<i>FfbHLH7</i> Transcription Factor Modulates Stipe Browning in <i>Flammulina filiformis</i> via Activation of Tyrosine-Dependent Melanin Biosynthesis

Tissue browning is particularly prevalent in mushrooms (Basidiomycetes), yet the regulatory mechanism of the process remains unclear. The chronological metabolic changes associated with stipe tissue browning, which result in a severe decline in the commercial quality of yellow Flammulina filiformis, were systematically investigated by integrating physiological assessment, multiomics analyses (transcriptomic and genomic), and subsequent molecular validation. The result of transcriptomic screening pinpointed tyrosine metabolism and melanin synthesis as the central pathway driving tissue browning, with potential modulation by bHLH transcription factors. Functional assays (EMSA, Y1H, and DLR) demonstrated that FfbHLH7 directly binds to the promoters of two key tyrosine metabolism genes, FfMIF and FfHGD, thereby activating melanin biosynthesis. Furthermore, overexpression of FfbHLH7 accelerated the browning degree and melanin accumulation in stipe tissues, whereas RNAi-mediated suppression delayed these processes. These findings reveal a novel transcriptional regulatory module, FfbHLH7-FfMIF/FfHGD-melanin, that governs tissue browning in mushrooms, offering potential targets for quality preservation in economically important macrofungal crops.