment in amino acid metabolism pathways reflects flavor compound biosynthesis and accumulation within the fruiting body, with minimal relevance to pileus morphogenesis. EBS vs MFB: Differential enrichment predominantly involves coenzyme A biosynthesis and methane metabolism pathways. These signatures likely arise from natural senescence processes in mature sporocarps. Specifically, elevated methanogenesis may be attributed to oxidative stress induction. Senescence inherently generates substantial reactive oxygen species (ROS), thereby activating methane metabolic genes as a ROS-scavenging response. Methane functions as a core signaling molecule in cellular redox adaptation a mechanism conserved in fungi (Boros and Keppler 2019). Consequently, these enriched pathways collectively represent senescence-countering mechanisms, phenotypically manifested as senescent spotting on lamellae rather than cap expansion. YFB vs MFB (critical veil rupture phase): Enriched pathways predominantly involve longevity regulation and endoplasmic reticulum (ER) protein processing. Longevity-associated transcription factors modulate antioxidant defense, energy metabolism, DNA repair, and autophagy (Mazheika et al. 2012). Irremediable ER stress can trigger apoptosis when cytoprotective mechanisms are overwhelmed. Pathway enrichment at this stage likely corresponds to tissue damage during universal veil rupture and may mechanistically underlie the distinctive cap-opening phenotype.

Physiological changes during pileus development

The antioxidant enzyme system is critically involved in the growth and development of mushrooms. As energy production required for strain growth and metabolic activity often generates deleterious oxidative substances, such as reactive oxygen species (Yaakoub et al. 2022), this system likely plays a regulatory role during mushroom development. Several studies indicate that antioxidant enzymes modulate key developmental stages, including mycelial expansion, fruiting body formation, and spore germination (Wang et al. 2017; Hu et al. 2023). Within fruiting body development, cellular aging and death are inevitable processes intrinsically linked to oxidative stress and cellular damage (Liu et al. 2025). Consequently, maintaining appropriate antioxidant enzyme activity enables strains to regulate senescence, preserving cellular function and viability. In this study, we observed a significant increase in superoxide dismutase (SOD) activity at the mature fruiting body (MFB) stage, while peroxidase (POD) activity concurrently decreased. This pattern aligns with findings reported by Liu (Liu et al. 2013) for Agaricus bisporus. The marked elevation in SOD activity during MFB may be closely associated with pileus senescence. Higher POD activity observed in the early button stage (EBS) and young fruiting body (YFB) stages potentially facilitates initial fruiting body differentiation and rapid cap expansion. Catalase (CAT) activity, however, showed no significant variation across these 3 stages. Collectively, these results demonstrate that the balance of the antioxidant system is disrupted during the later stages of cap development. Significant alterations in key antioxidant enzymes occur throughout the transition from impending veil rupture to complete cap expansion. Intriguingly, malondialdehyde (MDA) content exhibited minimal fluctuation during this process, suggesting that cap opening is not primarily driven by membrane disintegration but rather constitutes a programed aging process inherent to the veil tissue itself.

The function of candidate genes during pileus development

Glucose plays a pivotal role in the growth and development of fungal fruiting body. Its metabolism fuels critical biological processes including hyphal cell division, cell wall biosynthesis, and other essential cellular activities. Furthermore, glucose serves as a fundamental precursor for the synthesis and maintenance of cell wall components, thereby ensuring structural integrity and cellular stability (Chen et al. 2014; Villalba de la Peña et al. 2023). Additionally, glucose functions as a signaling molecule, participating in the regulation of fruiting body growth and development (Takeda et al. 2015; Zhang et al. 2017). Studies have demonstrated that fluctuations in glucose concentration can trigger a cascade of signaling pathways, thereby modulating critical physiological processes including cell cycle progression, mitotic activity, cellular differentiation, and morphogenesis (Takeda et al. 2015). However, the lignocellulosic substrates (e.g. sawdust) utilized in cultivation systems cannot be directly assimilated by fungal hyphae. These complex polymers require sequential enzymatic degradation: initial hydrolysis by endoglucanases and cellobiohydrolases liberates carbohydrate monomers from lignocellulose, which are subsequently hydrolyzed into metabolizable monosaccharides (e.g. via amylase/Amy activity) for ultimate utilization in hyphal cellular processes (Chen et al. 2014; Kabel et al. 2017).

