INTRODUCTION
High toughness in Flammulina filiformis fruiting bodies severely restricts consumption. Mechanical strength shows a significant positive correlation with chitin content in cell walls, and understanding the regulatory mechanism of bZIP transcription factors in regulating this polysaccharide synthesis has become pivotal for overcoming quality improvement bottlenecks in medicinal-edible fungi, as mechanical properties directly determine post-harvest processing suitability and consumer acceptance.
OBJECTIVES
To screen key bZIP transcription factors regulating chitin synthesis in F. filiformis and elucidate their mechanism in modulating fruiting body development and toughness through ROS signaling.
METHODS
Spatial distribution analysis of chitin content and mechanical properties, bZIP family expression profiling (mycelium vs. stipe), yeast one-hybrid binding validation, construction of OE/ knockdown strains, ROS metabolic enzyme assays, H2O2/NAC treatments, and transcriptomic analysis.
RESULTS
fap expression in stipes was 9.36-39.70-fold higher than in mycelia (P < 0.01), aligning with chitin gradients; knockdown strains showed intracellular ROS increased to 508-526% of WT and 10-68% higher chitin in upper stipes, causing malformed fruiting bodies; whereas OE strains exhibited reduced ROS (MDA content decreased to 18-35% of WT), 18-32-fold enhanced POD activity, 9-18% reduced chitin in middle/lower stipes with decreased toughness, and normal fruiting body development; H2O2/NAC treatments directly induced chitin increase/decrease (P < 0.05), confirming the ROS-chitin regulatory axis; transcriptomics further revealed fap significantly modulates genes in carbon metabolism, amino acid biosynthesis, and ribosomal pathways.
CONCLUSION
FAP controls intracellular ROS levels by regulating ROS metabolic enzymes, thereby indirectly suppressing chitin synthesis and ultimately reducing mechanical strength in F. filiformis. This mechanism provides a novel scheme for texture improvement in edible fungi.
Faqin Li, Hao Fan, Siyi Wang et al.· Journal of Advanced Research· 0 citations
ABSTRACT Thermotolerance is fundamental to fungal ecology and survival. Although heat stress triggers extensive metabolic reprogramming, the function of these changes for thermotolerance has remained poorly understood. Here, we identify glutamine synthetase (GS), a central nitrogen metabolism enzyme, as a critical determinant of thermotolerance in Ganoderma lucidum. Silencing of gs significantly enhanced fungal tolerance under heat stress and reduced the relative inhibition rate of mycelial growth to 9.71%, compared with 20.7% in the wild-type (WT) strain. Heat stress also increased reactive oxygen species and H₂O₂ levels by 1.49- and 1.38-fold in the WT strain, whereas the increments were markedly lower in gs-silenced strains. Subsequently, under heat stress, α-ketoglutarate contents in WT increased by 1.49-fold. Inhibition of GS further increased the accumulation, which was achieved by upregulating glutamate dehydrogenase to promote the conversion of glutamate to α-ketoglutarate. This metabolic response was correlated with the generation of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH). Moreover, under heat stress, the level of GS protein in WT strains decreased by 29.0%, compared with that under normal conditions, due to accelerated degradation via the 26S proteasome. Our findings reveal that a ubiquitin-dependent signal instantaneously rebalances central carbon-nitrogen metabolism, offering a direct and rapid link between central metabolism and thermotolerance in fungi. IMPORTANCE Understanding how organisms adapt to heat stress is of increasing urgency in the context of global warming. While the roles of heat-shock proteins and antioxidant systems are well established, how microbes actively reprogram central metabolism to survive thermal challenge remains a fundamental, unanswered question. This study reveals that the central nitrogen metabolism enzyme glutamine synthetase (GS) is degraded by the ubiquitin-proteasome system and that this degradation acts as a metabolic switch to enhance thermotolerance in Ganoderma lucidum. We discovered that heat stress induces ubiquitin-proteasome system-dependent GS degradation, leading to redirected central nitrogen flux that elevates α-ketoglutarate content. This metabolic shift boosts ATP and NADH production. In summary, our findings represent a significant advance beyond classical protein chaperone systems and reactive oxygen species-scavenging systems, highlighting a direct and rapid link between metabolic flux and thermotolerance. Understanding how organisms adapt to heat stress is of increasing urgency in the context of global warming. While the roles of heat-shock proteins and antioxidant systems are well established, how microbes actively reprogram central metabolism to survive thermal challenge remains a fundamental, unanswered question. This study reveals that the central nitrogen metabolism enzyme glutamine synthetase (GS) is degraded by the ubiquitin-proteasome system and that this degradation acts as a metabolic switch to enhance thermotolerance in Ganoderma lucidum. We discovered that heat stress induces ubiquitin-proteasome system-dependent GS degradation, leading to redirected central nitrogen flux that elevates α-ketoglutarate content. This metabolic shift boosts ATP and NADH production. In summary, our findings represent a significant advance beyond classical protein chaperone systems and reactive oxygen species-scavenging systems, highlighting a direct and rapid link between metabolic flux and thermotolerance.
Jinjin Qiao, Huajun Li, Yuzhen Yang et al.· Applied and Environmental Mi...· 0 citations
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