Flammulina velutipes is a widely cultivated edible mushroom in East Asia, recognized for its nutritional benefits and distinct morphology characterized by a long stipe and a compact, hemispherical pileus. The pileus not only plays a critical biological role in reproduction through spore formation but also serves as a key commercial trait influencing consumer preference and market value. Despite its economic importance, pileus development in F. velutipes is highly sensitive to environmental factors, among which carbon dioxide (CO₂) concentration is particularly influential under indoor cultivation conditions. While previous studies have reported that elevated CO₂ levels can inhibit pileus expansion in other mushroom species, the molecular mechanisms by which CO₂ affects pileus growth in F. velutipes remain poorly understood. In this study, we investigated the impact of CO₂ concentration on pileus morphology and gene expression in F. velutipes by cultivating fruiting bodies under two controlled atmospheric conditions: low (1000 ppm) and high (10,000 ppm) CO₂. Morphometric analysis revealed that elevated CO₂ levels significantly suppressed pileus expansion, reducing the average diameter by more than 50% compared to the low CO₂ condition. To elucidate the underlying genetic response, we conducted RNA sequencing and identified 102 differentially expressed genes (DEGs), with 78 being downregulated under elevated CO₂. Functional enrichment analysis highlighted the involvement of cyclin-dependent protein kinase regulatory pathways in this response. Two cyclin genes were found to be significantly downregulated under elevated CO₂ conditions, and their suppression was validated through quantitative real-time PCR. These genes, possessing conserved cyclin_N domains, are implicated in the regulation of the eukaryotic cell cycle, particularly in mitotic growth. These results indicate that CO₂-induced downregulation of cyclin genes may underlie cell cycle arrest, contributing to inhibited pileus development. This study is the first to provide transcriptomic evidence that elevated CO₂ concentrations specifically repress PHO80-like cyclin genes in F. velutipes, revealing a molecular mechanism by which CO₂ stress inhibits pileus development. These findings suggest that elevated CO₂ triggers a morphogenetic checkpoint by repressing PHO80-like cyclins, thereby modulating cell cycle progression during fruiting body development. This study provides the first evidence of such a transcriptional response in edible mushrooms and offers promising molecular targets for breeding CO₂-resilient strains and optimizing commercial cultivation conditions.

This study aimed to elucidate the molecular mechanisms underlying phenotypic divergence between two Lentinula edodes strains, Le_L and Le_S, which exhibit distinct fruiting body morphologies. While phenotypic variation among mushroom strains has been widely observed, the relative contributions of transcriptional regulation and structural genomic variation to these differences remain poorly understood. Comparative transcriptome analysis identified 8541 differentially expressed genes (DEGs), revealing clear functional divergence between the two strains. Genes upregulated in Le_S were predominantly enriched in ribosomal components and translation-related processes, indicating enhanced protein synthesis activity. In contrast, Le_L-upregulated genes were associated with transporters, transcription factors, and diverse metabolic pathways, suggesting broader regulatory and physiological functions. Protein–protein interaction network analysis further highlighted distinct regulatory architectures, with ribosomal proteins forming highly interconnected hub gene modules in Le_S, whereas Le_L hub genes were functionally diverse and included multiple members of the Major Facilitator Superfamily (MFS). Ortholog analysis across 33 L. edodes strains demonstrated that most hub genes were conserved, indicating their roles as core genetic components. Despite widespread genome-wide variation, including 7931 SNPs and 1149 INDELs, sequence variation within hub genes was limited, and allele-specific expression analysis revealed no significant allelic imbalance. In contrast, presence–absence variation (PAV) analysis identified structural differences affecting MFS transporter genes, which were absent in Le_S but present and upregulated in Le_L. Collectively, these findings suggest that structural genomic variation, particularly involving transporter genes, may play a more prominent role than sequence-level variation in driving phenotypic divergence. This study provides new insights into the genetic basis of strain-specific traits in L. edodes and highlights the importance of integrating multi-level genomic analyses.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of bioactive metabolites, particularly triterpenoids. Although most studies have focused on fruiting bodies and spores, the potential of cultured mycelial biomass as a source of functional secondary metabolites remains less explored. In this study, we isolated and characterized polycarpol from the mycelia of G. lucidum and evaluated its anti-melanogenic activity in B16F10 murine melanoma cells. Dried mycelia obtained after liquid culture were extracted with 70% ethanol, partitioned with ethyl acetate, and purified by repeated chromatographic separation. The isolated compound was identified as polycarpol by LC–ESI–MS, HR-MS/MS, and NMR spectroscopy, together with comparison with previously reported spectroscopic data. The anti-melanogenic activity of polycarpol was evaluated in α-MSH-stimulated B16F10 murine melanoma cells, a commonly used cellular model of melanogenesis. Polycarpol reduced α-melanocyte-stimulating hormone-induced cellular tyrosinase activity and melanin production at concentrations that did not markedly affect cell viability. Notably, 2 μg/mL (4.54 μM) polycarpol showed a melanin-inhibitory effect comparable to that of arbutin. Western blot analysis showed that polycarpol decreased the expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein-1. In addition, polycarpol suppressed CREB phosphorylation and modulated the MAPK signaling pathways. These findings suggest that polycarpol inhibits melanogenesis through regulation of MITF-associated melanogenic signaling. Overall, this study demonstrates that cultured G. lucidum mycelia can produce anti-melanogenic triterpenoids and highlights cultured mycelial biomass as a controllable fungal material for exploring anti-melanogenic triterpenoids, although further optimization is required to improve production efficiency.