Transcriptome Analysis Reveals an Essential Role of Exogenous Brassinolide on the Alkaloid Biosynthesis Pathway in Pinellia Ternata

Abstract

Pinellia ternata (Thunb.) Druce is a traditional medicinal plant containing a variety of alkaloids, which are important active ingredients. Brassinolide (BR) is a plant hormone that regulates plant response to environmental stress and promotes the accumulation of secondary metabolites in plants. However, the regulatory mechanism of BR-induced alkaloid accumulation in P. ternata is not clear. In this study, we investigated the effects of BR and BR biosynthesis inhibitor (propiconazole, Pcz) treatments on alkaloid biosynthesis in the bulbil of P. ternata. The results showed that total alkaloid content and bulbil yield was enhanced by 90.87% and 29.67% under BR treatment, respectively, compared to the control. We identified 818 (476 up-regulated and 342 down-regulated) and 697 (389 up-regulated and 308 down-regulated) DEGs in the BR-treated and Pcz-treated groups, respectively. Through this annotated data and the Kyoto encyclopedia of genes and genomes (KEGG), the expression patterns of unigenes involved in the ephedrine alkaloid, tropane, piperidine, pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis were observed under BR and Pcz treatments. We identified 11, 8, 2, and 13 unigenes in the ephedrine alkaloid, tropane, piperidine, and pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis, respectively. The expression levels of these unigenes were increased by BR treatment and were decreased by Pcz treatment, compared to the control. The results provided molecular insight into the study of the molecular mechanism of BR-promoted alkaloid biosynthesis.

Keywords: RNA-seq; alkaloid; brassinolide; ephedrine; secondary metabolism.

Figure 1

Functional annotations of the unigenes of the P. ternata bulbil transcriptome. (A) length distribution of assembled unigenes; (B) GO function annotation of P. ternata; (C) KEGG function annotation of P. ternata.

Figure 2

Identification and functional information of differential expression genes (DEGs) among control, BR, and Pcz treatments in P. ternata. (A) volcano of DEGs in control and BR groups; (B) volcano of DEGs in control and Pcz groups; (C) GO enrichment analyses with the DEGs generated in control and BR groups; (D) GO enrichment analyses with the DEGs generated in control and Pcz groups; (E) KEGG enrichment analyses with the DEGs generated in control and BR groups; (F) KEGG enrichment analyses with the DEGs generated in control and Pcz groups.

Figure 3

Effects of BR and Pcz treatments on the total alkaloid contents (A) and bulbil yield (B). The bars with different letters are significantly different from each treatment (p < 0.05). Values are means of four replicates ± SE. Proposed biosynthetic routes of ephedrine in P. ternata (C). Differentially expressed levels of unigenes related to ephedrine biosynthesis (D). Changes in expression level are represented by a change in color; blue indicates a lower expression level, whereas red indicates a higher expression level. All data shown reflect the average mean of three biological replicates.

Figure 4

Differential expression levels of unigenes related to tropane, piperidine, and pyridine alkaloid biosynthesis identified by KEGG annotation. Changes in expression level are represented by a change in color; blue indicates a lower expression level, whereas red indicates a higher expression level. All data shown reflect the average mean of three biological replicates.

Figure 5

Differential expression levels of unigenes related to indole alkaloid biosynthesis identified by KEGG annotation. Changes in expression level are represented by a change in color; blue indicates a lower expression level, whereas red indicates a higher expression level. All data shown reflect the average mean of three biological replicates.

Figure 6

Differential expression levels of unigenes related to isoquinoline alkaloid biosynthesis identified by KEGG annotation. Changes in expression level are represented by a change in color; blue indicates a lower expression level, whereas red indicates a higher expression level. All data shown reflect the average mean of three biological replicates.