Transcriptome and metabonomics combined analysis revealed the energy supply mechanism involved in fruiting body initiation in Chinese cordyceps

Abstract

Chinese cordyceps was one of most valuable traditional Chinese medicine fungi. To elucidate the molecular mechanisms related to energy supply mechanism involved in the initiation and formation of primordium in Chinese cordyceps, we performed the integrated metabolomic and transcriptomic analyses of it at pre-primordium period, primordium germination period and after-primordium period, respectively. Transcriptome analysis showed that many genes related to 'starch and sucrose metabolism', 'fructose and mannose metabolism', 'linoleic acid metabolism', 'fatty acids degradation' and 'glycerophospholipid metabolism' were highly up-regulated at primordium germination period. Metabolomic analysis showed many metabolites regulated by these genes in these metabolism pathways were also markedly accumulated at this period. Consequently, we inferred that carbohydrate metabolism and β-oxidation pathway of palmitic acid and linoleic acid worked cooperatively to generate enough acyl-CoA, and then entered TCA cycle to provide energy for fruiting body initiation. Overall, our finding provided important information for further exploring the energy metabolic mechanisms of realizing the industrialization of Chinese cordyceps artificial cultivation.

Figure 1

Sample collections of Chinese cordyceps for RNA-Seq. (A) Pre-primordium. (B) primordium. (C) After-primordium. (D) SEM of the pre-primordium mycelium (worm). (E) SEM of the primordium (the mycelium broke through the head). (F) SEM of the after primordium (the formation of stroma). (A–C) (Red shears) represented sampling site, respectively. (D) (1, 2), fusion of mycelium; (E) (1), slender and curved mycelium; (F) (1, 2), microscopic particles. Bar was 20 mm. (G) Total polysaccharide contents. (H) Polyphenol content. (I) Mannitol content. Bars represent mean ± SE (n = 3) and different lower-case letters represent significant difference at p < 0.05.

Figure 2

Differentially accumulated metabolites between three comparable groups. (A) Principal component analysis (PCA) of the variance-stabilized estimated raw counts of differentially accumulated metabolites. Bar plot showing numbers of DEMs in S2 vs S1 (B), S3 vs S2 (C), and S3 vs S1 (D) groups. The horizontal axis represents the differential expression multiple, and the vertical axis represents the degree of difference in metabolites meaning. The red dots indicated the upregulated expressed metabolites (Log₂ FC ≥ 2), the blue dots indicated the downregulated expressed metabolites (Log₂ FC ≤ 0.5).

Figure 3

TOP-20 enrichment diagram of metabolic pathways in three comparable groups. (A) S2 vs S1, (B) S3 vs S2, and (C) S3 vs S1 group. p-value in metabolic pathway was the significance of enrichment of this metabolic pathway. The red line indicated that the p value was 0.01, and the blue line indicated that the p value was 0.05. When the top of the bar was higher than the blue line, the signal pathway represented that it was significant.

Figure 4

Heatmap of different type DAMs metabolites before and after fruiting body initiation with TBtools software. (A) Lipid metabolism. (B) Carbohydrate metabolism. (C) Amino acid metabolism. (D) Neurotransmitter, active factor. DAMs are selected based on VIP > 1 and p < 0.05 in any of the comparison groups.

Figure 5

Bar plot showing numbers of DEGs in S2 vs S1 (A), S3 vs S2 (B), and S3 vs S1 (C) groups. Differential genes with p value < 0.05 and FC value > 2 were selected for KEGG enrichment pathways. Top 10 pathways as enrichment factor from DEGs in S2 vs S1 (D), S3 vs S2 (E), and S3 vs S1 (F) groups. The horizontal axis represents the differential expression multiple, and the vertical axis represents the degree of difference in gene meaning. KEGG enrichment pathways were created in http://www.kegg.jp/kegg/kegg1.html.

Figure 6

Integrated analyses of the transcriptome and metabolome of the KEGG pathways related to sugars and lipid. As shown in Suppl. Table S4. Three comparison groups of S2 vs S1 (A), S3 vs S2 (B), and S3 vs S1 (C) were selected. The specific name of the KEGG pathway was provided on the left of the bar chart, and the numbers of relative DEGs and DAMs were on the right of the bar. KEGG enrichment pathways were created in http://www.kegg.jp/kegg/kegg1.html.

Figure 7

Correlation network of DEGs and DAMs involved in sugars and lipid metabolism. (A) S2 vs S1, (B) S3 vs S2, (C) S3 vs S1. Green circles indicate genes, and orange circles indicate metabolites. Lines colored in “red” and “green” represent positive and negative correlations, respectively, as determined by a Pearson's correlation coefficient > 0.8 or < − 0.8 (q-value < 0.1), respectively.

Figure 8

The DEGs and DEMs involved in the carbohydrate metabolism and fatty acid degradation during the initiation process of fruiting body. The blue rectangle represented the significantly accumulated metabolites. The rectangle was divided into three equal parts (the left of the rectangle represents DEGs in S1, the middle represented DEGs in S2, and the right represented DEGs in S3. The color in the rectangle. The circle represented DEMs, three equal parts was similar to DEGs. HK hexokinase, NUDX14 ADP-sugar diphosphatase, rpiB ribose 5-phosphate isomerase B, TRE trehalose phosphate synthase, Glu beta-glucosidase, ACADM acyl-CoA dehydrogenase, fadA acetyl-CoA acyltransferase, MDH1 malate dehydrogenase, sucD succinyl-CoA synthetase, ST succinate thiokinase, ACO aconitase, IDP1:isocitrate dehydrogenase.

Figure 9

The DEGs and DEMs involved in the glycerophospholipid and linoleic acid metabolism during the initiation process of fruiting body. The blue rectangle represents the significantly accumulated metabolites. The rectangle was divided into three equal parts (the left of the rectangle represents DEGs in S1, the middle represented DEGs in S2, and the right represented DEGs in S3. The circle represented DEMs, three equal parts was similar to DEGs. glpA glycerol-3-phosphate dehydrogenase, AYR1 1-acylglycerone phosphate reductase, DPP1 diacylglycerol diphosphate phosphatase, AT2 acyltransferase, CK choline kinase, CCT cytidylyltransferase, EPC ethanolamine-phosphate cytidylyltransferase, AT1 acyltransferase1, EK ethanolamine kinase, CHPT1 CDP choline transferase, EHPT1 CDP ethanolamine transferase, LYPLA2 cytosolic phospholipase A2, SPLA2 ethanolaminephosphotransferase, CP1 cytosolic phospholipase, FAO fatty acid oxygenase.

Figure 10

Expression pattern validation (A–H) and linear dependence relation between the log₂ values of the key gene expression ratios obtained from RNA-seq and qRT-PCR (I).