The Regulation Mechanism Of The Nitric Oxide Signaling In Ganoderic Acid Biosynthesis Under Heat Stress In Ganoderma Lucidum

NO(Nitric oxide)is an important signal molecule in stress responses in fungi.However,little is known about the role and biosynthesis of NO in fungi.Ganoderma lucidum is one of the best-known medicinal basidiomycetes in the world,because it containing a wide range of immuno-modulatory and bioactive compounds.Ganoderic acid(GA)is one of the major secondary metabolites in Ganoderma lucidum(G lucidum),a basidiomycete with high commercial value.Previous studies have shown that HS increases ganoderic acid(GA)biosynthesis in G lucidum.However,whether the NO signals could involved in the regulatory process,as well as how to work in G lucidum,have yet be elucidated.Thus,in the present study,we studied the molecular mechanisms of NO on HS-induced GA biosynthesis in G lucidum.The mainly results are as follows:1.HS growth conditions(42?)could induce NO accumulation in G lucidum compared with that under control growth conditions(28?).Exogenous application of NO scavenger resulted in 25%in GA levels compared with heat treatment alone.The GA content was 30%lower after exogenous application of NO donor SNP than after heat treatment.To further explore the role of NO in the HS regulation of GA accumulation with genetic evidence,GA accumulation in the WT and NRi(nitrate reductase)gene-silenced strain in response to HS wa detected.the GA level in the NRi strains was 20%higher than that in the WT.These results from physiology and genetics studies demonstrate that the NO level is involved in GA biosynthesis following heat treatment,and NO relieves HS-induced GA accumulation in G lucidum.2.The previously research reported that HS induced a significant increase in cytosolic Ca2+concentration,to further examine the relationships between NO and Ca2+signaling pathway under HS conditions.The data demonstrate that NO can induce an increase in the concentration of Ca2+under HS,and Ca2+is also required for HS-induced NO production in G lucidum.After exogenous application of CaCl2,the GA contents of the NRi strains could be restored to the levels in the samples treated with only HS,despite the presence of EGTA.But,supplementation with SNP after pretreatment with PTIO had no significant effect on the GA content in the CaMi(CaM)silenced strains.These results suggest that Ca2+may have a more direct and significant effect than NO on the HS-induced GA increase.3.In the previous study have found that ROS participated in the HS regulation of GA biosynthesis.To determine the mechanism that NO relieves HS-induced GA accumulation in G.lucidum,the effect of NO on the ROS content was examined under heat treatment.The results showed that NO decreased the production of MitROS under heat treatment.The generation of ROS within the mitochondrial depends critically on the NADH/NAD+ratio.To detect the effect of NO on the NADH/NAD+ratio,the data show that NO can inhibit the activity NADH/NAD+ratio.The NADH/NAD+ratio which is mainly regulated by the TCA cycle in mitochondria.Further research found that NO reduced the NADH/NAD+ratio through inhibit the aconitase activity.In the Aconi strains,treatment with SNP showed no obviously effect on the mitROS content and NADH/NAD+ratio relative to the untreated sample.The GA content of the SNP-treated Aconi strain was not significantly different from that of the untreated Aconi strains.Combined with the prior results,these findings indicate that NO inhibits aconitase activity to reduced mitROS content and thereby relieved HS-induced GA overproduction in Ganoderma lucidum.4.Protein S-nitrosylation,the incorporation of a nitroso group to a Cys thiol,has been established as a significant route through which NO transmits its global cellular influence.To study the mechanism of the inhibition of the aconitase activity in G lucidum,the aconitase proteins S-Nitrosylation was detected.Biotin-switch technology(BST)and mass spectrometric analysis revealed that Acon was S-nitrosylated at amino acid residues,Cys 594.The substitution of Cys 594 with non-nitrosylatable Ser with site-directed mutagenesis causes the abolishes its responsiveness to the NO-reducted enzymatic activity.These findings demonstrate that NO inhibited the activity of aconitase through S-nitrosylation at Cys 594.