Unveiling Transcriptional Dynamics Across Five Developmental Stages of the Edible Mushroom Oudemansiella raphanipes

Abstract

Oudemansiella raphanipes is a prized edible mushroom renowned for its “three-high, one-low” nutritional profile (high protein, fiber, vitamins; low fat). However, the stage-specific molecular dynamics governing its development and their potential link to its superior nutrition remain unknown, hindering targeted genetic improvement. This study aimed to decipher the first comprehensive transcriptomic atlas across its five key developmental stages and to explore potential molecular signatures linked to its distinctive nutrition. We first confirmed the superior nutritional profile of O. raphanipes via comparative analysis with nine commercial mushrooms. RNA sequencing (RNA-seq) was performed on samples from five defined developmental stages (spores, mycelia, primordia, closed-cap and open-cap fruiting bodies), followed by de novo transcriptome assembly, functional annotation, and differential expression analysis. Results revealed extensive transcriptional reprogramming, with the most dramatic changes occurring at the spore-to-mycelium transition (19,827 differentially expressed genes). Stage-specific pathway enrichment highlighted regulators of germination (e.g., ribosome, transmembrane transport), primordium formation (e.g., glycerophospholipid metabolism, GTPase signaling), fruiting body development (e.g., starch/sucrose metabolism, terpenoid synthesis), and maturation (e.g., glycolysis, fatty acid biosynthesis, transcription factors MADS-box/bZIP). We identified 588 stage-exclusive genes in spores and 515 constitutively upregulated genes linked to energy metabolism and proteostasis. Crucially, integrating nutritional phenotypes with stage-resolved transcriptomics revealed that sustained transcriptional programs in mature fruiting bodies are associated with its nutritional excellence; e.g., upregulation of ribosomal/amino acid metabolic pathways aligns with high protein content, while active fatty acid degradation correlates with low fat levels. Our study provides the first multi-stage transcriptomic blueprint for O. raphanipes development, revealing stage-specific regulators and proposing molecular associations for its nutritional traits. This resource offers a foundational basis and candidate genetic targets for future breeding strategies aimed at enhancing agronomic and nutritional traits in this prized fungus.

1. Introduction

Edible fungi, commonly known as mushrooms, comprise a group of macrofungi capable of forming large fleshy or gelatinous fruiting bodies or sclerotial tissues for culinary and medicinal applications [1]. Advances in scientific identification have revealed over 6000 species of edible fungi globally, with commercially prominent varieties including Agaricus bisporus (button mushroom), Lentinula edodes (shiitake), Pleurotus ostreatus (oyster mushroom), and Flammulina velutipes (enoki mushroom) [2]. Edible fungi are valued for their rich nutritional profile, encompassing proteins, carbohydrates, dietary fiber, and vitamins [3,4]. Beyond basic nutrition, they exhibit significant bioactive properties and potential medicinal value, including immunomodulatory effects [5], antitumor properties [6], antioxidant activity [7], as well as hypoglycemic and hypolipidemic effects [8,9]. Food and Agriculture Organization (FAO) has endorsed the dietary structure of “one meat, one vegetable, and one mushroom” for the 21st century, underscoring the crucial role of edible fungi in global food systems [10].

Oudemansiella raphanipes (“Heipijizong” or “Changgengu”) [11], renowned as the “Edible Queen” [12], is taxonomically classified within the Basidiomycota, Agaricales, and Physalacriaceae [13]. Following successful breakthroughs in its artificial cultivation, research on O. raphanipes has focused on optimizing yield and characterizing its bioactive constituents [14,15,16]. A high-quality genome sequence is now available, revealing gene families related to secondary metabolism and lignocellulose degradation [17,18]. For instance, Zhu et al. (2023) reported the first whole-genome sequence of a monokaryotic strain (CGG-A-s1), identifying 21,308 protein-coding genes, including 56 involved in secondary metabolism (e.g., terpenes, non-ribosomal peptide synthase, type I polyketide synthase) and expanded CAZyme families (e.g., glycoside hydrolases, auxiliary activities enzymes), which support its efficient lignocellulose decomposition [17]. Comparative secretomics further defined substrate-specific extracellular enzyme profiles (e.g., laccases, xylanases) during growth on agricultural residues, underscoring its adaptive response to nutrient sources [18]. Despite these foundational resources, a critical knowledge gap persists: the molecular dynamics and regulatory networks underlying its developmental morphogenesis are still unknown. In contrast to well-studied model fungi such as Coprinopsis and Pleurotus, where key developmental regulators have been identified [19,20,21,22], the stage-specific transcriptional programs guiding O. raphanipes from spore to mature fruiting body are completely uncharacterized. This fundamental gap limits targeted breeding efforts for improved yield, disease resistance, and nutritional quality. Specifically, the gene expression patterns driving developmental transitions—spore germination, mycelial expansion, primordia initiation, and fruiting body maturation—are yet to be elucidated. Stage-resolved transcriptomics has proven to be a powerful approach for deciphering developmental networks in diverse edible fungi [23,24,25,26,27,28,29,30,31]. For example, studies in Auricularia polytricha and F. velutipes have linked differentially expressed genes to processes like protein biosynthesis, melanin accumulation, and metabolic reprogramming during the transition to fruiting bodies [11,23]. In other species such as L. edodes, Tricholoma matsutake, and Ganoderma lucidum, transcriptomic analyses have identified conserved regulators of primordia initiation, cell cycle control, and secondary metabolism [25,26,27]. Furthermore, integrated transcriptomic–metabolomic studies in fungi like Cordyceps militaris and Morchella sextelata have revealed how stage-specific metabolites and transcription factors coordinate development [30,31]. Applying such a stage-resolved transcriptional approach to O. raphanipes is therefore essential to uncover the molecular basis of its development and to support future genetic and breeding initiatives.

