A chromosome-level genome assembly of anesthetic drug-producing Anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids

Abstract

Tropane alkaloids (TAs), which are anticholinergic agents, are an essential class of natural compounds, and there is a growing demand for TAs with anesthetic, analgesic, and spasmolytic effects. Anisodus acutangulus (Solanaceae) is a TA-producing plant that was used as an anesthetic in ancient China. In this study, we assembled a high-quality, chromosome-scale genome of A. acutangulus with a contig N50 of 7.4 Mb. A recent whole-genome duplication occurred in A. acutangulus after its divergence from other Solanaceae species, which resulted in the duplication of ADC1 and UGT genes involved in TA biosynthesis. The catalytic activities of H6H enzymes were determined for three Solanaceae plants. On the basis of evolution and co-expressed genes, AaWRKY11 was selected for further analyses, which revealed that its encoded transcription factor promotes TA biosynthesis by activating AaH6H1 expression. These findings provide useful insights into genome evolution related to TA biosynthesis and have potential implications for genetic manipulation of TA-producing plants.

Keywords: AaWRKY11; Anisodus acutangulus; biosynthesis; hyoscyamine 6 β-hydroxylase; tropane alkaloid.

Figure 1

Anisodus acutangulus plants were used in ancient China for their medicinal properties. (A)A. acutangulus is an analgesic herb. (B)A. acutangulus genome assembly. The layers of the circos plot (outside to inside) represent chromosome length (I), repeat content (II), gene density (III), LTR content (IV), and syntenic blocks (V).

Figure 2

Evolutionary history of A. acutangulus. (A) Phylogenetic tree for A. acutangulus and 11 other plant species. The timing of the A. acutangulus WGD event is superimposed on the tree. Pie charts represent the expansion and contraction of gene families. (B) Distribution of 4DTv values indicated with colored lines. (C) Synteny maps of Solanum lycopersicum aligned with A. acutangulus and Vitis vinifera. Light gray lines indicate syntenic blocks. Different colors represent different WGD traces among the species.

Figure 3

Evolution of genes related to tropane alkaloid (TA) biosynthesis in Anisodus acutangulus. (A) TA metabolic pathways and TA biosynthetic gene expression as determined by transcriptomic analysis. Different A. acutangulus tissues were analyzed, including the leaf, stem, primary root (periderm, phloem, and xylem), secondary root (periderm, phloem, and xylem), and fibrous root. Source data are provided in the supplemental information. (B) Microsynteny analysis of H6H between A. acutangulus and other Solanaceae species. The species included in this analysis were A. acutangulus (Aac), Datura stramonium (Dst), Atropa belladonna (Abe), and Solanum lycopersicum (Sly). The syntenic H6H genes are highlighted in red. The genomic region and chromosome ID are indicated under the abbreviated species name. (C) Phylogenetic tree of H6H genes from different species. The NCBI accession numbers of the H6H genes are summarized in Supplemental Table 16.

Figure 4

Functional identification of H6Hs. (A) Purified His-tagged H6Hs from three Solanaceae plants. Lane 1, Escherichia coli BL21 total protein (without IsoPropyl b-D-ThioGalactoside [IPTG] induction); 2, AaH6H1 (chr04g01723.t1) from Anisodus acutangulus; 3, AaH6H2 (chr04g01695.t1) from A. acutangulus; 4, DsH6H (GenBank: KR006982.1) from Datura stramonium; 5, AbH6H (GenBank: JN415637.1) from Atropa belladonna. (B) Extracted-ion chromatogram (EIC) traces of TAs (hyoscyamine, anisodamine, and scopolamine). All detections were repeated with three biological replicates.

Figure 5

AaWRKY11 binds to the W-box element in the AaH6H1 promoter to activate expression. (A) Heatmap of WRKY gene expression levels in different Anisodus acutangulus tissues. The WRKY genes (AaWRKY11 and 12 other WRKY genes) with the highest expression levels in fibrous roots, which accumulate more tropane alkaloids (TAs) than other tissues, are indicated. (B) Relationships between the expression of TA biosynthetic genes and candidate AaWRKY genes with the highest expression levels in the fibrous root; a correlation coefficient >0.9 was used as the cutoff (P < 0.001). AaWRKY11 and AaH6H1 are co-expressed. (C) Subcellular localization of AaWRKY11 in Nicotiana benthamiana leaf epidermal cells. DAPI served as the positive control. (D) A transient dual-LUC assay in tobacco leaves showing that the AaWRKY11 transcription factor activates the AaH6H1 promoter. The effector vector contained the AaWRKY11 sequence under the control of the CaMV35S promoter. AaH6Hpro::LUC was used as the reporter construct. The empty vector served as the negative control. The LUC/REN ratio represents the activation of the AaH6H1 promoter by AaWRKY11. Error bars indicate the standard error of the mean. Asterisks represent significant difference as determined by a t-test (n = 3 biologically independent samples; ∗∗P < 0.01). (E) Validation of the binding of AaWRKY11 to the W-box element in the AaH6H1 promoter by a Y1H assay.

Figure 6

AaWRKY11 positively regulates tropane alkaloid biosynthesis in transgenic A. acutangulus hairy roots. (A and C) Expression of genes underlying tropane alkaloid biosynthesis in the AaWRKY11-OE and AaWRKY11-KO lines. Fold changes in relative gene expression in transgenic hairy roots are normalized to the expression level of AaActin. (B and D) Tropane alkaloid contents in AaWRKY11-OE and AaWRKY11-KO hairy root lines were detected by high-performance liquid chromatography. Error bars indicate the standard error of the mean. Asterisks on bars indicate significant differences (relative to the control) as determined by a t-test (n = 3 biologically independent samples; ∗P < 0.05). (E) Model showing that AaWRKY11 promotes tropane alkaloid biosynthesis by binding to the W-box cis-element in the AaH6H1 promoter to activate expression.