From dark-brown to yellow: metabolomics reveals ROS alterations regulating glutathione metabolism to control cap...

The cap color of Flammulina filiformis represents a significant commercial trait, directly affecting consumer purchasing preferences and market value, making it a primary target for genetic improvement. However, the biochemical mechanisms underlying pigmentation, particularly in newly developed dark-brown strains, remain insufficiently understood. In this study, dark-brown F. filiformis was used to investigate the role of reactive oxygen species (ROS) in cap color. Exogenous application of the ROS scavenger N,N'-Dimethylthiourea (DMTU) significantly induced the fading of dark-brown phenotype to yellow, concomitant with reduced activity of key enzymes involved in melanin biosynthesis and an increased pheomelanin/eumelanin ratio. Metabolomic analysis revealed that DMTU treatment significantly activated the glutathione metabolism pathway, with marked accumulation of pathway-associated metabolites. Further validation demonstrated that both oxidized glutathione (GSSG) and its reduced form (GSH) induced a cap fading phenotype similar to that induced by DMTU. Furthermore, quantitative real-time PCR (qRT-PCR) analysis revealed that DMTU treatment significantly upregulated the expression of genes associated with glutathione synthesis and regeneration, while downregulating the transcription levels of key genes involved in melanin synthesis. In conclusion, this study illustrates that alterations in ROS levels can profoundly affect the cap pigmentation process in dark-brown F. filiformis by modulating the glutathione-mediated redox balance. These findings elucidate the critical regulatory role of ROS-glutathione metabolism in the color formation of F. filiformis, offering a novel theoretical framework for the quality control and future breeding aimed at developing cultivars with optimized color traits and enhanced market appeal.