Heat stress-induced degradation of glutamine synthetase rebalances central carbon-nitrogen metabolism and promotes thermotolerance in Ganoderma lucidum
G 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, highlighting a direct and rapid link between metabolic flux and thermotolerance.
Abstract
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.
Global warming is increasing the frequency and intensity of high-temperature episodes, limiting plant productivity. However, the molecular mechanisms integrating primary metabolism with the heat stress response remains poorly understood. Here, we show that lysine biosynthesis contributes to the coordination of physiological, metabolic and transcriptional responses to heat stress in Arabidopsis thaliana. We compared wild-type, the lysine-biosynthesis mutant dapat, and the salicylic acid (SA)-biosynthesis and signaling mutants sid2-1 and npr1-3 under prolonged warming (6□°C above control for 7 days) and heat shock (38 °C for 6 h), followed by recovery. We assessed growth, gas exchange, photosynthetic performance, free SA, salicylic acid glucoside (SAG), salicylic acid glucose ester (SGE), and total SA content, primary metabolite profiles, heat-stress-responsive gene expression and transcriptome-wide changes by RNA sequencing. Before heat stress, dapat mutant presented a distinct metabolic state, marked by amino-acid accumulation, altered organic-acid profiles, reduced soluble sugars and elevated endogenous SA. This metabolic configuration persisted during prolonged warming, whereas WT and SA-pathway mutants underwent more dynamic reprogramming. Heat shock, by contrast, elicited a more convergent response across genotypes. Despite reduced basal PSII efficiency, dapat maintained photosynthetic performance during prolonged warming and recovered. Its transcriptional response, however, differed from that of WT and SA-pathway mutants: selected heat-responsive genes were constitutively or more strongly expressed, whereas some canonical heat- stress regulators showed weaker induction after heat shock. RNA-seq further revealed a largely conserved core heat-shock response but genotype-dependent regulation of defense, hormone and amino-acid-metabolism programs, particularly during recovery. Together, these findings indicate that impaired DAPAT activity establishes a metabolically primed but energetically constrained state that reshapes gas exchange, photosynthetic acclimation and heat-responsive transcription. Lysine homeostasis therefore emerges as a regulatory node linking primary metabolism and SA accumulation with SA-dependent and SA-independent components of heat-stress acclimation.
D. G. Gouveia, Philipp Westhoff, W. E. B. Barrios et al.· bioRxiv· 0 citations
This work resolves the fundamental oxygen contradiction between thermotolerance enhancement and product biosynthesis in high-temperature fermentation, providing a transformative strategy for cost-effective and sustainable industrial biomanufacturing.
Zhongmei Hu, Yanjie Li, Na Dong et al.· Bioresource Technology· 0 citations
Temperature stress is a major constraint on the productivity of microalgae used in aquaculture. γ-Aminobutyric acid (GABA) is well-established as a key player in the stress tolerance of higher plants, yet its role in microalgae remains largely unexplored. Here, we examined the effects of low (15 °C), optimal (25 °C), and high (35 °C) temperatures on the GABA shunt in Isochrysis zhanjiangensis during the initial and mid-exponential growth phases. The results demonstrated that temperature stress significantly inhibited cell growth and photosynthetic efficiency (assessed by Fv/Fm and Fv’/Fm’), with soluble protein decreasing and soluble sugar accumulating. During the initial exponential phase, both low and high temperature stress triggered marked GABA accumulation, accompanied by coordinated increases in glutamate decarboxylase (GAD) and diamine oxidase (DAO) activities. Interestingly, the transcript levels of IzGAD and IzDAO decreased under these conditions, suggesting that GABA accumulation at this stage is predominantly governed by post-translational activation rather than transcriptional upregulation. Upon entry into the mid-exponential phase, a distinct phase-dependent shift in GABA biosynthetic regulation emerged. Under low temperature stress, GAD activity and IzGAD expression were both suppressed, whereas DAO activity and IzDAO transcripts increased significantly, indicating the transition to DAO-mediated GABA production as the dominant route. Under high temperature stress, both GAD and DAO activities increased, yet their corresponding gene transcription remained repressed, revealing a persistent asynchrony between enzyme activities and gene expression across both phases. Meanwhile, the expression of catabolic genes (IzGABA-T, IzSSADH1, and IzSSADH2) was consistently downregulated, further facilitating the net accumulation of GABA. Promoter analysis revealed multiple stress- and hormone-responsive cis-elements in these genes, implying a complex regulatory network. Collectively, our findings uncover a growth-phase-dependent reconfiguration of GABA biosynthetic pathways in I. zhanjiangensis under temperature stress. These insights provide a mechanistic basis for strain-specific temperature management in aquaculture applications.