α-Amylase (α-Amy) is a hydrolytic enzyme ubiquitously present in plants, animals, and microorganisms (Wang et al. 2022). It primarily targets α-1,4-glucans such as soluble starch, amylose, and glycogen, catalyzing carbohydrate breakdown via hydrolysis of α-1,4-glycosidic bonds. Our study demonstrates that α-Amy expression is consistently upregulated during fruiting body development, particularly at the pileus expansion stage. We postulate that this expression pattern serves to meet the substantial energy and structural demands during rapid pileus expansion. Under the action of α-Amy, glycogen and starch are rapidly hydrolyzed into disaccharides, which are subsequently cleaved into monosaccharides by maltase-glucoamylase (MGAM). Consequently, these monosaccharides function as fundamental metabolic units for hyphal cellular activities, establishing the essential nutritional prerequisites for pileus opening (Fig. 7). To validate the potential quantitative relationship between α-Amy-driven glucose metabolism and pileus development, future investigations should quantify concentrations of glucose and its key metabolites in pileus tissues across distinct developmental stages. Furthermore, employing CRISPR-Cas9-mediated gene editing to generate α-Amy knockout strains would enable comparative analyses of fruiting body morphology, developmental chronology, and intracellular sugar content between wild-type and mutant strains. Such approaches would functionally establish the contribution of α-Amy-mediated saccharide metabolism to pileus morphogenesis. Collectively, these investigations will elucidate the molecular blueprint through which glucose metabolic networks regulate fruiting body formation in L. edodes at quantitative and mechanistic levels.

Functions exercised by candidate genes in pileus. The abbreviated content of the figure MGAM stands for maltase-glucoamylase, CTP for phosphatidate cytidylyltransferase, Psd for phosphatidyl-L-serine carboxy-lyase, PA for Phosphatidate, PS stands for Phosphatidylserine, PE stands for Phosphatidylethanolamine, PssA stands for PS synthase, Ub stands for ubiquitin protein, and BUQLN2 stands for ubiquinone protein 2. The light green bottom part is α-Amy in The main function exercised by the pileus opening process is to hydrolyze macromolecular carbohydrates to supply cells in the pileus site for rapid proliferation. The light blue bottom part is the main function exercised by Hsp70, which is mainly involved in endoplasmic reticulum-related degradation, binding to BUQLN2 and stretching unfolded proteins, and then using BUQLN2 as a shuttle for the proteasome to degrade insoluble ubiquitinated aggregates. The light pink bottom part is the main function exercised by the Psd, which converts PS to PE during lipid metabolism, and PE then allows the ATG gene to esterify to form the ATG-PE complex, which participates in the autophagic program of the cell.

Phospholipids constitute essential structural components of cellular membranes. Common phospholipids in fungal organisms include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). These lipids play critical roles in membrane architecture and stability, signal transduction, and metabolic regulation. PE represents a major membrane constituent; for instance, in Escherichia coli, PE accounts for 70% to 80% of membrane lipids and is synthesized primarily via 2 pathways: the CDP-ethanolamine pathway within the endoplasmic reticulum (ER) and the phosphatidylserine decarboxylase (PSD) pathway within mitochondria (Zhao and Wang 2020; Centola et al. 2021; Cho et al. 2021). Furthermore, PE generated via the mitochondrial PSD pathway is subsequently exported to other organelles. Notably, PE fulfills another vital physiological function by participating in autophagy. Following the initial discovery of autophagy-related genes (ATG) in yeast, PE was found to form ATG-PE conjugates essential for autophagic processes (Broeskamp et al. 2021; Maruyama et al. 2021; Sakamaki and Mizushima 2023). Our analysis of the annotated ATG-related gene expression changes within the current experimental sequencing dataset (Supplementary Fig. 3) revealed the upregulation of specific genes during the pileus expansion phase.

Phosphatidylserine (PS) is enzymatically converted to phosphatidylethanolamine (PE) by phosphatidylserine decarboxylase (PSD). Research indicates that PSD plays a critical role in phospholipid homeostasis, as well as in the growth and morphogenesis of filamentous fungi (Takagi et al. 2021). During pileus expansion, phosphatidic acid is progressively converted into substantial amounts of PS via the action of various enzymes. Concurrently, Psd gene expression is markedly upregulated, driving the conversion of PS to PE. Consequently, it is plausible that the highly expressed Psd gene facilitates the formation of ATG-PE conjugates (Wang et al. 2020; Sakamaki et al. 2022). Subsequently, the lipidated ATG-PE conjugates bind to and are retained on the phagophore membrane. This complex persists until fusion with hyphal vacuoles occurs, thereby promoting the completion of autophagy and facilitating pileus expansion (Fig. 7).

