Nitric oxide enhances ethanol stress tolerance in Wickerhamomyces anomalus through modulating cell membrane and cell wall homeostasis

Wickerhamomyces anomalus has garnered significant interest for its remarkable ability to shape wine flavor profiles. However, during fermentation, yeast cells are inevitably exposed to ethanol stress. Nitric oxide (NO) is a key signaling molecule that plays diverse physiological roles in living organisms. However, its impact on cell wall and membrane homeostasis under ethanol stress remains unclear. In this study, we investigated the regulatory effects of NO on the cellular integrity of W. anomalus under ethanol stress by supplementing with the NO donor (SNP) or the NO scavenger (Carboxy-PTIO) using physiological analyses focused on the cell wall and membrane. Our results demonstrated that elevated NO levels alleviated ethanol-induced morphological and ultrastructural alterations, primarily by maintaining cellular homeostasis, ultimately promoting cell survival and vitality. Specifically, NO mitigated ethanol stress-compromised cell wall integrity by activating the cell wall integrity (CWI) pathway and increasing intracellular levels of β-glucan and chitin. Furthermore, NO alleviated ethanol-induced disruption of membrane homeostasis by remodeling membrane composition, enhancing integrity, permeability, and fluidity, while simultaneously increasing ATPase activity, elevating intracellular K⁺ levels, and regulating fatty acid synthesis. These findings provide crucial insights into the mechanistic basis of NO-regulated stress responses in W. anomalus under ethanol stress and offer a foundation for developing novel strategies to improve its industrial utilization under fermentative stress conditions.