Genome-Wide Analysis of the Heat Shock Transcription Factor Gene Family in Flammulina filiformis and Its Response to CO2-Mediated Fruit Body Development

Flammulina filiformis is the key industrial edible fungus that requires elevated CO2 to promote the growth of long stipe and small pileus fruiting bodies. Heat shock transcription factors (HSFs) play vital roles in stress response and development regulation; yet the HSF gene family and its expression dynamics during fruiting body development in F. filiformis remain uncharacterized. This study aims to identify and characterize the HSF gene family in F. filiformis and to investigate their expression patterns during fruiting body development and in response to CO2 treatments. In this study, 7 FfHSFs were identified, and their structures, sequence features, and phylogenetics were further analyzed. Expression patterns under CO2 regulation were examined via qRT-PCR. The FfHSFs exhibited CDS lengths of 618–2298 bp, encoding 301–765 hydrophilic amino acids, with molecular weights ranging from 23.4 to 83.8 kDa and theoretical pI values between 4.75 and 9.15. All were predicted to be nuclear-localized. Cis-element analysis revealed motifs associated with growth regulation and stress responses such as low temperature, drought, and hypoxia. Phylogenetically, fungal HSFs were grouped into five clusters, with FfHSFs distributed across four. In this study, we examined the expression levels at four time points (0 h, 2 h, 12 h, and 36 h), under three different carbon dioxide concentrations (0.1%, 5%, and 20%) and in two types of tissues (pileus and stipe) for each six biological replicates. CO2 treatments showed that 5% CO2 significantly suppressed pileus expansion but not stipe elongation, while 20% CO2 inhibited both. Under 20% CO2 treatment, the pileus diameter decreased by approximately 40%, and simultaneously, the expression level of FfHSF1 decreased by about 70%. qRT-PCR indicated that FfHSF1 decreased with pileus expansion, whereas FfHSF4 increased. All FfHSFs were highly expressed in the stipe elongation zone. Elevated CO2 down-regulated FfHSF1 in pileus and FfHSF6 in stipes. Based on these findings, it could be proposed that FfHSF1 and FfHSF6 might be candidate regulators in CO2-mediated morphogenesis, providing insights into hormonal and environmental control of fruiting body development in F. filiformis.