Therefore, this study was designed to address two sequential biological questions. First, what are the stage-specific transcriptional programs that orchestrate the complete morphogenesis of O. raphanipes, from spore to mature fruiting body? To answer this, we decipher the transcriptional landscape across five definitive developmental stages, thereby constructing the first comprehensive molecular atlas of its development. Second, could specific transcriptional signatures within this developmental atlas be associated with its renowned “three-high, one-low” nutritional excellence? Building upon the core transcriptomic map, we then integrate it with a systematic nutritional phenotype analysis to explore potential molecular links to its distinctive traits. This integrated approach not only maps the core regulatory circuits of development but also provides testable hypotheses for how specific gene expression programs may contribute to traits of agricultural and nutritional importance.

2. Materials and Methods

2.1. Sample Collection and Preparation

Fruiting bodies of ten edible mushroom species (Figure S1), namely Hypsizygus marmoreus (brown and white strains), P. eryngii, P. ostreatus, L. edodes, A. bisporus, F. velutipes, A. delicata, and Tremella fuciformis, were purchased in three batches from a single commercial supplier (Hualian Supermarket Co., Ltd., Lanzhou Branch, Lanzhou, Gansu Province, China) in Gansu Province in May 2024 (Figure S2), to ensure consistency in cultivar and growth conditions. O. raphanipes was cultivated in-house by our research group. Samples were dried at 60 °C until constant weight was achieved, defined as a weight change of less than 0.1% between two consecutive measurements taken 24 h apart. The dried samples were then pulverized using a laboratory mill and passed through a 100-mesh sieve to obtain a homogeneous fine powder. The resulting powders were stored in airtight containers at room temperature until analysis.

2.2. Nutritional Component and Antioxidant Activity Analysis

All analyses were performed in triplicate on the powdered samples. Protein content was determined using the Coomassie Brilliant Blue method (Bradford assay) with bovine serum albumin as the standard [32]. Fat content was determined by Soxhlet extraction using petroleum ether as the solvent, following Method I of the Chinese National Standard GB 5009.6-2016 [33]. Vitamin content was determined according to the Chinese National Food Safety Standard GB 5009.86-2016 [34]. Crude fiber content was determined by the acid and alkali digestion method specified in the Chinese National Standard GB/T 5009.10-2003 [35]. In addition, total antioxidant capacity (TAC) was assessed using the Total Antioxidant Capacity Assay Kit (Catalog No. S0119, Beyotime Biotechnology, Shanghai, China) with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) method following the manufacturer’s instructions [36]. Results for TAC are expressed as Trolox equivalents (TE) per gram dry weight (DW).

