Full-length transcriptome and metabolite analysis reveal reticuline epimerase-independent pathways for benzylisoquinoline alkaloids biosynthesis in Sinomenium acutum

Abstract

Benzylisoquinoline alkaloids (BIAs) are a large family of plant natural products with important pharmaceutical applications. Sinomenium acutum is a medicinal plant from the Menispermaceae family and has been used to treat rheumatoid arthritis for hundreds of years. Sinomenium acutum contains more than 50 BIAs, and sinomenine is a representative BIA from this plant. Sinomenine was found to have preventive and curative effects on opioid dependence. Despite the broad applications of S. acutum, investigation on the biosynthetic pathways of BIAs from S. acutum is limited. In this study, we comprehensively analyzed the transcriptome data and BIAs in the root, stem, leaf, and seed of S. acutum. Metabolic analysis showed a noticeable difference in BIA contents in different tissues. Based on the study of the full-length transcriptome, differentially expressed genes, and weighted gene co-expression network, we proposed the biosynthetic pathways for a few BIAs from S. acutum, such as sinomenine, magnoflorine, and tetrahydropalmatine, and screened candidate genes involved in these biosynthesis processes. Notably, the reticuline epimerase (REPI/STORR), which converts (S)-reticuline to (R)-reticuline and plays an essential role in morphine and codeine biosynthesis, was not found in the transcriptome data of S. acutum. Our results shed light on the biogenesis of the BIAs in S. acutum and may pave the way for the future development of this important medicinal plant.

Keywords: Sinomenium acutum; benzylisoquinoline alkaloid; biosynthetic pathway; transcriptome; weighted gene co-expression network analysis.

Figure 1

Photos and HPLC profiles of fresh tissues of S. acutum. (A–D): the photos of fresh root (A), stem (B), leaf (C), and seed (D). (E): HPLC profiles of the four tissues and the mixed standards of 12 BIAs from S. acutum. (F): Chemical structures of 12 selected BIAs, including sinomenine N-oxide (1), sinomenine (2), acutumine (3), dauricumidine (4), bianfugenine (5), magnoflorine (6), 8-demethoxyrunanine (7), sinoacutine (8), sinoracutine (9), menisperine (10), stepharanine (11), and tetrahydropalmatine (12).

Figure 2

The length distribution and functional annotation of the unigenes in S. acutum. (A) Length distribution of the 60,675 unigenes. (B) Annotated unigenes from different public databases. (C) NR homologous species distribution analysis.

Figure 3

The number and pathway terms of differentially expressed genes among the root, stem, and leaf. (A) Venn diagram of DEGs in the three tissues. (B), (C): pathway enrichment of genes predominantly expressed in the root (B), stem (C), and leaf (D).

Figure 4

Weighted gene co-expression network analysis (WGCNA) of DEGs identified in the root, stem, leaf, and seed of S. acutum. (A) Determination of soft-thresholding power in WGCNA. (B) Hierarchical cluster tree showing seven modules of co-expressed genes. (C) Correlations of the modules and the seven BIAs. (D) Heatmap of the correlation between different modules in the weighted gene co-expression network. The symbol * denotes correlation coefficient smaller than -0.5.

Figure 5

Expression patterns of candidate unigenes in the common biosynthetic pathway of BIAs.

Figure 6

Phylogenetic analysis and expression patterns of CYPs and methyltransferases in S. acutum. (A) Phylogenetic tree of the candidate CYPs with known CYPs involved in BIA biosynthesis. (B) Expression levels of the screened CYP-encoding genes in the root, stem, leaf, and seed of S. acutum. (C) Phylogenetic tree of the candidate methyltransferases for BIA biosynthesis. (D) Expression levels of genes encoding the methyltransferases for BIA biosynthesis in the root, stem, leaf, and seed of S. acutum..

Figure 7

Proposed biosynthetic pathways for sinomenine, magnoflorine, and tetrahydropalmatine in S. acutum.