Integrative analysis of the transcriptome and metabolome provides insights into polysaccharide accumulation in Polygonatum odoratum (Mill.) Druce rhizome

Abstract

Background: Polygonatum odoratum (Mill.) Druce is a traditional Chinese herb that is widely cultivated in China. Polysaccharides are the major bioactive components in rhizome of P. odoratum and have many important biological functions.

Methods: To better understand the regulatory mechanisms of polysaccharide accumulation in P. odoratum rhizomes, the rhizomes of two P. odoratum cultivars 'Y10' and 'Y11' with distinct differences in polysaccharide content were used for transcriptome and metabolome analyses, and the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were identified.

Results: A total of 14,194 differentially expressed genes (DEGs) were identified, of which 6,689 DEGs were down-regulated in 'Y10' compared with those in 'Y11'. KEGG enrichment analysis of the down-regulated DEGs revealed a significant enrichment of 'starch and sucrose metabolism', and 'amino sugar and nucleotide sugar metabolism'. Meanwhile, 80 differentially accumulated metabolites (DAMs) were detected, of which 52 were significantly up-regulated in 'Y11' compared to those in 'Y10'. The up-regulated DAMs were significantly enriched in 'tropane, piperidine and pyridine alkaloid biosynthesis', 'pentose phosphate pathway' and 'ABC transporters'. The integrated metabolomic and transcriptomic analysis have revealed that four DAMs, glucose, beta-D-fructose 6-phosphate, maltose and 3-beta-D-galactosyl-sn-glycerol were significantly enriched for polysaccharide accumulation, which may be regulated by 17 DEGs, including UTP-glucose-1-phosphate uridylyltransferase (UGP2), hexokinase (HK), sucrose synthase (SUS), and UDP-glucose 6-dehydrogenase (UGDH). Furthermore, 8 DEGs (sacA, HK, scrK, GPI) were identified as candidate genes for the accumulation of glucose and beta-D-fructose 6-phosphate in the proposed polysaccharide biosynthetic pathways, and these two metabolites were significantly associated with the expression levels of 13 transcription factors including C3H, FAR1, bHLH and ERF. This study provided comprehensive information on polysaccharide accumulation and laid the foundation for elucidating the molecular mechanisms of medicinal quality formation in P. odoratum rhizomes.

Keywords: Metabolome; Polygonatum Odoratum (Mill.) Druce; Polysaccharide accumulation; Transcriptome.

Figure 1. Polysaccharides content (A) and representative images (B) of rhizome in two P. odoratum cultivars ‘Y10’ and ‘Y11’.

All date are means (±SD), n = 3. Significant differences were determined using one-way ANOVA: * P < 0.05.

Figure 2. Functional enrichment analysis of differentially expressed genes (DEGs) by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based enrichment analysis.

(A) The top 20 pathway classifications selected by KEGG pathway-based enrichment analysis based on the DEGs between ‘Y11’ and ‘Y10’. (B) The number of up-regulated or down-regulated DEGs related to the 15 pathway classifications for carbohydrate metabolism in the ‘Y10’ vs ‘Y11’ group.

Figure 3. Heatmap visualization of differentially accumulated metabolites (DAMs) of rhizome between ‘Y11’ and ‘Y10’.

M10-1 M10-6: The six simples of ‘Y10’ for metabolome analysis; M11-1 M11-6: The six simples of ‘Y11’ for metabolome analysis.

Figure 4. Functional enrichment analysis of differentially accumulated metabolites (DAMs) by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based enrichment analysis.

(A) The top 20 pathway classifications selected by KEGG pathway-based enrichment analysis based on the DAMs between ‘Y11’ and ‘Y10’ group. (B) The number of up- or down-regulated DAMs related to the 11 pathway classifications for carbohydrate metabolism in the ‘Y10’ vs ‘Y11’ group. (C) Heatmap visualisation of up-regulated and down-regulated DAMs related to the 11 pathway classifications for carbohydrate metabolism.

Figure 5. Correlation analysis of DAMs and DEGs related to carbohydrate metabolism (A) Genes-metabolites interaction network. (B) Heat map of the correlation coefficients between four DAMs and 17 DEGs.

Triangle: DAMs; Circle marked in red: the up-regulated genes in ‘Y10’ compared with those in ‘Y11’; Circle marked in pink: the down-regulated genes in ‘Y10’ compared with those in ‘Y11’. Asterisks represent statistical significance determined by Student’s t-test (*P ¡ 0.05, **P ¡ 0.01).

Figure 6. (A–B) Expression pattern analysis of SUS, HK, UGDH and UGP2 at different tissues and different genotypes.

Different letters indicate significant difference at P < 0.05.

Figure 7. Proposed biosynthetic pathways of polysaccharide in P. odoratum rhizome.

The up-regulated DAMs in ‘Y11’, compared with ‘Y10’, were marked in red; key enzymes encoded by nine DEGs related to the above-mentioned DAMs were marked in purple.

Figure 8. The transcription factors (TFs)-metabolite association network constructed according to the differentially expressed TFs and differentially accumulated metabolites involved in proposed pathways for polysaccharide biosynthesis in P. odoratum.

(A) Type and number of the differentially expressed TFs in P. odoratum rhizome. (B) TFs-metabolite association network analysis. The red lines indicate TFs that were positively significantly associated with the three metabolites, while the blue lines indicate TFs that were negatively significantly associated with the three metabolites. (C) Heatmap of the correlation coefficients between glucose, levan and beta-D-fructose 6-phosphate and the expression levels of TFs based on the Pearson’s correlation coefficient.

Figure 9. (A–I) The relative expression levels of nine selected DEGs were compared by RNA-seq and qRT PCR.

The line chart shows the gene expression level from the transcriptome (FPKM).

Figure 10. A model explaining the key genes and metabolites involved in polysaccharide accumulation in P. odoratum.

In the process of polysaccharide accumulation in the rhizome of P. odoratum, three metabolites including glucose, Beta-D-Fru-6P and levan were the key metabolites for polysaccharide accumulation. Among the these metabolites, glucose was regulated by the enzyme genes sacA, and beta-D-fru-6P was regulated by the genes HK, GPI and scrK. In addition, the transcription factors C3H, FAR1, bHLH, ERF, HB, HSF, MADS and NAC were also involved in regulating the accumulation of the two metabolites, glucose and beta-D-fru-6P. Meanwhile, two other key metabolites, maltose and 3-beta-D-galactosyl-sn-glycerol, involved in an unknown pathway to account for the accumulation of polysaccharide, and 3-beta-D-galactosyl-sn-glycerol accumulated in the rhizome may be associated with the expression of the gene UGLA.