2.3. Fungal Strain and Cultivation Conditions

The experiments employed the monokaryotic strain O. raphanipes ZW130. This strain was originally isolated from a natural fruiting body collected in Lanzhou, Gansu Province, China, and has been deposited in the Specimen and Culture Collection Center, College of Life Sciences, Northwest Normal University, with the internal accession number (No.) ZW130. Strain ZW130 was maintained on Potato Dextrose Agar (PDA, Beijing Aoboxing Biotechnology Co., Ltd., Beijing, China; 200 g/L potato infusion, 20 g/L dextrose and 15 g/L agar) slants at 4 °C. Stock cultures were activated by subculturing onto fresh PDA plates followed by 10-day incubation at 28 °C. The cultivation substrate for fruiting body production consisted of 38% cottonseed hull, 20% sawdust, 20% corncob, 20% wheat bran, 1.5% calcium carbonate, and 0.5% lime (dry weight basis), adjusted to 60–65% moisture content. Substrates were packed into polypropylene bags (17 × 35 × 0.04 cm), sterilized at 121 °C for 120 min, and aseptically inoculated with 5 mycelial plugs (5 mm diameter) per bag. Inoculated bags were incubated at 28 °C in darkness for 60 days until full colonization. For fruiting induction, colonized bags were removed from polypropylene packaging and cased with a 3–5 cm layer of sterilized garden soil. Subsequently, colonized bags were transferred to cultivation chambers maintained at 18–22 °C with 85–90% relative humidity under a 12 h photoperiod (200 lux) according to the cultivation protocol established by Zhu et al. [17]. with some modification. Primordia emerged after 40 days of casing, developing into closed-cap fruiting bodies within 48 h and open-cap fruiting bodies after an additional 48 h. Harvest occurred at 112 days post-inoculation.

2.4. RNA Sequencing and Bioinformatic Analysis

(i) Sample preparation

Samples representing key developmental stages of O. raphanipes were collected as follows (Figure 1): (stage A) Spores obtained by washing fully expanded mature fruiting bodies with distilled water. (stage B) Mycelia harvested after spreading spores on PDA followed by 10-day incubation at 28 °C. (stage C) Primordia collected from substrate bags. (stage D) Closed-cap fruiting bodies sampled at 110 days post-inoculation. (stage E) Open-cap fruiting bodies collected at 112 days post-inoculation. Three biological replicates per stage yielded 15 total samples. All samples were flash-frozen in liquid nitrogen and stored at −80 °C prior to RNA extraction.

(ii) RNA extraction, cDNA library construction and sequencing

Total RNA was isolated from above cryopreserved samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration and purity (A260/A280 and A260/A230 ratios) were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Strand-specific cDNA libraries were constructed from 1 mg of high-quality total RNA per sample using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Catalog No. NR612-02, Vazyme Biotech, Nanjing, China) following the manufacturer’s protocol. Library quality control was performed using the Agilent 2100 Bioanalyzer. Qualified libraries were pooled in equimolar amounts and sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) to generate 150 bp paired-end reads.

(iii) Bioinformatic analysis

Although a reference genome for O. raphanipes has been reported [17], the raw sequencing data or assembled genome sequence is not publicly available for alignment and analysis. Therefore, a de novo transcriptome assembly strategy was necessitated. This approach also ensures the comprehensive capture of transcripts specific to our experimental conditions and developmental stages. RNA-seq data processing commenced with raw read refinement using Trimmomatic for adapter trimming and low-quality base filtration, generating high-quality clean reads [37], which were subsequently assembled into transcripts via Trinity (v2.4) with paired-end methodology [38]. The unigene set was constructed by clustering sequences based on both sequence similarity and length parameters, with the longest transcript per cluster retained as the representative unigene for downstream analysis. Functional annotation [39,40] was performed through basic local alignment search tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 24 July 2024)) alignments (e < 1 × 10⁻⁵) against four reference databases, including non-redundant protein database (NR, https://www.ncbi.nlm.nih.gov/), Swiss-Prot (http://www.uniprot.org/), non-supervised Orthologous Groups (eggNOG, http://eggnog.embl.de/), and clusters of orthologous groups (KOG/COG, https://www.ncbi.nlm.nih.gov/research/COG/ (accessed on 24 July 2024)). Based on these alignments, Swiss-Prot identifiers were mapped to gene ontology (GO, http://www.geneontology.org) terms for functional categorization, while pathway annotations were derived from kyoto encyclopedia of genes and genomes (KEGG, http://www.genome.jp/kegg/) alignments using KEGG automatic annotation server (KAAS).

For quantitative expression profiling and differential expression analysis, RNA-seq reads were first aligned to the unigene reference using Bowtie2 (v2.4.2) with default parameters, followed by transcript quantification via eXpress (v1.5.1) to generate both normalized expression values (FPKM) and raw read counts [41,42]. Differential expression analysis was performed using a negative binomial distribution model implemented in the nbinomTest function (R package DESeq2, 4.2.2), which calculated statistical significance (adjusted p-value) and expression fold changes (FC) between experimental groups [43]. To minimize false positives from large datasets, DEGs were identified through dual-threshold filtering: |FC| > 2 with false discovery rate (FDR)-adjusted p < 0.05 using the Benjamini–Hochberg procedure, and DESeq2’s median-of-ratios normalization was applied to account for library size differences.

