Transcriptomic analysis of genes related to alkaloid biosynthesis and the regulation mechanism under precursor and methyl jasmonate treatment in Dendrobium officinale

Abstract

Dendrobium officinale is both a traditional herbal medicine and a plant of high ornamental and medicinal value. Alkaloids, especially terpenoid indole alkaloids (TIAs), with pharmacological activities are present in the tissues of D. officinale. A number of genes involved in alkaloid biosynthetic pathways have been identified. However, the regulatory mechanisms underlying the precursor and methyl jasmonate (MeJA)-induced accumulation of alkaloids in D. officinale are poorly understood. In this study, we collected D. officinale protocorm-like bodies (PLBs) and treated them with TIA precursors (tryptophan and secologanin) and MeJA for 0 (T0), 4 (T4) and 24 h (T24); we also established control samples (C4 and C24). Then, we measured the total alkaloid content of the PLBs and performed transcriptome sequencing using the Illumina HiSeq 2,500 system. The total alkaloid content increased significantly after 4 h of treatment. Go and KEGG analysis suggested that genes from the TIA, isoquinoline alkaloid, tropane alkaloid and jasmonate (JA) biosynthetic pathways were significantly enriched. Weighted gene coexpression network analysis (WGCNA) uncovered brown module related to alkaloid content. Six and seven genes related to alkaloid and JA bisosynthetic pathways, respectively, might encode the key enzymes involved in alkaloid biosynthesis of D. officinale. Moreover, 13 transcription factors (TFs), which mostly belong to AP2/ERF, WRKY, and MYB gene families, were predicted to regulate alkaloid biosynthesis. Our data provide insight for studying the regulatory mechanism underlying TIA precursor and MeJA-induced accumulation of three types of alkaloids in D. officinale.

Keywords: Dendrobium officinale; alkaloid biosynthesis; methyl jasmonate; precursors; transcriptome.

FIGURE 1

Determination of total alkaloid content in D. officinale PLBs under Trp + S + MeJA treatment. (A) The PLBs used for Trp + S + MeJA treatment. (B) The total alkaloid content in PLBs treated with TIA precursors and MeJA for 0, 4 and 24 h. Data represent means ± SD from three biological replicates. Asterisks shows significant differences based on the Student’s t-test (*p < 0.05).

FIGURE 2

Expression patterns of four key genes involved in the TIA biosynthetic pathway of D. officinale PLBs under precursor and MeJA treatment.

FIGURE 3

Differentially expressed genes (DEGs) identified by RNA-Seq analysis in Trp + S + MeJA-treated and control samples after induction for 4 and 24 h. (A) Volcano plot of the RNA-Seq data showing DEGs in red and green. The x-axis indicates the fold change in C4 vs. T4 and C24 vs. T24. The y-axis represents the negative -log₁₀-transformed p-values (FDR < 0.01) for differences between the samples. (B) DEG numbers in the C4 vs. T4 and C24 vs. 24 groups. (C) Venn diagram of all DEGs in the C4 vs. T4 and C24 vs. T24 groups. (D) Venn diagram of upregulated and downregulated DEGs in the C4 vs. T4 and C24 vs. T24 groups.

FIGURE 4

GO enrichment of DEGs in Trp + S + MeJA-treated and non-treated PLBs of D. officinale. The x-axis indicates the percentage of DEGs in the subcategories of each main category. The y-axis indicates the number of DEGs in each subcategory.

FIGURE 5

KEGG enrichment analysis of DEGs in Trp + S + MeJA-treated and non-treated PLBs of D. officinale. (A) KEGG enrichment analysis of upregulated DEGs. (B) KEGG enrichment analysis of downregulated DEGs. The y-axis and x-axis represent the KEGG pathways and the enrichment factors, respectively. The greater the enrichment factor is, the more obvious the enrichment level of DEGs in the pathway. The color of the block represents the q-value, and the q-value is the p-value corrected by multiple hypothesis tests. The smaller the q-value is, the more reliable the enrichment of DEGs in this pathway is. The size of the circle indicates the number of genes enriched in the pathway.

FIGURE 6

Weighted gene coexpression network analysis (WGCNA) and modules related to alkaloid biosynthesis in D. officinale PLBs. (A) Dendrogram with color annotation. (B) Pearson correlation coefficient (r) and the p-value for alkaloid content and each module. The color scale on the right shows module-trait correlation from −1 (blue) to 1 (red).

FIGURE 7

Expression patterns of genes putatively involved in the biosynthesis of alkaloids in D. officinale PLBs under precursor and MeJA treatment. The enzymes are marked in red, indicating that genes encoding the enzymes showed different expressions in the C4 vs. T4 or C24 vs. T24 groups. The genes marked in red in the heat map indicates the DEGs identified from the C4 vs. T4 or C24 vs. T24 groups. Blue numbers in brackets represent the correlation coefficient which analyzed between alkaloid content and biosynthesis-related genes. ^**p < 0.01 and —r— > 0.8. The red triangle indicates that the gene was found in the brown module.

FIGURE 8

Expression pattern analysis of genes involved in JA biosynthesis in D. officinale PLBs under the precursor and MeJA treatment. (A) JA biosynthesis pathway. The enzymes are marked in red, indicating that genes encoding the enzymes were differentially expressed in the C4 vs. T4 or C24 vs. T24 groups. (B) Heat map of genes associated with JA biosynthesis. The genes marked in red in the heat map indicates the DEGs identified from the C4 vs. T4 or C24 vs. T24 groups. Blue numbers in brackets represent the correlation coefficient which analyzed between alkaloid content and JA biosynthesis-related genes. ^**p < 0.01 and —r— > 0.8. The red triangle indicates that the gene was found in the brown module.

FIGURE 9

Coexpression network of TFs and structure genes related to alkaloid biosynthesis pathway in D. officinale PLBs under the precursor and MeJA treatment. (A) Heat map displaying the expression of 66 TFs in different samples. (B) Construction of regulatory networks of TFs and structure genes related to alkaloid biosynthesis. **p < 0.01 and |r| > 0.8.

FIGURE 10

qRT-PCR validation of DEGs related to alkaloid biosynthesis and metabolic regulation in D. officinale PLBs. The left y-axis represents the relative expression levels corresponding to the histogram. The right y-axis represents expression levels calculated by the FPKM method corresponding to the line plots. The relationship between the RNA-Seq and qRT-PCR in T0, C4, T4, C24, and T24 samples was analyzed based on Pearson’s correlation analysis and r represents correlation coefficients. *p < 0.05, ^**p < 0.01.