Original articleGlsnf1-mediated metabolic rearrangement participates in coping with heat stress and influencing secondary metabolism in Ganoderma lucidum
Graphical abstract
Glsnf1 activity was activated under heat stress in Ganoderma lucidum. Activated Glsnf1 mediated shifts in metabolism from respiration to glycolysis to cope with ROS damage. Reduced glycolysis resulted in higher ratios of NADP/NADPH and GSSG/GSH. Furthermore, strains impaired in enzymes involved in glycolysis showed reduced expression levels of genes involved in the antioxidant system. In addition, Glsnf1 inhibited GA synthesis by removing ROS.
Introduction
Temperatures above optimum are sensed as heat stress by all living organisms. As a major environmental factor, heat stress influences almost all biological processes, such as disturbing cellular homeostasis, leading to severe retardation of growth and development and even to organism death. Heat stress is also considered to be one of the major limiting factors for crop production [1]. Heat stress induces the rapid production and accumulation of reactive oxygen species (ROS), such as superoxide anion (O2−), hydroxyl radical (OH−), and hydrogen peroxide (H2O2). When the cellular ROS is at normal levels, it can function as second messenger. However, the abnormal ROS accumulation can provoke damage to organisms by oxidizing macromolecules such as DNA, lipids and proteins [2]. The detoxification of these ROS is consequently very important in coping with heat stress, and living organisms have evolved complex strategies to address these stresses [3].
The AMP-activated protein kinase (AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1 (Snf1) pathway is highly conserved among different organisms and functions to monitor cellular energy status in response to nutritional environmental variations. AMPK is essential in balancing glycolysis and mitochondrial metabolism based on the availability of energy [4]. Under glucose restriction conditions, AMPK induces mitochondrial respiration, which is known as a more efficient type of metabolism [5]. The study of the regulatory mechanisms of Snf1/AMPK in S. cerevisiae provided references for other eukaryotes [6]. In S. cerevisiae, Snf1 is one part of the SNF complex and has catalytic function. Once ScSnf1 is phosphorylated, it can regulate the activities of rate-limiting enzymes of downstream metabolism pathways and influence the energy metabolism [7]. In C. albicans, mitochondrial respiration and cellular fermentation can be regulated by Snf1 according to the available of glucose [8].
In addition to the response to energy availability, other environmental stresses such as oxidative stress, sodium ion stress and so on, can also activate AMPK/Snf1 [7]. Similarly, osmotic stress and hydrogen peroxide have been suggested to activate AMPK/Snf1 without any change in cellular nucleotides [[9], [10], [11]]. It seems that AMPK/Snf1 plays a wider role in the cellular stress response than was previously understood. However, the functions of AMPK/Snf1 in heat stress adaptability have not yet been investigated thoroughly. In Cancer irroratus, AMPK responded to temperature stress and regulated the expression of one HSP gene [12]. In Caenorhabditis elegans, heat stress activates AMPK to inactivate CREB-regulated transcriptional coactivators (CRTCs) and then increases longevity [13]. However, in human and rodent cells, heat shock inhibits AMPK activity [14]. Compared with the functions of AMPK/Snf1 in animals, fewer studies have been done in microorganisms. In Cryptococcus neoformans, a snf1 mutant showed a profound growth defect at elevated temperatures compared to those of the wild-type strain. However, the regulatory mechanism is unclear [15].
To better understand the relationship among heat stress, AMPK/Snf1 and energy homeostasis, we used Ganoderma lucidum, an important medicinal fungus in China and worldwide, as a research material. G. lucidum is a basidiomycete with high commercial value because it contains many pharmacologically active compounds and has received much research interest [16]. Among them, ganoderic acid (GA) is of great value in the global mushroom industry, and the overall quality of G. lucidum is often assessed by the content of GA [17]. G. lucidum transcriptome sequencing showed that AMPK/Snf1 signal gene transcription responses were increased significantly under heat stress [18]. Therefore, we hypothesized that Glsnf1 has certain functions under heat stress in G. lucidum. The results showed that Glsnf1 regulated the change in metabolic flow to anaerobic glycolysis and helped G. lucidum cope with ROS damage caused by heat stress.
