Elsevier

Free Radical Biology and Medicine

Volume 147, 1 February 2020, Pages 220-230
Free Radical Biology and Medicine

Original article
Glsnf1-mediated metabolic rearrangement participates in coping with heat stress and influencing secondary metabolism in Ganoderma lucidum

https://doi.org/10.1016/j.freeradbiomed.2019.12.041Get rights and content

Highlights

  • Glsnf1 mediates the balance between respiration and glycolysis under HS and inhibites secondary metabolism in G. lucidum..

Abstract

The AMP-activated protein kinase (AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1 (Snf1) plays an important role in metabolic remodelling in response to energy stress. However, the role of AMPK/Snf1 in responding to other environmental stresses and metabolic remodelling in microorganisms was unclear. Heat stress (HS), which is one important environmental factor, could induce the production of reactive oxygen species and the accumulation of ganoderic acids (GAs) in Ganoderma lucidum. Here, the functions of AMPK/Snf1 were analysed under HS condition in G. lucidum. We observed that Glsnf1 was rapidly and strongly activated when G. lucidum was exposed to HS. HS significantly increased intracellular H2O2 levels (by approximately 1.6-fold) and decreased the dry weight of G. lucidum (by approximately 45.6%). The exogenous addition of N-acetyl-l-cysteine (NAC) and ascorbic acid (VC), which function as ROS scavengers, partially inhibited the HS-mediated reduction in biomass. Adding the AMPK/Snf1 inhibitor compound C (20 μM) under HS conditions increased the H2O2 content (by approximately 2.3-fold of that found in the strain without HS treatment and 1.5-fold of that found in the strain under HS treatment without compound C) and decreased the dry weight of G. lucidum (an approximately 28.5% decrease compared with that of the strain under HS conditions without compound C). Similar results were obtained by silencing the Glsnf1 gene. Further study found that Glsnf1 meditated metabolite distribution from respiration to glycolysis, which is considered a protective mechanism against oxidative stress. In addition, Glsnf1 negatively regulated the biosynthesis of GA by removing ROS. In conclusion, our results suggest that Glsnf1-mediated metabolic remodelling is involved in heat stress adaptability and the biosynthesis of secondary metabolites in G. 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.

Image 1
  1. Download : Download high-res image (200KB)
  2. Download : Download full-size image

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)

  • R. Liu et al.

    Heat stress-induced reactive oxygen species participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in Ganoderma lucidum

    Microbiol. Res.

    (2018)
  • J.L. Tian et al.

    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.

    (2019)
  • R.Y. Zhang et al.

    Anoxia and anaerobic respiration are involved in “spawn-burning” syndrome for edible mushroom Pleurotus eryngii grown at high temperatures

    Sci. Hortic.

    (2016)
  • R. Liu et al.

    SA inhibits complex III activity to generate reactive oxygen species and thereby induces GA overproduction in Ganoderma lucidum

    Redox Biol.

    (2018)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method

    Methods

    (2001)
  • G. Zhang et al.

    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.

    (2017)
  • R.C. Rabinovitch et al.

    AMPK maintains cellular metabolic homeostasis through regulation of mitochondrial reactive oxygen species

    Cell Rep.

    (2017)
  • Y. Zhao et al.

    ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway

    Mol. Cancer

    (2017)
  • S.B. Wu et al.

    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.

    (2012)
  • A. Le et al.

    Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells

    Cell Metabol.

    (2012)
  • I. Manoli et al.

    Mitochondria as key components of the stress response

    Trends Endocrinol. Metab.

    (2007)
  • Y. Nishida et al.

    SIRT5 regulates both cytosolic and mitochondrial protein malonylation with glycolysis as a major target

    Mol. Cell

    (2015)
  • P.S. Ward et al.

    Metabolic reprogramming: a cancer hallmark even warburg did not anticipate

    Cancer Cell

    (2012)
  • K.C. Patra et al.

    The pentose phosphate pathway and cancer

    Trends Biochem. Sci.

    (2014)
  • I.S. Harris et al.

    Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression

    Cancer Cell

    (2015)
  • A.S. Marsin et al.

    Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia

    Curr. Biol.

    (2000)
  • K. Dong et al.

    Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes

    Free Radical Biol. Med.

    (2016)
  • View full text