Nitrogen is the main macronutrient for fungal structural and energy requirements. G. lucidum suffers the nitrogen limiting conditions during its growth. In this study, we detected the intracellular amino acid contents under the different concentrations of nitrogen. The content of each detected amino acid decreased under the low nitrogen condition (Table 1), compared with that under the high nitrogen condition, except for threonine. As shown in Fig. 1A, the content of amino acids reached 120.51 mg/g in the WT strain under the 60 mM asparagine condition. However, the content of amino acids under the 3 mM asparagine condition decreased by 62.96% compared with that under the 60 mM asparagine condition (Fig. 1A). Further, we calculated the content of essential amino acids, MSG-like taste amino acids, sweet amino acids, bitter amino acids and tasteless amino acids respectively. Under the low concentration of nitrogen, the contents of essential amino acids decreased by 52.63% compared with the high concentration of nitrogen (Fig. 1B). The contents of MSG-like, sweet, bitter and tasteless amino acids under 3 mM asparagine decreased by 79.06%, 14.65%, 47.35% and 44.23%, respectively, compared with those under 60 mM asparagine (Fig. 1C-F). Taken together, these results showed that high concentration of nitrogen benefits the amino acids accumulation while low concentration of nitrogen resulted in a decrease of amino acid contents in G. lucidum.
GCN4 contributes to maintaining the intracellular amino acid contents under the low concentration of nitrogen
GCN4 has been widely reported in response the amino acid starvation in yeast [34]. Our previous study found GCN4 was highly induced in response to the low concentration of nitrogen condition in G. lucidum [35]. To assess the regulation of GCN4 on amino acid contents in G. lucidum, we examined the amino acid contents in gcn4-silenced strains. Almost each amino acid content in the gcn4-silenced strains was significantly lower than that in WT strain under the different nitrogen concentrations (Table 1). Silencing of gcn4 resulted in a 54.2% decrease of intracellular amino acid contents under the 60 mM asparagine condition. However, under the 3 mM asparagine condition, the contents of amino acids decreased by 59.8% and 62.3% in the gcn4i-1 and gcn4i-22 strains, respectively, compared with those in WT (Fig. 1A). After silencing of gcn4, significantly decrease of essential and tasteless amino acids contents was observed under the 3 mM asparagine, compared with 60 mM asparagine (Fig. 1B, F). Under the 3 mM asparagine, the contents of MSG-like taste amino acids decreased by 79.8% and 88.3% in gcn4i-1 and gcn4i-22 strains compared with those in WT, while 69.8% and 68.3% decrease in gcn4i-1 and gcn4i-22 strains were found under the 60 mM asparagine (Fig. 1C). Either under high or low nitrogen conditions, the content of sweet amino acids in the gcn4-silenced strains had no significant change compared with that in the WT strain (Fig. 1D). In conclusion, our results indicated that the GCN4 facilitated the accumulation of amino acids, especially the essential and MSG-like taste amino acids, under low nitrogen condition.
Silencing of gcn4 decreased the expression of amino acid metabolism related genes under the low concentration of nitrogen
The intracellular amino acids are obtained through many pathways, and GCN4 is one of the masters responsible for regulating genes involved in amino acid transport and biosynthesis [22]. We further investigated the expression of genes related to amino acid metabolism. The glutamic acid and aspartic acid were the abundant amino acids in mushroom, as well as in G. lucidum (Table 1). Therefore, the genes including the asparagine synthetase gene (asns), glutamine synthetase gene (gs), glutamate synthase gene (gogat), glutamic oxaloacetic-transaminase gene (got1 and got2) and glutamic-pyruvic transaminase gene (gpt) were detected. The expression of all these genes in the WT strain under the 3 mM asparagine was significantly induced, especially the asns gene, which was 2.41-fold higher than that under the 60 mM asparagine (Fig. 2A). Silencing of gcn4 under 3 mM asparagine markedly inhibited the expression of almost all these genes, and resulted in the reduction for approximately 58.71%-90.83% compared with WT. While under 60 mM asparagine, the expression of these genes decreased by 24.64%-62.88% in the gcn4 silenced strains compared with WT (Fig. 2A-F). However, the expression of gogat was unchanged either in the WT or in the gcn4-silecend strains (Fig, 2C). Furthermore, the expression of 7 genes responsible for amino acid transporting (Gl23068, Gl29937, Gl20736, Gl21744, Gl23271, Gl28933, and Gl23088) were decreased by 71.29–92.59% in gcn4-silenced strains compared with that in the WT strain under the 3 mM asparagine (Fig. 2G-M). While under the 60 mM asparagine, the expression of these genes decreased by 22.84%-60.1% in the gcn4 silenced strains compared with WT. These results indicated that silencing of gcn4 decreased the expression of genes related to amino acid metabolism, especially under the low nitrogen conditions.
Silencing of gcn4 decreased TCA and glycolysis pathway under the low concentration of nitrogen
Amino acids are also replenished through the TCA cycle and glycolysis pathway [36, 37]. Therefore, we further investigated the effect of GCN4 on the TCA cycle and glycolysis pathway. As shown in Fig. 3, we examined the activities of two key enzymes in the TCA cycle. Under the 3 mM asparagine, the activities of mitochondrial isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (KGDH) in the WT strain were significantly up-regulated by 1.9 and 2.4 folds, respectively, compared to that under the 60 mM asparagine condition (Fig. 3A). The activities of hexokinase (HK), pyruvate kinase (PK), and phosphofructokinase (PFK) activities in the WT strain also increased 1.65, 1.52 and 2.2 folds, respectively, compared to 60 mM asparagine condition (Fig. 3C). These suggested that low nitrogen condition promotes the TCA cycle and glycolysis pathway in G. lucidum.
Further, under the 60 mM asparagine condition, after silencing gcn4, the enzyme activities of ICDH and KGDH decreased by 56% and 92.1%, respectively, compared with the WT strain. Silencing of gcn4 also led to 54.63%, 83.4% and 62.39% decrease of PFK, HK and PK activities, respectively, compared with WT. However, under the 60 mM asparagine condition, the enzyme activities of ICDH and three key enzymes involved in glycolysis pathway showed no difference between WT and gcn4-silenced strains (Fig. 3A, B). The expression of genes in gcn4-silenced strains corresponded to the enzyme activities and exhibited a significant decrease for 35.38–83.15% under the 3 mM asparagine condition (Fig. 3B and 3D). The above results suggest that GCN4 enhanced TCA cycle and glycolysis pathway under the low nitrogen conditions.
Silencing of gcn4 increased the activity of TORC1 under the low concentration of nitrogen
The TORC1 plays a central role in sensing and regulating metabolism and availability of amino acid [38]. To comprehensively analyze the regulation of GCN4 on the amino acid homeostasis, the activity of TORC1 (characterized by the phosphorylation state of S6K) in WT and gcn4-silenced strains under the 3 mM or 60 mM asparagine conditions were tested (Fig. 4A). The phosphorylation level of S6K in WT strain significantly decreased by 66.4% at 3 mM asparagine condition compared with that at 60 mM asparagine condition. However, silencing of gcn4 increased the S6K phosphorylation compared with WT, with the 71.4% and 22% increased under 3 mM asparagine and 60 mM asparagine, respectively (Fig. 4B). This result suggests that GCN4 exerts an inhibitory effect on TORC1 activity, especially under the low nitrogen conditions.