2.5. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA isolation and cDNA synthesis were performed as described in the above-mentioned method. Gene-specific primers (Table S1) for target genes and the reference gene (18S ribosomal RNA, 18S rRNA) were designed with Primer3Plus (https://www.primer3plus.com/) and synthesized by Beijing Tsingke Biotech Co., Ltd. (Beijing, China). qRT-PCR was carried out in 20 mL reaction volumes containing 2 mL cDNA template (500 ng), 0.4 mL ROX Reference Dye, 10 mL 2× FastReal qRT-PCR PreMix (SYBR Green), 0.4 mL each forward/reverse primer (10 mM), and 6.8 mL nuclease-free ddH₂O. Amplification was performed on QuantStudio 1 Plus (Thermo Fisher Scientific, Waltham, MA, USA) under the thermal cycling protocol consisted of initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s (denaturation), 60 °C for 10 s (annealing), and 72 °C for 20 s (extension). Relative gene expression levels were calculated using the 2^−ΔΔCt method with cycle threshold (Ct) values normalized against 18S rRNA.

2.6. Statistical Analysis

All experiments were performed with three independent biological replicates. Results are presented as mean ± standard deviation (SD). To manage biological variability, consistent sample collection, cultivation, and sequencing protocols were strictly followed, with rigorous quality control measures (Q30 > 96.7%, RNA integrity > 8.0) to minimize technical variation. Data were subjected to one-way analysis of variance (ANOVA). For the comparative nutritional profiling of ten mushroom species, post hoc pairwise comparisons were performed using Duncan’s multiple range test. For other analyses, Fisher’s Least Significant Difference (LSD) test was used. Statistical significance was defined at p < 0.05. Analyses were conducted in SPSS v26.0, with data visualization performed using GraphPad Prism v9.0 and Microsoft Excel 365.

3. Results

3.1. Nutritional Profile of O. raphanipes in Comparison with Other Edible Mushrooms

Comparative analysis of ten commercially cultivated edible mushrooms (Figure S1) revealed significant nutritional variation. O. raphanipes ZW130 exhibited notably high protein (47.77 mg/g DW; Figure 2a), crude fiber (140.74 mg/g DW; Figure 2b), and vitamin (6596.10 mg/g DW; Figure 2d) content, ranking among the top three species for these parameters. Conversely, its fat content (7.22 mg/g DW; Figure 2e) was significantly lower than in T. fuciformis, A. delicata, L. edodes, H. marmoreus (HM1, brown strain), P. ostreatus, and F. velutipes. In addition, total antioxidant capacity across all species ranged from 3.49 to 5.14 mmol TE/g DW (Figure 2c), with O. raphanipes ZW130 demonstrating moderate value. These findings position O. raphanipes as a nutritionally advantageous mushroom characterized by high protein, fiber, and antioxidant levels coupled with low fat content.

3.2. Stage-Ordered Transcriptional Profiles of O. raphanipes

O. raphanipes development encompasses five distinct morphological stages: spores (stage A), mycelia (stage B), primordia (stage C), closed-cap fruiting bodies (stage D), and open-cap fruiting bodies (stage E). Mature fruiting bodies produced ovoid basidiospores (Figure 3a), which germinated under suitable conditions to form white, flocculent mycelia (Figure 3b). Following inoculation of mycelial plugs and complete colonization of cultivation bags, de-bagging and soil-covering for 40 days induced local mycelial aggregation and swelling, leading to the formation of white or light-brown primordia (Figure 3c). Environmental factors promoted the differentiation of these primordia into fruiting bodies characterized by an undifferentiated, smooth stipe and pileus (Figure 3d). Further maturation involved significant stipe elongation, complete pileus expansion, and the development of lamellae, culminating in the typical agaric morphology of mature, sporulation-capable fruiting bodies (Figure 3e).

