Full-length transcriptome profiling of Acanthopanax gracilistylus provides new insight into the kaurenoic acid biosynthesis pathway

Abstract

Acanthopanax gracilistylus is a deciduous plant in the family Araliaceae, which is commonly used in Chinese herbal medicine, as the root bark has functions of nourishing the liver and kidneys, removing dampness and expelling wind, and strengthening the bones and tendons. Kaurenoic acid (KA) is the main effective substance in the root bark of A. gracilistylus with strong anti-inflammatory effects. To elucidate the KA biosynthesis pathway, second-generation (DNA nanoball) and third-generation (Pacific Biosciences) sequencing were performed to analyze the transcriptomes of the A. gracilistylus leaves, roots, and stems. Among the total 505,880 isoforms, 408,954 were annotated by seven major databases. Sixty isoforms with complete open reading frames encoding 11 key enzymes involved in the KA biosynthesis pathway were identified. Correlation analysis between isoform expression and KA content identified a total of eight key genes. Six key enzyme genes involved in KA biosynthesis were validated by real-time quantitative polymerase chain reaction. Based on the sequence analysis, the spatial structure of ent-kaurene oxidase was modeled, which plays roles in the three continuous oxidations steps of KA biosynthesis. This study greatly enriches the transcriptome data of A. gracilistylus and facilitates further analysis of the function and regulation mechanism of key enzymes in the KA biosynthesis pathway.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-024-01436-7.

Keywords: Acanthopanax gracilistylus; Differentially expressed genes; Ent-kaurene oxidase; Full-length transcriptome; Kaurenoic acid.

Fig. 1

Determination of KA content in A. gracilistylus. a Morphological characteristics of A. gracilistylus. b Results of KA determination in three tissues of A. gracilistylus (error bars indicate standard error from three biological replicates). **p < 0.01, ****p < 0.0001

Fig. 2

Expression of isoforms in the three tissues of A. gracilistylus. a Distribution of the number of isoforms with different expression levels in the three tissues. b Boxplots of isoforms expressed in three tissues. L, leaf; R, root; S, stem

Fig. 3

Annotations and functional classification of isoforms in A. gracilistylus. a Gene functional annotation in the KOG database. b Gene functional annotation in the GO database

Fig. 4

Annotation of A. gracilistylus isoforms in the KEGG database. a Five categories of biosynthesis pathways and the number of related isoforms. b The biosynthesis pathway of terpenoids and polyketones and the number of related isoforms

Fig. 5

Heatmap of isoforms related to KA biosynthesis in A. gracilistylus. a The expression level of key enzyme isoforms in various tissues of the KA biosynthesis pathway. b Correlation analysis between the content of KA and the expression of isoforms involved in the KA biosynthesis pathway. L, leaf; R, root; S, stem. *0.01 < p < 0.05, **0.001 < p < 0.01, ***p ≤ 0.001

Fig. 6

Quantity and enrichment analysis of DEGs. a Up-regulated and down-regulated DEGs in different tissues. b Venn diagram of DEGs in different comparison groups. c Enrichment of KEGG pathways for DEGs in the leaves compared to the roots. d Enrichment of KEGG pathways for DEGs in the stems compared to the leaves. e Enrichment of KEGG pathways for DEGs in the roots compared to the stems

Fig. 7

Sequence alignment and protein structure model of KO in A. gracilistylus. a Sequence alignment and secondary structure of KO in A. gracilistylus. The substrate-binding domain ETLR, redox site PERF, and heme-binding domain FGGGKRVCAG are labeled with green, blue, and black boxes, respectively. b Cartoon model of the structure of KO in A. gracilistylus; α-helices and β-sheets are represented in cyan and red, respectively. c Active site of KO in A. gracilistylus. The redox sites, substrate-binding domains, and heme-binding domains are indicated as yellow, green, and red spheres, respectively

Fig. 8

Identification of TFs and the interaction network with key enzymes in the KA biosynthesis pathway of A. gracilistylus. a Classification of TF families to which isoforms belong. b Network interactions between key enzymes in the KA biosynthesis pathway and the FHA family. The size of the circle and the thickness of the line represent the strength of the interaction between proteins

Fig. 9

Gene expression analysis of six isoforms related to KA biosynthesis in A. gracilistylus (error bars indicate standard error from three biological replicates). n = 3. L, leaf; R, root; S, stem. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, p > 0.05