Carbon dioxide is commonly used as one of the significant environmental factors to control pileus expansion during mushroom cultivation. However, the pileus expansion mechanism related to CO₂ is still unknown. In this study, the young fruiting bodies of a popular commercial mushroom Flammulina filiformis were cultivated under different CO₂ concentrations. In comparison to the low CO₂ concentration (0.05%), the pileus expansion rates were significantly lower under a high CO₂ concentration (5%). Transcriptome data showed that the up-regulated genes enriched in high CO₂ concentration treatments mainly associated with metabolism processes indicated that the cell metabolism processes were active under high CO₂ conditions. However, the gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with cell division processes contained down-regulated genes at both 12 h and 36 h under a high concentration of CO₂. Transcriptome and qRT-PCR analyses demonstrated that a high CO₂ concentration had an adverse effect on gene expression of the ubiquitin–proteasome system and cell cycle–yeast pathway, which may decrease the cell division ability and exhibit an inhibitory effect on early pileus expansion. Our research reveals the molecular mechanism of inhibition effects on early pileus expansion by elevated CO₂, which could provide a theoretical basis for a CO₂ management strategy in mushroom cultivation.

• Lyophyllum decastes is a mushroom prone to browning upon prolonged storage. • Ultrasonication (US) treatment retarded browning through enzyme inhibition. • US treatment maintained a higher energy charge and decreased NADH/NAD⁺. • US treatment reduced LdPpo1, LdPpo2, and LdPpo3 genes expression level. • US treatment may be a common strategy for prolonging mushroom shelf life.

In real-time quantitative PCR (RT-qPCR), internal control genes (ICGs) are crucial for normalization. This study screened 6 novel ICGs: Pre-mRNA-splicing factor cwc15 (Cwf15); ER associated DnaJ chaperone (DnaJ); E3 ubiquitin-protein ligase NEDD4 (HUL4); ATP-binding cassette, subfamily B (MDR/TAP), member 1 (VAMP); Exosome complex exonuclease DIS3/RRP44 (RNB); V-type H⁺-transporting ATPase sub-unit A (V-ATP) from the 22-transcriptome data of 8 filamentous fungi. The six novel ICGs are all involved in the basic biological process of cells and share the different transcription levels from high to low. In order to further verify the stability of ICGs candidates, the six novel ICGs as well as three traditional housekeeping genes: β-actin (ACTB); β-tubulin (β-TUB); glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and the previously screened reference genes: SPRY-domain-containing protein (SPRYp); Ras-2 protein (Ras); Vacuolar protein sorting protein 26 (Vps26) were evaluated by geNorm and NormFinder statistical algorithms. RT-qPCR of 12 ICGs were performed at different developmental stages in Flammulina filiformis and under different treatment conditions in Neurospora crassa. The consistent results of the two algorithms suggested that the novel genes, RNB, V-ATP, and VAMP, showed the highest stability in F. filiformis and N. crassa. RNB, V-ATP, and VAMP have high expression stability and universal applicability and therefore have great potential as ICGs for standardized calculation in filamentous fungi. The results also provide a novel guidance for the screening stable reference genes in RT-qPCR and a wide application in gene expression analysis of filamentous fungi.

Stipe elongation is an important process in the development of the fruiting body and is associated with the commodity quality of agaric fungi. In this study, F. filiformis was used as a model agaric fungus to reveal the function of the chromatin modifier gene containing the JmjC domain in stipe elongation. First, we identified a JmjC domain family gene (FfJmhy) with a 3684 bp length open reading frame (ORF) in F. filiformis. FfJmhy was predicted to have a histone H3K9 demethylation function, and was specifically upregulated during stipe rapid elongation. Further investigation revealed that the silencing of FfJmhy inhibited the mycelial growth, while overexpression of this gene had no effect on the mycelial growth. Comparative analysis revealed that the stipe elongation rate in FfJmhy overexpression strains was significantly increased, while it was largely reduced when FfJmhy was silenced. Taken together, these results suggest that FfJmhy positively regulates the mycelial growth and controls the elongation speed and the length of the stipe. Moreover, cell wall-related enzymes genes, including three exo-β-1,3-glucanases, one β-1,6-glucan synthase, four chitinases, and two expansin proteins, were found to be regulated by FfJmhy. Based on the putative functions of FfJmhy, we propose that this gene enhances the transcription of cell wall-related enzymes genes by demethylating histone H3K9 sites to regulate remodeling of the cell wall in rapid stipe elongation. This study provides new insight into the mechanism of rapid stipe elongation, and it is important to regulate the commodity quality of agaric fungi.