Bo Wang

Protein phosphorylation modification plays a role in cells’ response to oxidative stress, a key factor leading to postharvest browning of Flammulina filiformis. However, the molecular mechanism by which protein phosphorylation contributes to postharvest browning of F. filiformis remains unclear. This study aimed to characterize the basal phosphoproteomic landscapes associated with variations in different browning phenotypes of F. filiformis. Using data-independent acquisition (DIA) mass spectrometry, we comprehensively profiled the phosphorylation dynamics in susceptible-to-browning (SB) and resistant-to-browning (RB) cultivars at harvest and after 24 h storage. We identified 84,244 phosphorylation sites on 4494 phosphoproteins, with the SB cultivar displaying more altered sites (21,195) than the RB (16,087). Functional enrichment analysis revealed that the differential phosphorylation was significantly implicated in kinases and energy metabolism pathways. Notably, the SB cultivar exhibited a more pronounced phosphorylation profile on key proteins involved in ATP synthesis and glycolysis. Protein–protein interaction (PPI) network analysis further indicated a kinase-mediated regulatory network targeting core energy metabolism components, including ATP synthase and 6-phosphofructokinase. This distinct phosphosignature in the SB cultivar correlated with its more severe browning phenotype and a sharper decline in ATP content during storage. Our findings suggest that divergent phosphorylation-mediated regulation of energy metabolism is strongly associated with the differential postharvest browning susceptibility between these two cultivars, providing a valuable molecular resource for future functional studies.

Flammulina filiformis is a widely cultivated edible mushroom valued for its taste and nutrition. However, its stipe often develops a fibrous and stringy texture that unpleasantly lodges between teeth during chewing. Texture analysis confirmed a distinct toughness gradient, with the upper stipe being more brittle and less tough than the lower part. UHPLC-MS/MS-based metabolomics of these regions identified 953 metabolites, predominantly spanning lipids and lipid-like molecules, organic acids and derivatives, and nucleosides, nucleotides, and analogues. Comparative analysis revealed that the tender upper stipe was characterized by a widespread downregulation of primary metabolites, including severe depletion of key signaling molecules (cAMP, cGMP) and amino acids such as L-tryptophan. In contrast, the tough lower stipe was enriched with metabolites indicative of an oxidative environment, notably a broad spectrum of oxidized lipids and phenolic compounds. KEGG pathway analysis attributed this dichotomy to distinct metabolic programs. While the upper stipe exhibited downregulation in tryptophan and purine metabolism, the lower stipe was enriched for pathways associated with redox homeostasis and lipid peroxidation, including glutathione metabolism and lipid peroxidation. The co-accumulation of oxidized lipids and phenolics suggests a potential mechanism for oxidation-driven tissue fortification. This study reveals a spatially programmed metabolic basis for the textural differentiation in F. filiformis stipes, providing a framework for understanding tissue development and highlighting potential regulatory targets for breeding varieties with improved eating quality.

The growth of Flammulina filiformis is strongly dependent on low-temperature cues for the initiation of primordia formation. To obtain a comprehensive understanding of the molecular mechanisms that govern the mycelial response to cold stress, de novo genome sequencing of the F. filiformis monokaryon and multi-omics data (transcriptome and metabolome) analyses of the mycelia, primordia, and fruiting bodies were conducted in the present study. Genome sequencing based on PacBio HiFi and Hi-C resulted in a 36.3 Mb genome sequence that mapped to 12 chromosomes, comprising 11,886 protein-coding genes. A total of 25 cold-responsive (COR) genes and 520 cold-adapted enzymes were identified in the genome. Multi-omics analyses showed that the pathways related to carbohydrate metabolism in the mycelia under low temperature (10 °C) were significantly enriched. Further examination of the expression profiles of carbohydrate-active enzymes (CAZymes) involved in carbohydrate metabolism revealed that out of 515 CAZyme genes in F. filiformis, 58 were specifically upregulated in mycelia under low-temperature conditions. By contrast, the expression levels of these genes in primordia and fruiting bodies reverted to those prior to low-temperature exposure. These indicate that CAZyme genes are important for the low-temperature adaptation of F. filiformis. This research contributes to the targeted breeding of F. filiformis.

The enoki mushroom (Flammulina filiformis) is one of the most important and popular edible mushrooms commercially in China. However, traditional mushroom cultivar identification is challenging due to poor accuracy, heavy workloads, and low reproducibility. To overcome this challenge, we developed a method for identifying F. filiformis strains using multiple nucleotide polymorphism sequencing (MNP-seq). This involved screening 179 universal MNP markers based on whole-genome sequencing data, constructing an MNP sequence library, and performing multiplex PCR amplification and high-sequencing. We further screened 69 core MNP markers and used them to build a neighbor-joining (NJ) phylogenetic tree of 232 cultivated and wild strains. Our analysis showed that all cultivars could be accurately separated by computing genetic similarity values and that the cultivars could be separated into 22 distinct evolutionary pedigrees. The specific value of genetic similarity can be used as the standard to distinguish F. filiformis cultivars, however, it needs to be comprehensively defined by the additional phenotype and biological characteristics of those strains in the future work.