Oderic acid 24) (B) and total ganoderic acids (total GAs) (C) were evaluated. The means of three independent samples with standard deviations are presented. doi:10.1371/journal.pone.0053616.gsitive reaction [33]. A few studies have indicated that fungal elicitors are able to induce cell apoptosis and the production of secondary metabolites, including taxol, artemisinin, and bthujaplicin, in Taxus chinensis, Artemisia annua, and Cupressus lusitanica, respectively [34?6]. In addition to biotic inducers, SPDB abiotic stress has been widely used to increase plant secondary metabolite production [37]. However, whether abiotic stress induces secondary metabolites biosynthesis during cell apoptosis remains unknown. Recently studies have indicated that methyl jasmonate and ROS, which were previously used to enhance plants secondary metabolites production [38], also increased GA biosynthesis in G. lucidum [16,19,20]. These findings suggest that one or more common regulatory components may control secondary metabolite biosynthesis in fungi and plants. Thus, it is quite possible that apoptosis induction by abiotic stress may be an alternative approach to inducing plant secondary metabolite production.Xu et al. showed that GAs production and expression of SQS, LS, and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) was increased by static liquid culture as compared with shaking culture [13]. Methyl jasmonate and phenobarbital have also been demonstrated to increase GAs biosynthesis and expression of various biosynthetic genes [15,16]. Over expression of HMGR in G. lucidum enhanced GA production indicating that HMGR play critical role for GA biosynthesis [39]. However, in this study, aspirin induced GAs production but reduced transcript of the LS and SQS. Our previous study has shown that high doses of ROS, which induce GAs biosynthesis, also reduce SQS and LS mRNA expression [19]. These findings support the idea that aspirin, as well as high doses of ROS, may up-regulate the GA biosynthetic genes down-stream of lanosterol biosynthesis [19]. However, the role of HMGR in apoptosis-induced GA biosynthesis is unknown.Effect of aspirin on reactive oxygen species productionReactive oxygen species (ROS) has been proved to be an important regulator that is able to induce apoptosis. The putative role of ROS in aspirin-induced apoptosis in G. lucidum was evaluated. Fungal mycelium was incubated with aspirin and ROS production was SC-66 chemical information evaluated using 29,79-dichlorofluorescin diacetate (DCFH-DA). No visible enhancement of fluorescence was detectedEffect of aspirin on expression of squalene synthase and lanosterol synthase mRNABoth squalene synthase (SQS) and lanosterol synthase (LS) have been proposed to be involved in the biosynthesis of GAs. Gene expression of the SQS and LS in response to aspirin was assessed by Northern blotting analysis. The application of aspirin to G. lucidum cultures significantly reduced the levels of the SQS and LS gene transcripts (Figure 6). Our data also indicated that gene expression of the SQS and LS were reduced by acetic acid and zinc chloride (data not shown).Figure 6. Transcription level of the squalene synthase (SQS) and lanosterol synthase (LS) in response to aspirin. Fungal mycelium of Ganoderma lucidum was incubated with 2 mM aspirin. Expression of SQS and LS coding region was determined by northern blotting. Gel stained with ethidium bromide was shown to indicate the relative loadings of the total RNA. doi:10.1371/journal.pone.0.Oderic acid 24) (B) and total ganoderic acids (total GAs) (C) were evaluated. The means of three independent samples with standard deviations are presented. doi:10.1371/journal.pone.0053616.gsitive reaction [33]. A few studies have indicated that fungal elicitors are able to induce cell apoptosis and the production of secondary metabolites, including taxol, artemisinin, and bthujaplicin, in Taxus chinensis, Artemisia annua, and Cupressus lusitanica, respectively [34?6]. In addition to biotic inducers, abiotic stress has been widely used to increase plant secondary metabolite production [37]. However, whether abiotic stress induces secondary metabolites biosynthesis during cell apoptosis remains unknown. Recently studies have indicated that methyl jasmonate and ROS, which were previously used to enhance plants secondary metabolites production [38], also increased GA biosynthesis in G. lucidum [16,19,20]. These findings suggest that one or more common regulatory components may control secondary metabolite biosynthesis in fungi and plants. Thus, it is quite possible that apoptosis induction by abiotic stress may be an alternative approach to inducing plant secondary metabolite production.Xu et al. showed that GAs production and expression of SQS, LS, and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) was increased by static liquid culture as compared with shaking culture [13]. Methyl jasmonate and phenobarbital have also been demonstrated to increase GAs biosynthesis and expression of various biosynthetic genes [15,16]. Over expression of HMGR in G. lucidum enhanced GA production indicating that HMGR play critical role for GA biosynthesis [39]. However, in this study, aspirin induced GAs production but reduced transcript of the LS and SQS. Our previous study has shown that high doses of ROS, which induce GAs biosynthesis, also reduce SQS and LS mRNA expression [19]. These findings support the idea that aspirin, as well as high doses of ROS, may up-regulate the GA biosynthetic genes down-stream of lanosterol biosynthesis [19]. However, the role of HMGR in apoptosis-induced GA biosynthesis is unknown.Effect of aspirin on reactive oxygen species productionReactive oxygen species (ROS) has been proved to be an important regulator that is able to induce apoptosis. The putative role of ROS in aspirin-induced apoptosis in G. lucidum was evaluated. Fungal mycelium was incubated with aspirin and ROS production was evaluated using 29,79-dichlorofluorescin diacetate (DCFH-DA). No visible enhancement of fluorescence was detectedEffect of aspirin on expression of squalene synthase and lanosterol synthase mRNABoth squalene synthase (SQS) and lanosterol synthase (LS) have been proposed to be involved in the biosynthesis of GAs. Gene expression of the SQS and LS in response to aspirin was assessed by Northern blotting analysis. The application of aspirin to G. lucidum cultures significantly reduced the levels of the SQS and LS gene transcripts (Figure 6). Our data also indicated that gene expression of the SQS and LS were reduced by acetic acid and zinc chloride (data not shown).Figure 6. Transcription level of the squalene synthase (SQS) and lanosterol synthase (LS) in response to aspirin. Fungal mycelium of Ganoderma lucidum was incubated with 2 mM aspirin. Expression of SQS and LS coding region was determined by northern blotting. Gel stained with ethidium bromide was shown to indicate the relative loadings of the total RNA. doi:10.1371/journal.pone.0.