Hsp70, the most abundant and highly inducible heat shock protein in biological cells, possesses diverse biological functions and is consequently designated as the major heat shock protein. L. edodes (shiitake mushroom) is a typical thermogenic edible fungus, requiring a specific temperature differential stimulus for primordium differentiation. Studies indicate that Hsp70 facilitates adaptation to thermal stress during the early stages of fruiting body development in commercially significant edible fungi such as Flammulina filiformis, Morchella importuna, and Flammulina velutipes (Liu et al. 2017, 2020; Hao et al. 2019). During fruiting body development in the Le.M-sd2 strain, Hsp70 is also abundantly expressed at the primordium stage (Fig. 4), which is closely associated with the thermoinduction of primordium differentiation. Under optimal cultivation conditions, Hsp70 expression would be expected to gradually decrease following the thermoinductive stimulus; indeed, the relative expression change from EBS to YFB aligns with this expectation. However, we observed a resurgence of high Hsp70 expression during the transition from YFB to MFB, prompting us to associate this phenomenon with the regulation of pileus expansion. Previous research on Hsp70 in edible fungi has predominantly focused on its roles in fruiting body morphogenesis and stress resistance, largely overlooking another crucial function: its auxiliary role in the proteasomal degradation pathway for protein aggregates (Behl 2016). While the proteasome can only accommodate single unfolded polypeptide chains and was historically considered incapable of degrading insoluble ubiquitinated aggregates, recent studies reveal that Ubiquilin-2 (UBQLN2) acts synergistically with Hsp70. This collaboration enables the proteasomal degradation of such insoluble ubiquitinated protein aggregates (Hjerpe et al. 2016; Renaud et al. 2019). UBQLN2 is a shuttle protein associated with the ubiquitin-proteasome system (UPS) but remains inactive in its resting state. Only upon binding to Hsp70 is UBQLN2 activated. The activated complex then engages the proteasome; concurrently, Hsp70 mediates the remodeling of aggregated proteins. UBQLN2 subsequently acts as a proteasomal shuttle, delivering ubiquitinated proteins to the proteasome for hydrolysis, thereby eliminating misfolded or superfluous proteins and maintaining cellular homeostasis. In our experiments, Hsp70 expression increased concomitantly with pileus expansion from YFB to MFB (Fig. 4). Therefore, it is highly plausible that Hsp70 participates in endoplasmic reticulum-associated degradation processes, collaborating with UBQLN2 to facilitate the hydrolysis of ubiquitinated proteins, thereby ensuring the physiological progression of pileus maturation (Fig. 7).

The ubiquitin-proteasome system (UPS) and autophagy represent the 2 primary intracellular protein degradation mechanisms. These interconnected pathways cooperatively maintain proteostasis within the cell (Ayala-Torres et al. 2025), primarily through the timely degradation of misfolded, damaged, and unnecessary proteins. Fruiting body development results from the coordinated action of multiple genes. We hypothesize that during the rapid expansion of the mushroom cap toward maturity, significant morphological changes occur alongside vigorous cellular growth and division. The substantial expression of α-amylase (α-Amy) likely provides abundant nutrients to fuel rapid cellular proliferation (Chen et al. 2019), concurrently facilitating the swift initiation of the spore dispersal program (reproductive maturity). To complete its life cycle, the mushroom cap undergoes rapid expansion, resulting in the tearing of the partial veil to expose the spore-bearing gills. Consequently, this process necessitates the abundant expression of Psd and Hsp70. This upregulation prepares the system for the efficient hydrolysis of ubiquitinated proteins, thereby mediating programmed cell death within the partial veil region and facilitating cap expansion.

Conclusion

In summary, comparative transcriptomic analysis employing RNA sequencing technology was conducted across 3 key developmental stages of the L. edodes pileus. This approach generated a substantial volume of transcriptomic data, which was subsequently analyzed to elucidate the biological mechanisms underpinning pileus maturation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differentially expressed genes (DEGs) identified critical candidate genes potentially regulating maturation. These genes encode α-Amy, Heat shock protein 70 (Hsp70), and Phosphatidylserine decarboxylase (Psd). Furthermore, the study quantified temporal variations in resistance enzyme activities and membrane lipid peroxidation levels within the pileus across these 3 stages. Collectively, these findings provide crucial insights into the molecular mechanisms governing shiitake pileus development and yield precise genetic datasets applicable to molecular breeding and subsequent research. These results may offer valuable insights for the edible mushroom industry regarding cultivar development strategies dependent on accelerated pileus maturation requirements during production and for further enhancing fruiting body quality.

Supplementary Material

jkaf316_Supplementary_Data