Global transcriptome analysis profiles metabolic pathways in traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao

Abstract

Background: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao (A. mongolicus, family Leguminosae) is one of the most important traditional Chinese herbs. Among many secondary metabolites it produces, the effective bioactive constituents include isoflavonoids and triterpene saponins. The genomic resources regarding the biosynthesis of these metabolites in A. mongolicus are limited. Although roots are the primary material harvested for medical use, the biosynthesis of the bioactive compounds and its regulation in A. mongolicus are not well understood. Therefore, a global transcriptome analysis on A. mongolicus tissues was performed to identify the genes essential for the metabolism and to profile their expression patterns in greater details.

Results: RNA-sequencing was performed for three different A. mongolicus tissues: leaf, stem, and root, using the Illumina Hiseq2000 platform. A total of 159.5 million raw sequence reads were generated, and assembled into 186,324 unigenes with an N50 of 1,524bp. Among them, 129,966 unigenes (~69.7%) were annotated using four public databases (Swiss-Prot, TrEMBL, CDD, Pfam), and 90,202, 63,946, and 78,326 unigenes were found to express in leaves, roots, and stems, respectively. A total of 8,025 transcription factors (TFs) were identified, in which the four largest families, bHLH, MYB, C3H, and WRKY, were implicated in regulation of tissue development, metabolisms, stress response, etc. Unigenes associated with secondary metabolism, especially those with isolavonoids and triterpene saponins biosynthesis were characterized and profiled. Most genes involved in the isoflavonoids biosynthesis had the lowest expression in the leaves, and the highest in the stems. For triterpene saponin biosynthesis, we found the genes in MVA and non-MVA pathways were differentially expressed among three examined tissues, indicating the parallel but compartmentally separated biosynthesis pathways of IPP and DMAPP in A. mongolicus. The first committed enzyme in triterpene saponin biosynthesis from A. mongolicus, cycloartenol synthase (AmCAS), which belongs to the oxidosqualene cyclase family, was cloned by us to study the astragalosides biosynthesis. Further co-expression analysis indicated the candidate CYP450s and glycosyltransferases (GTs) in the cascade of triterpene saponins biosynthesis. The presence of the large CYP450 families in A. mongolicus was further compared with those from Medicago truncatula and Arabidopsis thaliana, and the diversity and phylegenetic relationships of the CYP450 families were established.

Conclusion: A transcriptome study was performed for A. mongolicus tissues to construct and profile their metabolic pathways, especially for the important bioactive molecules. The results revealed a comprehensive profile for metabolic activities among tissues, pointing to the equal importance of leaf, stem, and root in A. mongolicus for the production of bioactive compounds. This work provides valuable resources for bioengineering and in vitro synthesis of the natural compounds for medical research and for potential drug development.

Figure 1

A. mongolicus used in this study. A) whole-plant. The name of each tissue was labeled with black words. B) A. mongolicus in normal growth conditions.

Figure 2

Length distribution of assembled unigenes. Out of the 186,324 unigenes, 95,274 unigenes (51.1%) were longer than 500 bp and 55,988 (30%) were longer than 1000 bp

Figure 3

Unigenes expressed number and level in different tissues. A) The number of expressed genes (RPKM ≥ 0.5) in each tissue was labeled over the bars. B) Expressional levels of unigenes of three tissues from A. mongolicus. We took the logarithm of each RPKM value of all unigenes.

Figure 4

Venn diagram of expressed unigenes in three tissues. A total of 134,445 unigenes (72.15%) were expressed, of which 37,490 genes were common expressed in all the three tissues. Unigenes with RPKM≥0.5 were considered as expressed in each tissue.

Figure 5

COG function classifications of unigenes. A total of 65,989 unigenes were classified into 24 COG categories. A: RNA processing and modification; B: Chromatin structure and dynamics; C: Energy production and conversion; D: Cell cycle control, cell division, chromosome partitioning; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; G: Carbohydrate transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination and repair; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; O: Posttranslational modification, protein turnover, chaperones; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; R: General function prediction only; S: Function unknown; T: Signal transduction mechanisms; U: Intracellular trafficking, secretion, and vesicular transport; V: Defense mechanisms; Y: Nuclear structure; Z: Cytoskeleton.

Figure 6

Venn diagram showing expressed TFs in three tissues. A total of 6,359 TFs (79.23%) were significantly expressed, of which 2,676 TFs were commonly expressed in all three tissues. Unigenes with RPKM≥0.5 were considered as expressed in each tissue.

Figure 7

Classification of TF families. Number of unigenes related to TFs in each TF family. Among the TF families, bHLH, MYB, C3H, and WRKY proteins were the most abundant.

Figure 8

Related unigenes of the top TFs in three tissues. Unigenes with RPKM≥0.5 were considered as expressed in each tissue. A, Venn diagram showing expressed bHLHs in three tissues. B, Venn diagram showing expressed MYBs in three tissues. C. Venn diagram showing expressed C3Hs in three tissues D. Venn diagram showing expressed WRKYs in three tissues

Figure 9

Two secondary metabolic pathways in A. mongolicus. A) Enzymes involved in the pathway of isoflavonoid biosynthesis. B) Enzymes involved in the putative pathway of triterpenoid biosynthesis. Blue boxes indicate the highly expressed enzymes in root and stem, while green boxes indicate the highly expressed in leaf. Red boxes indicate important compounds in the pathway. C) Structural formulas of compounds synthesized in the putative downstream pathway of astragalosides biosynthesis.

Figure 10

Expression level of genes involved in isoflavonoids biosynthesis in three tissues. Nine genes involved in the isoflavonoids biosynthesis showing different expression patterns.

Figure 11

Tissue-differential expressed genes involved in triterpenoid saponins biosynthesis. ACAT, HMGCS, HMGCR, MVK and MVD from MVA pathway were highly expressed in the root and stem. DXR, ispF and ispH from non-MVA pathway were highly expressed in the leaf.

Figure 12

Protein sequence alignment of CAS from A. mongolicus, Glycyrrhiza glabra, Lotus japonicas, Medicago truncatula and Pisum sativum. AmCAS and Glycyrrhiza glabra CAS showing 93% identity, AmCAS and Lotus japonicas CAS showing 90% identity, AmCAS and CAS from Medicago truncatula and Pisum sativum showing 88% identity. Red box indicates the terpene synthases signature.

Figure 13

The neighbor-joining bootstrapped phylogenetic tree of A. mongolicus P450 genes. Bootstrap values which have been converted into the percentage obtained after 1000 replications are indicated on the branches.

Figure 14

Proportion of each CYP450 clan of A. mongolicus, Medicago truncatula and Arabidopsis thaliana. Proportion of nine CYP450 clans of three species were compared, respectively. The distribution trend of CYP450 clans is basically consistent in all three species.

Figure 15

Venn diagram showing expressed CYP450s in three tissues. A total of 85 CYP450s (89.47%) are expressed in three tissues, of which 31 CYP450s were commonly expressed in all three tissues. Each tissue had 57 expressed CYP450 unigenes. Unigenes with RPKM≥0.5 were considered as expressed in each tissue.