To elucidate stage-specific gene expression profiles, RNA was extracted from biological replicates of each developmental stage. High-throughput sequencing generated 104.39 Gb of clean data following stringent quality control (Q30, 96.71–97.23%; Valid Bases, >90%; Average GC content, 53.61%) (Table S2), confirming high sequencing and library quality suitable for downstream analysis. De novo transcriptome assembly yielded 29,766 unigenes (total length > 300 bp; min, 301 bp; max, 12,031 bp) (Table S2). Database annotation assigned functions to unigenes predominantly via eggNOG (47.77%), followed by Pfam (36.44%), NR (31.55%), Swiss-Prot (15.78%), GO (14.55%), and KEGG (6.98%) (Table S3). Principal component analysis (PCA) of FPKM values revealed clear transcriptional distinctions between stages, with PC1 and PC2 explaining 95.19% of the variance (Figure 3f). Notably, biological replicates within each stage showed minimal dispersion and clustered tightly, indicating high reproducibility of the transcriptional profiles. Stage B (mycelia) samples formed a tight cluster distinct from stage A (spores), indicating pronounced transcriptional divergence. Samples from stages C (primordia), D (closed-cap fruiting bodies), and E (open-cap fruiting bodies) occupied overlapping regions in the central PC1 and lower PC2 quadrants, suggesting closer transcriptional relatedness during later development, with high intra-stage reproducibility.

Differential gene expression analysis identified significant transcriptional reprogramming across developmental transitions (Table S4): During the transition from spores to mycelia (B vs. A), 19,827 DEGs were identified (9858 upregulated; 9969 downregulated; Figure 3g). The subsequent shift from mycelia to primordia (C vs. B) involved 11,831 DEGs with 5260 upregulated and 6571 downregulated (Figure 3h). Transitioning from primordia to closed-cap fruiting bodies (D vs. C) yielded 5150 DEGs with 1967 upregulated and 3183 downregulated (Figure 3i), while the final maturation from closed-cap to open-cap fruiting bodies (E vs. D) revealed 6553 DEGs with 3595 upregulated and 2958 downregulated (Figure 3j).

Stage-specific expression analysis across all genes (Figure S3) revealed distinct gene expression patterns during the development of O. raphanipes. Specifically, 588 genes exhibited exclusive expression during the sporulation stage A, while 82 genes were specific to the mycelial stage B. Expression specificity was further observed in the primordium stage C (34 genes), closed-cap fruiting body stage D (3 genes), and open-cap fruiting body stage E (5 genes). The relatively low number of exclusive genes identified in later stages (D and E) should be interpreted with caution, as it may be influenced by the detection sensitivity of the method, the higher complexity and mixed cell types within fruiting body tissues, or genuinely low expression levels of stage-specific regulators. In contrast, 24,531 genes were constitutively expressed throughout all developmental stages analyzed.

3.3. Transcriptional Dynamics Across O. raphanipes Developmental Stages

(i) B (mycelium) vs. A (spore), global biosynthetic activation during spore germination and hyphal growth

The transition from dormant spores to vegetative mycelia (B vs. A) represented the most dramatic transcriptional reprogramming in the life cycle (19,827 DEGs; Figure 3g), marking a shift from dormancy to active growth. GO functional enrichment revealed a concerted activation of cellular biosynthetic machinery (Figure 4a; Table S5). This was underscored by the significant upregulation of the ribosome pathway (ko03010), alongside pathways for one-carbon metabolism and sulfur assimilation (Figure 4b; Table S6), collectively enabling robust protein synthesis and cofactor production for rapid cellular expansion. Concurrently, pathways associated with the dormant spore state, such as oxidative phosphorylation, were downregulated (Figure 4c; Table S6), indicating a metabolic rewiring. Key upregulated genes facilitating this transition included those involved in spore germination, cell wall modification (e.g., glycosyl hydrolases), and hyphal morphogenesis regulators like fungal hydrophobin (Table S7).

(ii) C (primordium) vs. B (mycelium), signaling and membrane remodeling in primordium formation

The formation of reproductive primordia (C vs. B) involved 11,831 DEGs (Figure 3h), highlighting a shift from vegetative growth to developmental morphogenesis. GO analysis emphasized processes related to transmembrane transport and signaling (Figure 5a; Table S5). KEGG enrichment showed upregulated genes significantly involved in glycerophospholipid metabolism (ko00564) and the citrate cycle (Figure 5b; Table S6), supporting the extensive membrane biosynthesis and energy production required for tissue differentiation. In contrast, several secondary metabolic and xenobiotic processing pathways were suppressed (Figure 5c). Crucially, we observed the upregulation of key developmental regulators identified from our transcriptome data, including the sexual development transcription factor NsdD, meiosis-related proteins (e.g., Mei2), and GTPase signaling components (e.g., SEC4), which are pivotal for initiating fruiting body development (Table S8).

