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The carbon-to-nitrogen (C/N) ratio in the cultivation medium significantly influences the growth rate, vigor of mycelium, yield of fruiting bodies, and their nutritional composition. Recently, agricultural and forestry wastes have been increasingly used in cultivating Flammulina velutipes. However, systematic research on how these materials affect the nutritional and functional properties of the fruiting bodies is lacking. This study investigated the effects of different C/N ratios on F. velutipes cultivation. We evaluated the agronomic traits, nutritional composition, and flavor compounds of the fruiting bodies. Our findings reveal that an optimal C/N ratio of 27:1 in the composted substrates enhances the total yield of fruiting bodies, with 25.1% soybean straw as the primary raw material. This ratio also significantly increases the levels of crude protein, total amino acids, and essential amino acids in the fruiting bodies (p < 0.05). Fruiting bodies from the high-nitrogen (HN) treatment showed the highest content of umami amino acids and equivalent umami concentration value. Additionally, we employed an untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomics approach to analyze the metabolite profiles of fruiting bodies cultivated in high-nitrogen (HN), medium-nitrogen (MN), and low-nitrogen (LN) substrates. We found that the carbon–nitrogen ratio can affect the flavor and quality of fruiting bodies by regulating amino acid biosynthesis and metabolism and other related pathways. Our results suggest that a C/N ratio of 27:1 offers numerous benefits for the cultivation of F. velutipes with comprehensive analyses and has promising application prospects. [ABSTRACT FROM AUTHOR]

• Heat stress response mechanism of Grifola frondosa is proposed by multiomics analysis. • Heat shock proteins play a central role in heat stress response in Grifola frondosa. • Polyamines serve as protection agents in response to heat stress in Grifola frondosa. • Grifola frondosa uses mTOR and Ca2+ pathways to sense and transmit heat-stress signal. High temperature can lead to severe retardation in the growth and development of mushrooms, which influence the quality of fruiting bodies. Grifola frondosa is a popular and wide-cultivated mushroom in Asia; however, little information about the heat response mechanism in this commercially cultivated mushroom has been reported. The growth test was performed to determine the heat tolerance of different strains. Then, the TMT-based proteome and transcriptome analysis of major G. frondosa cultivar Qinghui-151 under different temperatures were performed. High temperature seriously affected the growth and recovery growth of mycelia. Thirty-four heat shock proteins (HSPs) were identified in the multiomics data and the expressions of most HSPs were induced by high temperature. The accumulation of HSPs under the control of HSFs is assumed to play a central role in the heat stress response (HSR) in G. frondosa. mTOR and Ca2+ signaling pathways were found to be activated under high temperature and might involved in heat stress signaling transduction. Polyamines synthesis enzymes were also up-regulated, suggesting the accumulation of stress protector polyamines under heat stress. In addition, expression of an L-phenylalanine ammonia-lyase (GfPAL) gene was significantly up-regulated under high temperature, which might be related to secondary metabolites synthesis. Taken together, these findings improve our understanding of the molecular mechanisms underlying the response to heat stress in G. frondosa , which could promote the breeding of new heat-tolerant G. frondosa cultivars. [Display omitted] [ABSTRACT FROM AUTHOR]