The Function Of S-nitrosogluthathione Reductase In Ganderic Acids Biosynthesis Under Heat Stress In Ganoderma Lucidum

From the perspective of taxonomy,Ganoderma lucidum belongs to Eumycota and Basidiomycotina,referring as a large medicinal basidiomycete containing various pharmacologically active substances.Ganoderic acids（GAs）is one of the important kind of secondary metabolites in G.lucidum,which has a variety of biological actives.During the growth of G.lucidum,it is inevitable influenced by heat stress.In the process of responding to heat stress,a variety of signal molecules such as nitric oxide（NO）and reactive oxygen species（ROS）.Previous studies have shown that nitrate reductase（NR）,a key enzyme that produces NO,inhibits heat stress-induced GA biosynthesis by regulating intracellular NO synthesis.However there is still little research on whether NO metabolism participates in heat stress.Therefore,the paper discusses the role of S-nitrosogluthathione reductase（GSNOR）,a key enzyme in the process of NO metabolism,in response to heat stress.GSNOR mainly affects the S-nitrosylation level of intracellular protein in response to various biological stress processes by reducing the level of GSNO which is the main storage form of intracellular NO.The paper firstly detected the effect of heat stress on the activity of GSNOR in order to explain the molecular mechanism of GSNOR participating in response to heat stress.The results showed that compared with normal growth,GSNOR activity decreased significantly under heat stress.Since GSNOR is a key enzyme that regulates the level of S-nitrosylation,further proteomics technology was used to detect the effect of heat stress on the level of S-nitrosylation in G.lucidum.The results illustrated that compared with normal growth,heat stress increased the modification level of S-nitrosylation in G.lucidum,but the specific impact mechanism remained unclear.Then,the paper constructed GSNOR-silenced strains and determined their response to heat stress,which indicated that silencing GSNOR could alleviate the inhibition of mycelium growth and GA increase caused by heat stress in G.lucidum.Compared to wild type（WT）,the level of GA in GSNORi strains reduced by about 26%.This result indicated that GSNOR may participate in the heat stress response process of G.lucidum.Previous studies have found that ROS has an important role in GA biosynthesis,thence ROS level in GSNORi strains were further tested.Compared with WT,the intracellular ROS level of the GSNORi strains decreased significantly by about 77%.The above results showed that GSNOR could inhibit heat stress-induced GA biosynthesis and reduce intracellular ROS content in G.lucidum.In order to further explore the molecular mechanism of GSNOR in reducing intracellular ROS content,the enzyme activities of four major antioxidant enzymes were tested.The results showed that silencing GSNOR could cause a significant increase in catalase（CAT）activity,while the other three enzyme activities have no obvious change.In order to verify the role of GSNOR-regulated S-nitrosylation in affecting CAT activity,GPS-SNO and protein three-dimensional structure analysis were used to infer the possible modification of S-nitrosylation in CAT1 protein.Combined with biotin switch method,it was found that CAT1 was S-nitrosylated in vitro and in vivo.Besides that,the enzyme activity of CAT1-SNO increased significantly.The above results indicated that silencing GSNOR can cause S-nitrosylation of CAT1 protein and increased CAT enzyme activity.Furthermore,the paper constructed CAT1i strains and found that in heat-stressed CAT1i strains,ROS level increased by about two times and GA content increased by about 35%.And exogenous addition of CAT can restore the phenotype,that is,alleviating the inhibition of mycelium growth and GA rise caused by heat stress growth in G.lucidum.In order to verify whether GSNOR can regulate GA biosynthesis by affecting the activity of CAT1,exogenous addition of GSNO（GSNOR substrate,equivalent to silencing GSNOR）,the results showed that it had no significant effect on GA in CAT1i strains,but it could greatly reduce GA levels in WT strains under heat stress.The above results indicated that CAT1 is an important part in the process of GSNOR affecting the growth of G.lucidum under heat stress,in other words,GSNOR may increase the activity of CAT1 through S-nitrosylation modification,decreasing the ROS content and thus affecting the growth and the biosynthesis of GA in G.lucidum.In summary,GSNOR plays an important role in responding to heat stress.This research also contributes to the exploration of the signaling mechanism between NO and ROS under heat stress.It also provides a new research idea for further research on the molecular mechanism of S-nitrosylation modification regulating fungal secondary metabolism.