Jian-Sen Luo, Lin Zhang, Ji-Chang Han et al.· Microorganisms· 0 citations
Heterologous expression of CsAlaDC establishes a functional ethylamine-theanine metabolic branch in tomato and enhances thermotolerance through coordination with the GABA metabolic network, offering a promising strategy to improve both stress resilience and nutritional quality in crops.
Qianying Wang, Jingbo Yu, Peng Mao et al.· Plant Physiology· 0 citations
Sesame is an important oilseed crop, but its germination stage growth is increasingly limited by heat stress under climate warming. However, the molecular responses underlying heat tolerance in sesame germination stage remain unclear. In this study, a heat-tolerant genotype (G14) and a heat-sensitive genotype (G9) were compared under different durations of high-temperature treatment using phenotypic, metabolomic, and transcriptomic analyses. The results showed that G14 maintained better growth than G9 under both normal and heat stress conditions, with a more pronounced advantage under high temperature. G14 also accumulated lower levels of hydrogen peroxide (H
2
O
2
) under heat stress, suggesting reduced oxidative damage and enhanced heat tolerance. Metabolomic analysis showed that differentially accumulated metabolites (DAMs) in G14 were mainly enriched in lipid metabolism, antioxidant systems, and secondary metabolism, which were activated at early stages and maintained throughout heat stress. In contrast, DAMs in G9 were primarily associated with hormone signaling, carbon metabolism, energy metabolism, and vitamin metabolism. Transcriptomic analysis further showed that heat stress induced extensive changes in gene expression. KEGG enrichment analysis suggested that differentially expressed genes (DEGs) in G14 were mainly involved in maintaining key physiological processes, including photosynthesis, protein processing, and lipid metabolism, whereas those in G9 were predominantly associated with defense and signaling pathways. Integrated metabolomic and transcriptomic analyses supported these patterns and indicated a close coordination between gene expression and metabolic changes. Transcription factor analysis identified members of the
ERF
and
bHLH
families as candidate transcription factors associated with the heat stress response in sesame. These findings provide new insights into the molecular responses underlying heat tolerance during sesame germination.
Pan Zeng, Yan-Xin Deng, Xiao-Wen Yan et al.· Frontiers in Plant Science· 0 citations
Soil salinisation limits agricultural productivity by disrupting microbial homeostasis and plant–microbe interactions. Osmoprotectants, including glycine betaine, play a crucial role in protecting microbial cells against osmotic stress by stabilising cellular structures and maintaining osmotic balance. To optimise microbial production of glycine betaine, adaptive laboratory evolution (ALE) was applied to
Bacillus velezensis
S_51 under increasing sodium chloride (NaCl) concentrations, providing insight into the underlying metabolic and genomic adaptations. After 10 sequential passages (500 generations), the evolved strain exhibited an adaptive shift in growth preference towards higher salinity from 0% to 4% (0.68 M) NaCl, indicating niche specialisation. This adaptation was accompanied by a pronounced increase in osmoprotection, with intracellular glycine betaine levels rising by ~262-fold relative to the ancestral strain, reaching to 882.25 ± 68.58 µM g⁻¹ DM. Metabolic profiling revealed a non-uniform reorganisation of substrate utilisation, with differentiation between ancestral (P1) and evolved (P10) strains largely driven by a limited subset of compounds (~62% contribution of key substrates). The evolved strain showed a ~ 4–5-fold increase in the number of effectively utilised carbon (C) and nitrogen (N) sources, with selective enhancement of specific amino acids (e.g., L-serine, L-aspartic acid) and reduced utilisation of several carbohydrates and hexose phosphates, indicating redistribution of substrate utilisation profiles under osmotic constraints. Functional trait analysis showed increased biofilm formation and elevated indole-3-acetic acid (IAA) production (from 0.1 to 0.13 µg mL⁻¹), while siderophore production remained stable. Genomic analysis identified mutations in key regulatory genes, including
kapB
,
spo0A
, and
comP
, suggesting an association between phenotypic shifts and regulatory network reconfiguration. Together, these results demonstrate that ALE drives coordinated metabolic, physiological, and regulatory adaptation, enhancing microbial fitness under saline conditions and supporting its application in saline soil biotechnologies.
A. Goszcz, M. Musiałowski, Karol Ciuchcinski et al.· Microbial Cell Factories· 0 citations
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