Section snippets
Strains and medium
The parental G. lucidum strain (ACCC53264, dikaryotic) was provided by the Agricultural Culture Collection of China. The wild-type (WT) strain, the two Glsnf1-silenced strains (Glsnf1i-7 and Glsnf1i-12) and the empty-vector controls (Si-control) have been described previously [19]. The G. lucidum strains were grown in mushroom complete media (CYM: 20 g L−1 glucose, 4.6 g L−1 KH2PO4, 0.5 g L−1 MgSO4·7H2O, 2 g L−1 tryptone, and 2 g L−1 yeast extract) at 28 °C.
Treatments
The G. lucidum strains were treated
Glsnf1 responds to HS
The responses of AMPK/Snf1 to HS a widespread environmental stress response factor, has not yet been investigated thoroughly. As shown in Fig. 1A, HS increased the expression levels of Glsnf1 (p < 0.05, Fig. 1A). In addition, the increase in the phosphorylation of Glsnf1 in response to HS was transient and rapid. Glsnf1 was rapidly and strongly activated in response to HS when G. lucidum was exposed to HS for 10 min, 30 min and 60 min, but activity returned to basal levels after 2 h (Fig. 1B).
Discussion
AMPK/Snf1 is essential for balancing glycolysis and mitochondrial metabolism to control cell stress and survival based on the availability of energy. Other environmental stresses, such as sodium ion stress, alkaline pH, and oxidative stress, have been suggested to activate AMPK/Snf1 without any change in adenine nucleotides [7,10]. The association of energy metabolism with environmental stress adaptation is not well understood. Previous studies in our laboratory have found that heat stress,
Acknowledgments
This work was financially supported by the earmarked fund for China Agriculture Research System (Project No. CARS20), National Natural Science Foundation of China (Project No. 31972059) and a Postgraduate Research Innovation Project of Jiangsu Province (Project No. KYCX18-0741).
References (54)
- et al.
Physiological and proteome studies of responses to heat stress during grain filling in contrasting wheat cultivars
Plant Sci.
(2015) - et al.
Effect of heat stress on polyamine metabolism in proline-over-producing tobacco plants
Plant Sci.
(2012) - et al.
AMPK is essential to balance glycolysis and mitochondrial metabolism to control T-ALL cell stress and survival
Cell Metab.
(2016) AMPK: positive and negative regulation, and its role in whole-body energy homeostasis
Curr. Opin. Cell Biol.
(2015)- et al.
Respiratory stress in mitochondrial electron transport chain complex mutants of Candida albicans activates Snf1 kinase response
Fungal Genet. Biol.
(2018) - et al.
The regulation of AMP-activated protein kinase by H2O2
Biochem. Biophys. Res. Commun.
(2001) - et al.
The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways
J. Biol. Chem.
(2002) - et al.
Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase
J. Biol. Chem.
(2010) - et al.
Regulation of virulence factors, carbon utilization and virulence by SNF1 in Cryptococcus neoformans JEC21 and divergent actions of SNF1 between cryptococcal strains
Fungal Genet. Biol.
(2010) - et al.
Secondary metabolites from Ganoderma
Phytochemistry
(2015)
Heat stress-induced reactive oxygen species participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in Ganoderma lucidum
Microbiol. Res.
Hydrogen sulfide, a novel small molecule signalling agent, participates in the regulation of ganoderic acids biosynthesis induced by heat stress in Ganoderma lucidum
Fungal Genet. Biol.
Anoxia and anaerobic respiration are involved in “spawn-burning” syndrome for edible mushroom Pleurotus eryngii grown at high temperatures
Sci. Hortic.
SA inhibits complex III activity to generate reactive oxygen species and thereby induces GA overproduction in Ganoderma lucidum
Redox Biol.
Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method
Methods
The mitogen-activated protein kinase GlSlt2 regulates fungal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in Ganoderma lucidum
Fungal Genet. Biol.
AMPK maintains cellular metabolic homeostasis through regulation of mitochondrial reactive oxygen species
Cell Rep.
ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway
Mol. Cancer
AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases
Biochim. Biophys. Acta (BBA) - Mol. Basis Dis.
Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells
Cell Metabol.
Mitochondria as key components of the stress response
Trends Endocrinol. Metab.
SIRT5 regulates both cytosolic and mitochondrial protein malonylation with glycolysis as a major target
Mol. Cell
Metabolic reprogramming: a cancer hallmark even warburg did not anticipate
Cancer Cell
The pentose phosphate pathway and cancer
Trends Biochem. Sci.
Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression
Cancer Cell
Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia
Curr. Biol.
Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes
Free Radical Biol. Med.
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