Near-freezing temperature storage extends the shelf life of shiitake mushrooms by modulating the homeostasis of endogenous reactive oxygen species

ABSTRACT To develop more effective preservation strategies for edible fungi, this study investigated the role of reactive oxygen species (ROS) in quality changes of shiitake mushrooms ( Lentinula edodes cv. T2) during storage at 5 °C and −1 °C (near-freezing temperature, NFT), as well as the underlying metabolic mechanisms of ROS. Compared with storage at 5 °C, NFT storage significantly maintained mushroom quality, as evidenced by higher hardness, glutathione (GSH), ascorbic acid (AsA), and total phenolics contents, as well as lower pileus opening degree (PD) and browning index (BI). These changes were accompanied by reduced levels of O 2 • - , H 2 O 2 , and malondialdehyde (MDA). NFT storage suppressed NADPH oxidase (NOX) gene expression and reduced NOX activity, thereby decreasing ROS production. Meanwhile, NFT storage upregulated the expression of antioxidant-related genes, including LeSOD2 , LeCAT1 , LePOD1 , LeAPX1 , LeMDHAR1 , LeDHAR1 , LeGR1 , and LeGPX1 , resulting in increased activities of the corresponding enzymes and enhanced AsA-GSH cycle-related antioxidant activity in shiitake mushrooms during NFT storage. Correlation and cluster analyses revealed that O 2 • - and H 2 O 2 levels in shiitake mushrooms showed a significant negative correlation with hardness and a significant positive correlation with PD and BI. Combined with gene silencing and dual-luciferase reporter assays, these results demonstrate that NFT storage maintains endogenous ROS homeostasis in shiitake mushrooms by initiating intrinsic transcriptional regulation, thereby reducing NOX gene expression and activity while upregulating antioxidant enzyme-related genes and their corresponding enzyme activities, thereby preserving mushroom quality. This study demonstrates the feasibility of non-freezing NFT storage of shiitake mushrooms at –1 °C. This nonchemical, low-cost physical technique extends the shelf life of shiitake mushrooms from 16 days (under conventional storage at 5 °C) to 32 days, thereby markedly reducing postharvest losses and facilitating long-distance transportation and off-season supply. These findings offer both a novel molecular framework for postharvest preservation and a practical low-temperature storage protocol for the edible mushroom cold-chain industry.