Abstract
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 (CO(2)) concentration is particularly influential under indoor cultivation conditions. While previous studies have reported that elevated CO(2) levels can inhibit pileus expansion in other mushroom species, the molecular mechanisms by which CO(2) affects pileus growth in F. velutipes remain poorly understood. In this study, we investigated the impact of CO(2) 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) CO(2). Morphometric analysis revealed that elevated CO(2) levels significantly suppressed pileus expansion, reducing the average diameter by more than 50% compared to the low CO(2) condition. To elucidate the underlying genetic response, we conducted RNA sequencing and identified 102 differentially expressed genes (DEGs), with 78 being downregulated under elevated CO(2). 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 CO(2) 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 CO(2)-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 CO(2) concentrations specifically repress PHO80-like cyclin genes in F. velutipes, revealing a molecular mechanism by which CO(2) stress inhibits pileus development. These findings suggest that elevated CO(2) 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 CO(2)-resilient strains and optimizing commercial cultivation conditions.