Integrated Metabolomics and Transcriptomics Analysis of Flavonoid Biosynthesis Pathway in Polygonatum cyrtonema Hua

Abstract

Flavonoids, a class of phenolic compounds, are one of the main functional components and have a wide range of molecular structures and biological activities in Polygonatum. A few of them, including homoisoflavonoids, chalcones, isoflavones, and flavones, were identified in Polygonatum and displayed a wide range of powerful biological activities, such as anti-cancer, anti-viral, and blood sugar regulation. However, few studies have systematically been published on the flavonoid biosynthesis pathway in Polygonatum cyrtonema Hua. Therefore, in the present study, a combined transcriptome and metabolome analysis was performed on the leaf, stem, rhizome, and root tissues of P. cyrtonema to uncover the synthesis pathway of flavonoids and to identify key regulatory genes. Flavonoid-targeted metabolomics detected a total of 65 active substances from four different tissues, among which 49 substances were first study to identify in Polygonatum, and 38 substances were flavonoids. A total of 19 differentially accumulated metabolites (DAMs) (five flavonols, three flavones, two dihydrochalcones, two flavanones, one flavanol, five phenylpropanoids, and one coumarin) were finally screened by KEGG enrichment analysis. Transcriptome analysis indicated that a total of 222 unigenes encoding 28 enzymes were annotated into three flavonoid biosynthesis pathways, which were "phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "flavone and flavonol biosynthesis". The combined analysis of the metabolome and transcriptome revealed that 37 differentially expressed genes (DEGs) encoding 11 enzymes (C4H, PAL, 4CL, CHS, CHI, F3H, DFR, LAR, ANR, FNS, FLS) and 19 DAMs were more likely to be regulated in the flavonoid biosynthesis pathway. The expression of 11 DEGs was validated by qRT-PCR, resulting in good agreement with the RNA-Seq. Our studies provide a theoretical basis for further elucidating the flavonoid biosynthesis pathway in Polygonatum.

Keywords: Polygonatum cyrtonema Hua; differential metabolites; flavonoid biosynthesis; qRT-PCR; transcriptomics–metabolomics combined analysis.

Figure 1

Metabolite analysis on the basis of principal component analysis (PCA).

Figure 2

Metabolite analysis based on orthogonal partial least-squares discriminant analysis (OPLS-DA). (a–f) OPLS-DA model plots for the comparison group Leaf vs. Stem, Leaf vs. Rhizome, Leaf vs. Root, Stem vs. Rhizome, Stem vs. Root, Rhizome vs. Root, respectively. The colorful shading represents the confidence ellipse.

Figure 3

Statistics of differently expressed metabolites. The statistical analysis of the number of differential metabolites for all different groups is presented, with the red color indicating upregulated metabolites and the blue color indicating downregulated metabolites.

Figure 4

KEGG pathway enrichment of the differential metabolites in each comparison: (a) Leaf vs. Stem, (b) Leaf vs. Rhizome, (c) Leaf vs. Root, (d) Stem vs. Rhizome, (e) Stem vs. Root, (f) Rhizome vs. Root.

Figure 5

Transcription metabolism conjoint analysis data plot. (a) Correlation heatmap between differentially accumulated metabolites and differentially expressed genes. * p < 0.05, ** p < 0.01, *** p < 0.001; (b) The flavonoids biosynthesis and accumulation pathway of P. cyrtonema. The circle represents metabolite, and the purple circle indicates metabolite annotated by KEGG enrichment analysis. The box represents genes, and the red box represents the gene selected for joint analysis. The solid line indicates the metabolic reactions in only one step, the dash line presents more than one step of the metabolic reaction.

Figure 6

qRT-PCR validation of 11 DEGs. The X-axis means four tissues of P. cyrtonema. The left Y-axis represents the relative expression of each gene in four tissues by qRT-PCR, and the right Y-axis represents the FPKM value of each gene in four tissues by transcriptomics.