(iii) D (closed-cap fruiting body) vs. C (primordium), metabolic shifts during fruiting body expansion

The transition from primordia to expanding, closed-cap fruiting bodies (D vs. C) involved 5150 DEGs (Figure 3i), marking a shift towards rapid tissue growth and initial differentiation. GO enrichment highlighted the central roles of carbohydrate metabolism, transmembrane transport, and intracellular signaling in this process (Figure 6a; Table S5). KEGG pathway analysis further revealed that upregulated genes were significantly involved in starch and sucrose metabolism (ko00500) and alanine, aspartate, and glutamate metabolism (ko00250) (Figure 6b; Table S6), indicating active provisioning of carbon skeletons and nitrogen for biosynthesis. Concurrently, pathways such as amino sugar metabolism and glycerolipid metabolism were downregulated (Figure 6c; Table S6), suggesting a reprioritization of metabolic flux. This stage also saw the upregulation of key genes involved in terpenoid synthesis (e.g., octaprenyl-diphosphate synthase) and specific GTPase signaling components, while genes related to earlier sporulation programs were suppressed (Table S9).

(iv) E (open-cap fruiting body) vs. D (closed-cap fruiting body), maturation and preparation for sporulation

The final maturation into open-cap, sporulation-competent fruiting bodies (E vs. D) encompassed 6553 DEGs (Figure 3j) and was characterized by a distinct metabolic and regulatory reprogramming to complete the life cycle. GO analysis showed strong enrichment for processes including carbohydrate metabolism, oxidoreductase activity, and DNA repair (Figure 7a; Table S5). Correspondingly, KEGG pathways for glycolysis/gluconeogenesis (ko00010) and fatty acid biosynthesis (ko00061) were significantly upregulated (Figure 7b; Table S6), likely supporting energy production and fat storage for spore development. In contrast, pathways related to melanogenesis and several amino acid metabolisms (e.g., tyrosine and tryptophan) were downregulated (Figure 7c; Table S6), potentially fine-tuning the biochemical properties of mature tissues. Crucially, this stage featured the marked upregulation of master regulators for sporulation, including transcription factors such as MADS-box, bZIP, and fungal Zn(2)-Cys(6) binuclear cluster proteins, alongside meiosis-specific genes (e.g., SPO22/ZIP4) (Table S10).

3.4. Sustained and Stage-Exclusive Transcriptional Programs

(i) Constitutively expressed genes underpin core developmental energetics and homeostasis

Beyond stage-transition-specific changes, we identified gene sets with sustained expression trends across the entire developmental continuum, revealing fundamental processes that fuel morphogenesis. A cluster of 515 constitutively upregulated genes (Figure 8a) was significantly enriched for functions in energy metabolism and proteostasis (Figure 8c; Table S11). This included strong enrichment for glycolytic process, ATP binding, and proteasomal protein catabolic process. This persistent transcriptional signature indicates that continuous energy supply coupled with active protein turnover is a fundamental requirement supporting all phases of growth, from spore germination to fruiting body maturation. Conversely, a set of 1124 constitutively downregulated genes (Figure 8b) was enriched for broad signaling and regulatory functions, such as GTPase activity, intracellular signal transduction, and cyclic nucleotide biosynthesis (Figure 8d; Table S11). This gradual attenuation suggests a developmental progression from a state reliant on generalized environmental sensing and response during early growth, towards the execution of more specialized, hard-wired developmental programs in later stages.

(ii) Stage-exclusive genes execute phase-specific adaptive functions

The identification of genes expressed exclusively in a single developmental stage (Figure S3) highlights the precise regulatory partitioning of the life cycle. Spores (stage A), equipped with 588 exclusive genes, invested in stress resilience and dormancy management. This is exemplified by genes for metallo-proteases and chitin biosynthesis, crucial for wall integrity, and antioxidant systems like cytochrome P450s. Mycelia (stage B) expressed 82 unique genes, with a focus on vegetative expansion and nutrient assimilation, including F-box proteins for cell cycle regulation and beta-galactosidase for carbohydrate utilization. Primordia (stage C) activated 34 specific genes, notably those ensuring genomic fidelity during rapid cell division, such as DNA repair Rad50 ATPase, and mitochondrial ATP synthase for localized energy production. The few exclusive genes in closed-cap (stage D) and open-cap (stage E) fruiting bodies (3 and 5 genes, respectively) included enzymes like a pectate lyase, potentially fine-tuning substrate interaction or cell wall remodeling during final maturation. These stage-exclusive genetic arsenals (Table S12) provide a clear molecular demarcation of each phase, deploying specialized tools for stage-specific challenges.

In addition, qRT-PCR validation of 20 DEGs selected from various stages confirmed that their expression patterns were consistent with those derived from the transcriptome data (Figure S4).

