Integrated metabolomics and transcriptomics study of traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao reveals global metabolic profile and novel phytochemical ingredients

Abstract

Background: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is one of the most common herbs widely used in South and East Asia, to enhance people's health and reinforce vital energy. Despite its prevalence, however, the knowledge about phytochemical compositions and metabolite biosynthesis in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is very limited.

Results: An integrated metabolomics and transcriptomics analysis using state-of-the-art UPLC-Q-Orbitrap mass spectrometer and advanced bioinformatics pipeline were conducted to study global metabolic profiles and phytochemical ingredients/biosynthesis in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. A total of 5435 metabolites were detected, from which 2190 were annotated, representing an order of magnitude increase over previously known. Metabolic profiling of Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao tissues found contents and synthetic enzymes for phytochemicals were significantly higher in leaf and stem in general, whereas the contents of the main bioactive ingredients were significantly enriched in root, underlying the value of root in herbal remedies. Using integrated metabolomics and transcriptomics data, we illustrated the complete pathways of phenylpropanoid biosynthesis, flavonoid biosynthesis, and isoflavonoid biosynthesis, in which some were first reported in the herb. More importantly, we discovered novel flavonoid derivatives using informatics method for neutral loss scan, in addition to inferring their likely synthesis pathways in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao.

Conclusions: The current study represents the most comprehensive metabolomics and transcriptomics analysis on traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. We demonstrated our integrated metabolomics and transcriptomics approach offers great potentials in discovering novel metabolite structure and associated synthesis pathways. This study provides novel insights into the phytochemical ingredients, metabolite biosynthesis, and complex metabolic network in herbs, highlighting the rich natural resource and nutritional value of traditional herbal plants.

Keywords: Astragalus membranaceus Bge. Var. mongolicus; Biosynthesis; Flavonoid derivative; Non-targeted metabolomics; Phytochemical compositions; Secondary metabolites.

Fig. 1

Functional annotation and classification of differentially expressed unigenes between different tissues of AMM. a The reads number and mapped ratio to the reference transcriptome of different tissues (L1-L3:leaf; R1-R3:root; S1-S3: stem). The bar plot represents the clean reads number in different tissues, and the dots plot represents mapped ratio of different tissues to the reference transcriptome in different tissues. b The expression level of unigenes in different tissues from AMM (FPKM ≥0.5). We took the logarithm of each FPKM value of all unigenes. c Gene Ontology (GO) functional classifications of differentially expressed unigenes (Only list significantly enriched GO terms, P value≤0.05). Red: CC Cellular Component; Green: MF Molecular Function; Blue: BP Biological Process. d Significantly enriched KEGG pathways between differentially expressed unigenes. The Q value denoted the corrected P-value (Significant pathways were identified by Q value≤0.05). Count denoted the number of differentially expressed unigenes mapped to a certain pathway according to KEGG database. The pathways with ‘**’ denoted the significantly enriched pathways that included in both between the root vs leaf and root vs stem analysis

Fig. 2

The relative content (GroupArea) of metabolites. a phenylpropanoids, b alkaloids, c terpenoids, and d flavonoids in different tissues of AMM

Fig. 3

The inferred pathways for Terpenoids biosynthesis in Mongolicus. a The Terpenoids biosynthesis pathway. The enzymes confirmed by transcriptomics data are shown in red. Blue indicate the highly expressed enzymes in root and stem, while green indicate the highly expressed in leaf. ACAT: acetyl-CoA C-acetyltransferase; HMGCS: hydroxymethylglutaryl-CoA synthase; HMGCR: hydroxymethylglutaryl-CoA reductase; MVK: mevalonate kinase; PMVK: phosphomevalonate kinase; MVD: diphosphomevalonate decarboxylase; DXS: 1-deoxy-D-xylulose-5-phosphate synthase;DXR:1-deoxy-D-xylulose-5-phosphate reductoisomerase; ispD:2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; ispE: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; ispF: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ispG:(E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; ispH: 4-hydroxy-3-methylbut-2-en-1-yl diphosphatereductase; IDI: isopentenyl-diphosphate Delta-isomerase. FDPS:farnesyldiphosphate synthase; ZFPS:(2Z,6Z)-farnesyldiphosphate synthase; FDFT1: farnesyl-diphosphatefarnesyltransferase; SQLE: squalenemonooxygenase. CAS1: cycloartenol synthase; P450: cytochromeP450; GT: glycosyltransferase. b The expression of the genes (enzymes) in the pathway measured by FPKM value

Fig. 4

The inferred pathways for phenylpropanoid biosynthesis, flavonoids biosynthesis, and isoflavonoids biosynthesis in AMM. a The phenylpropanoid,flavonoids and isoflavonoids biosynthesis pathway. The relative contents (log2GroupArea) for each metabolite in the three tissues were denoted with juxtaposed colored boxes (left: root; middle: leaf; and right: stem). The metabolites that significantly higher in root than the other two tissues were marked by ‘**’, while the metabolites that significantly lower in root than the other two tissues were marked by ‘*’. The enzymes confirmed by transcriptomics data are shown in red. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalconeisomerase; CHR, chalconereductase; IFS, isoflavone synthase; IOMT, Isoflavone O-methyltransferase; I3’H, isoflavone 3′-hydroxylase; I2’H, isoflavone 2′-hydroxylase; UCGT, under calycosin 7-O-glucosyltransferase [2]; HIDH,2-hydroxyisoflavanone dehydratase; HI4OMT,isoflavone 4′-O-methyltransferase; IF7GT, isoflavone 7-O-glucosyltransferase . b The expression of the genes (enzymes) in the pathway measured by FPKM value

Fig. 5

Derivatives of isoflavonoids for some bioactive components from AMM. a Derivatives of isoflavonoids of the modifications by glycoside-, malonyl-, and Rha-hexoses- groups are illustrated. b-e Mass spectra and structures of novel derivatives from modifications with malonylglucoside, or Rha-hexoses groups in AMM. b Derriscanoside A (m/z: 575.1779) is a derivative of formononetin (m/z: 267.0664) with modification of a Rha-hexose-group. c Linarin (m/z: 593.1862) is a derivative of acacetin (m/z: 285.0757) with modification of a Rha-hexose-group. d Glycitein 6″-O-Malonylglucoside (m/z: 533.1291) is a derivative of glycitein with malonylglucoside modification. e Biochanin A 7-O-glucoside-6″-O-malonate (m/z: (533.1288) is a derivative of Biochanin A with malonylglucoside modification

Fig. 6

Mass spectra and structures of novel derivatives of Chrysoeriol. a AMM06537p (m/z 797.1767) is a derivative of Chrysoeriol (m/z [M + H]+: 301.0705) with modification of two malonylglucoside group. b AMM05484n (m/z 841.2196) a derivative of Chrysoeriol (m/z [M-H]-: 299.0563 with modification of an acetyl-hexose-group. c The inferredbiosynthetic pathway of the novel derivatives of Chrysoeriol. CHI, chalconeisomerase; FNSI, flavone synthase I