Study On The Regulation Mechanism Of Gaseous Signal Molecule Hydrogen Sulfide In The Ganoderic Acids Biosynthesis Under Heat Stress In Ganoderma Lucidum

Ganoderma lucidum is traditional medicinal basidiomycetes,which contains many secondary metabolites with pharmacological effects.With the completion of the whole genome sequencing,the construction and improvement of the genetic system,G lucidum is gradually becoming a model fungus to study the secondary metabolic regulation mechanism.Hydrogen sulfide(H2S)is the third gas signaling molecule after nitric oxide(NO)and carbon monoxide(CO).H2S can be involved in a variety of physiological processes in the organism,including influencing signal transduction,responding to external environmental pressure and participating in metabolic processes.However,less research has been done on H2S in basidiomycetes.In this paper,the role of H2S in the biosynthesis of secondary metabolites under the heat stress(HS)of G.lucidum was systematically studied for the first time.In this study,we found the content of H2S in G lucidum wild-type(WT)strains increased by approximately 1.7 times under HS,and the content of triterpene compounds(also known as the Ganoderma lucidum acids,GAs)increased by approximately 1.8 times.When treated scavenger hypotaurine(HT),the intracellular H2S content decreased and GAs content increased.When the donor sodium hydrosulfide(NaHS)of H2S was treated,intracellular H2S content increased and GAs content decreased.In addition,if WT strains pretreated H2S donor NaHS,the elevated GAs content induced by HS could restore to the non-HS WT strains level.Further studies showed that the expression level of Cystathionineβ-synthase(CBS)gene significantly increased under HS,so CBS gene was cloned in G lucidum and the CBS-silenced strains were constructed using RNAi technology.Silencing CBS gene caused intracellular H2S content decreased,resulting in GAs content increased by approximately 65%.Then the content of GAs in CBS-silenced strains under HS was determined,it was found that the content of GAs in CBS-silenced strains increased by approximately 1.5 times under HS,and the content of GAs could be restored to WT strains level after H2S exogenous donor NaHS pretreatment.These results showed that H2S generated by CBS could inhibit GAs biosynthesis induced by HS.Subsequent systematic transcriptome studies have shown that H2S can be involved in multiple signal transduction processes and physiological metabolism processes(primary and secondary metabolism)in G.lucidum.The results showed that the transcriptional levels of 1367 genes in CBS-silenced strains were significantly changed,among which 609 genes and 758 genes were significantly up-regulated and down-regulated,respectively.In addition,the transcriptional levels of 586 and 2280 genes were significantly up-regulated and down-regulated in CBS-silenced strains under HS,respectively.Further analysis showed that H2S can be involved in various primary intracellular metabolic processes,including glycolysis,kreb’s cycle(TCA),oxidative phosphorylation(OXPHOS)and pentose phosphate pathway(PPP).H2S can also affect secondary metabolic processes such as steroid synthesis and terpenoids synthesis.In addition,H2S can affect a variety of intracellular signal transduction processes,including calcium signal,reactive oxygen signal(ROS),nitric oxide signal,etc.Further studies showed that intracellular Ca2+increased by approximately 2.4 times in CBS-silenced strains under HS,while NaHS pretreatment could restore intracellular Ca2+to WT strains level in CBS-silenced strains under HS.When the intracellular Ca2+chelating agent Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid(EGTA)or Ca2+channel blocker lanthanum chloride(LaCl3)were treated,the elevated GAs content in CBS-silenced strains could be restored to WT strains level under HS.These above results showed that H2S generated by CBS under HS could inhibit GAs biosynthesis by negatively regulating intracellular calcium under HS.It is found that H2S can alleviate the damage of the mitochondria of G.lucidum under HS,and can maintain the stability and energy generation of mitochondria under HS.In CBS-silenced strains,mitochondrial membrane potential(MMP)was significantly reduced by approximately 54%under HS,and mitochondrial DNA copy number(mtDNAcn)was also significantly reduced by approximately 60%under HS.Subsequently,it was found that mitochondrial respiratory was further inhibited in CBS-silenced strains under HS.In addition,the damage of mitochondria of G.lucidum under HS could be alleviated by pretreating donor NaHS.Further studies showed that H2S could regulate mitochondrial function through mitochondrial sulfide quinone reductase(SQR).H2S could alleviate the damage of mitochondria of G lucidum under HS and inhibit the GAs biosynthesis under HS through SQR.In SQR-silenced strains,NaHS pretreatment failed to restore mitochondrial homeostasis and respiratory productivity under HS.Subsequently,it was found that the inhibition of GAs biosynthesis by pretreatment NaHS in WT strains under HS disappeared in SQR-silenced strains,suggesting that H2S can negatively regulate the biosynthesis of GAs by regulating mitochondrial energy metabolism through SQR protein.