4. Discussion

4.1. An Integrated Transcriptomic Atlas Elucidates Developmental and Nutritional Determinants in O. raphanipes

Our study provides the first multi-stage transcriptomic map of the prized mushroom O. raphanipes, capturing the gene expression dynamics that guide its journey from spore to mature fruiting body. The profound transcriptional shifts we uncovered, especially during spore germination (19,827 DEGs; Figure 3g), demonstrate that each morphological change is driven by a distinct genetic program. By integrating this developmental atlas with a systematic analysis of its mature nutritional profile, we connect these molecular programs to the distinctive “three-high, one-low” nutritional traits that define its market value [4,12]. This approach moves beyond cataloging genes to propose how the execution of late-stage developmental plans actively shapes its final nutritional quality.

4.2. Developmental Transitions: Metabolic Activation to Reproductive Commitment

The transition from quiescent spores to vegetative mycelia (stage B vs. A) represents a wholesale metabolic activation. Significant enrichment of ribosomal biogenesis, translation, and transmembrane transport pathways (Figure 4) underscores the rapid deployment of cellular machinery for nutrient assimilation and biomass production—a conserved hallmark of fungal germination [44,45]. The subsequent shift to primordium formation (stage C vs. B) marks the commitment to reproduction. While conserved regulators like NsdD and SEC4 are involved in primordiation across fungi [21,46], a distinctive feature in O. raphanipes is the pronounced and specific enrichment of glycerophospholipid metabolism (ko00564) as a core pathway supporting this transition (Figure 5b). This strong enrichment, which appears more prominent than typically reported in studies of other edible fungi such as F. velutipes and L. edodes [11,25], coordinates with NsdD/SEC4 signaling, suggesting a potential species-specific emphasis on membrane biogenesis for primordium initiation. This regulatory logic aligns with conserved signaling frameworks but highlights O. raphanipes’ unique metabolic prioritization. For O. raphanipes, this study provides the first report of these conserved regulatory and metabolic dynamics, establishing a foundational transcriptomic atlas that was previously unavailable for this species.

4.3. Maturation Phase: Transcriptional Coordination of Structural and Nutritional Trait Development

Our analysis reveals that the final stages of fruiting body development (stages D and E) are not only periods of morphological expansion and sporulation preparation but also the critical phase for active accumulation of nutritional compounds. The transcriptomic signatures during maturation provide a coherent, mechanistic hypothesis for the superior nutritional profile observed in the mature sporocarp. This coordinated expression pattern across multiple nutritional axes presents a notable feature of O. raphanipes. In contrast to the more pathway-specific nutritional adaptations reported in some edible fungi [25,47], our data suggest that O. raphanipes achieves its “three-high, one-low” profile through a broader transcriptional coordination involving:

High protein content (47.77 mg/g DW; Figure 2a) is potentially associated with the sustained upregulation of genes involved in nitrogen assimilation and amino acid biosynthesis in stages D and E. Key enzymes such as glutamine synthetase and various amino acid permeases reached peak expression in mature fruiting bodies, suggesting enhanced capacity for nitrogen assimilation (Table S13) [48] and precursor supply during the phase of maximal protein deposition—a process also emphasized in protein-rich fungi like L. edodes [25].

High crude fiber content (140.74 mg/g DW; Figure 2b) may be linked to the active biosynthetic program for cell wall assembly during tissue expansion. We observed significant upregulation of genes encoding chitinases, β-glucan synthesis-associated proteins, and other glycosyl hydrolases specifically in late stages (Table S13). This active remodeling of structural polysaccharides likely contributes to the dietary fiber matrix, paralleling the role of cell wall biogenesis genes in other edible basidiomycetes [49,50].

Low fat content (7.22 mg/g DW; Figure 2e) aligns with a potential transcriptional balance shifted toward catabolism. Genes associated with fatty acid β-oxidation were upregulated in stage E, while expression of core de novo fatty acid biosynthesis genes remained stable or declined (Table S13). This pattern suggests a metabolic state that may favor fat turnover over storage lipid accumulation in mature tissues, a potential distinguishing feature from other fat-rich fungi [47].

Antioxidant capacity is potentially correlated with the strong induction of a suite of oxidoreductases, peroxidases, and cytochrome P450s in mature stages (Figure 7a; Tables S10 and S13). This enhanced antioxidant machinery likely serves a dual purpose: protecting the developing sporulating tissues and enriching the edible portion with redox-active compounds, as observed in G. lucidum and other medicinal fungi [7,27].

