Combined Analysis of Volatile Terpenoid Metabolism and Transcriptome Reveals Transcription Factors Related to Terpene Synthase in Two Cultivars of Dendrobium officinale Flowers

Abstract

Dendrobium officinale is a kind of traditional Chinese herbal medicine. Its flowers could be used as health care tea for its aroma flavor and medicinal value. Most recent studies demonstrated that terpenoids are the main components of the aromatic compounds in the flowers, but the biosynthesis of terpenoids is poorly understood in D. officinale. In the experiment, the flowers from two cultivars of D. officinale with different smells were collected. The transcriptome analysis and combined volatile terpenoids determination were performed to identify the genes related to the biosynthesis of the terpenoids. The results showed that the different products of volatile terpenoids are α-thujene, linalool, α-terpineol, α-phellandrene, γ-muurolene, α-patchoulene, and δ-elemene in two cultivar flowers. The transcriptome analysis detected 25,484 genes in the flowers. And 18,650 differentially expressed genes were identified between the two cultivars. Of these genes, 253 genes were mapped to the terpenoid metabolism pathway. Among these genes, 13 terpene synthase (TPS) genes may have correlations with AP2/ERF, WRKY, MYB, bHLH, and bZIP transcription factors by weighted gene co-expression network analysis (WGCNA). The transcription factors have regulatory effects on TPS genes. These results may provide ideas for the terpenoid biosynthesis and regulatory network of D. officinale flowers.

Keywords: Dendrobium officinale; WGCNA; terpene synthase gene; terpenoid; transcription factors.

FIGURE 1

Two cultivars of Dendrobium officinale flowers. (A) Wanhu No.5. (B) Wanhu No.6.

FIGURE 2

GC-MS analysis of the products formed in two cultivars of Dendrobium officinale flowers. (A) Wanhu No.5. (B) Wanhu No.6.

FIGURE 3

Volatile terpenoids compounds of two cultivars of Dendrobium officinale flowers. (A) Wanhu No.5. (B) Wanhu No.6. The same color represents the same terpenoid.

FIGURE 4

RNA-seq and different expression genes (DEGs) analysis. (A) Statistical histogram of the number of different genes in the different comparison combinations. The numbers on the columns indicate the number of differential genes. (B) KEGG pathway classification map of DEGs.

FIGURE 5

Expression pattern of genes related to terpenoid biosynthesis. Gene expression levels in the fully opened flowers of Wanhu No.5 and Wanhu No.6 are represented by color gradations. AACT, acetyl-CoA acetyltransferase; CMK, 4-(cytidine 59-diphospho)-2-C-methyl-D-erythritol kinase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, DXP synthase; FPPS, farnesyl diphosphate synthase; GPPS, geranyl diphosphate synthase; HDS, 4-hydroxy-3-methyl-2-butenyl pyrophosphate synthase; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IDI, isopentenyl diphosphate isomerase; IDS, isoprenyl diphosphate synthase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; ME, ME-CDP synthase; MPDC, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; TPS, terpene synthase.

FIGURE 6

Clustering dendrogram of expressed genes. Gene modules were identified by dynamic hierarchical tree cut and shown in different colors. Height cut = 0.25, minimal module size = 30.

FIGURE 7

Correlation coefficients between terpene synthase (TPS), module, and volatile compound. Each row represents a module, and each column represents a terpenoid. The color of each block at the row–column intersection indicates the correlation coefficient: red for high positive correlation and green for high negative correlation, with a scale shown on the right of the panel.

FIGURE 8

Phylogeny tree analysis of Dendrobium officinale TPSs. Triangles represent finger citron TPSs, and filled colors indicate their location in modules shown in Figure 6. Sequence alignment was performed by ClustalX. Phylogeny tree was visualized by MEGA7.

FIGURE 9

Gene co-expression subnetwork of the turquoise module. Network was reconstructed by edge weight cutoff = 0.25 and visualized by Cytoscape.

FIGURE 10

Gene co-expression subnetwork of the blue module. Network was reconstructed by edge weight cutoff = 0.25 and visualized by Cytoscape.

FIGURE 11

Expression patterns of eight genes as verified by qRT-PCR. (A) The relative expression levels of Wanhu No.5 and Wanhu No.6. (B) FPKM values from RNA-Seq. Values shown are mean ± SE of three replicates. “*” indicates that the difference is significant, “**” indicates that the difference is very significant.