Additionally, the 588 spore-specific genes (stage A) involved in stress resilience (e.g., metallo-proteases, cytochrome P450s) are more abundant than reported in spores of F. velutipes or A. bisporus [11,51], reflecting O. raphanipes’ unique adaptive strategy for dormancy and germination.

4.4. Core Processes and Conserved Regulators Across Development

Looking across all stages, we identified two fundamental expression patterns. A set of 515 constitutively upregulated genes (Figure 8a) acts as a continuous engine, providing essential energy (glycolysis, ATP synthesis) and maintaining protein quality (proteasome function) throughout development [51,52]. Conversely, 1124 constitutively downregulated genes (Figure 8b) are involved in general signaling (e.g., GTPase activity), suggesting the fungus transitions from broad environmental sensing to executing a determined developmental script [53,54]. Genes active only in one stage (Figure S3; Table S12) serve as specialized tools: spores employ metallo-proteases and chitin synthases for stress resistance [55,56], while primordia activate DNA repair proteins like Rad50 to maintain genome integrity during rapid division. Furthermore, the upregulation of transcriptional regulators such as MADS-box and bZIP proteins during maturation (Table S10) underscores a conserved toolkit for coordinating complex development and sporulation across mushrooms, from C. cinerea to Schizophyllum commune [21,57,58].

4.5. Implications, Study Limitations and Future Directions

This first multi-stage transcriptomic atlas transforms O. raphanipes into a genetically tractable organism. We have moved from describing its superior traits to identifying the specific developmental stages and molecular pathways likely responsible, providing a direct scientific basis for targeted breeding. The candidate genes we identified—for growth regulation (e.g., hydrophobins, GTPases), cell wall synthesis (CAZymes), and stress resilience—constitute a valuable resource for programs aimed at improving yield, nutritional content, and cultivation robustness. Beyond pathway enrichment, we functionally prioritized DEGs by focusing on stage-specific expression patterns, constitutive expression trends, and 20 key genes validated by qRT-PCR, alongside literature-supported regulators of fungal development, to refine insights from extensive DEG sets.

It is important to emphasize that the connections we propose between gene expression and nutritional quality are correlative and serve as strong, testable hypotheses. Transcript levels indicate potential, not guaranteed metabolic output. Furthermore, while our functional analysis focused on annotated genes within known pathways, approximately half of the assembled unigenes (e.g., ~52% not annotated in the best-covered database, eggNOG) lacked functional assignments. This inherent limitation of de novo transcriptomics means that our pathway-based interpretation captures a substantial, but incomplete, picture of the transcriptional landscape. Some true biological signals, particularly those involving novel or lineage-specific genes, may reside within this unannotated fraction, which represents a valuable resource for future gene discovery in this non-model fungus. Therefore, the primary future direction arising from this work is functional validation. This includes protein and metabolite profiling across the same stages to validate these associations, and crucially, genetic manipulation (e.g., gene knockout or overexpression) to establish causality. Additionally, measuring nutritional dynamics across all five developmental stages under controlled conditions would map the complete nutrient flux. Finally, the many uncharacterized, stage-specific genes we discovered represent fertile ground for uncovering novel biology unique to this prized mushroom.

5. Conclusions

This study establishes the first comprehensive transcriptomic framework for the development of the high-value edible mushroom O. raphanipes. By analyzing five key developmental stages, we have decoded the stage-specific gene expression programs that orchestrate its morphogenesis, from spore germination to the production of a mature fruiting body. Our work bridges a critical gap by linking these molecular dynamics to the mushroom’s renowned nutritional excellence. We provide correlative evidence and propose that the distinct “three-high, one-low” nutritional profile (high in protein, fiber, and vitamins, low in fat) may not be a passive trait but could be actively shaped by transcriptional programs executed during late-stage maturation. Specifically, our data suggests potential associations where high protein accumulation correlates with upregulated nitrogen metabolism, high fiber with active cell wall biosynthesis, and low fat with a transcriptional bias toward fatty acid degradation.

Beyond these specific insights, our atlas helps illuminate fundamental principles of its development, including the constitutive energy systems that power the entire life cycle and the phased deployment of specialized genes for stage-specific challenges. The discovery of conserved regulatory factors (e.g., MADS-box, bZIP) is consistent with shared genetic logic across edible fungi. This resource provides a foundational tool for the science-driven improvement of O. raphanipes. The candidate genes and pathways identified here offer concrete targets for precision breeding strategies aimed at enhancing agronomic traits such as growth rate, yield, and nutritional composition, ultimately supporting the sustainable cultivation and expanded utilization of this exceptional fungal resource.