The Transcriptome Profiling of Flavonoids and Bibenzyls Reveals Medicinal Importance of Rare Orchid Arundina graminifolia

Abstract

Orchids are very important flowering plants that spend long juvenile phases before flowering. Along with aesthetic importance, they are rich sources of medicinal components. However, their long reproductive cycle is the major hurdle to study the medicinal efficacy. Arundina graminifolia is a rare orchid that grows fast, unlike other orchids, and this characteristic makes it an ideal plant to study the medicinal enrichment of orchids. Therefore, this study presents the identification of important medicinal components in various parts of A. graminifolia. Transcriptome analysis was performed for five stages (FD1-FD5) of flower development and four tissue types (mature flower, silique, root, and leaf) to ascertain genetic regulators of flavonoids and bibenzyls. Most of the genes showed the highest expression in roots as compared with other tissues. Weighted gene coexpression network analysis (WGCNA) was performed to identify the coexpression modules and the candidate genes involving biosynthesis pathways of these chemicals. MEyellow module contained the highly coexpressed genes. Moreover, the concentrations of phenylpropanoid, bibenzyls, and flavone were ascertained through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Phenylpropanoid and bibenzyl were comparatively high in the leaf, while flavone showed a high concentration in the stem. The selected candidate genes [bibenzyl biosynthesis (BIBSY212), CYP84A1, CYP73A4, 4CLL7, UGT88B1, UGT73C3, anthocyanin synthase (ANS), phenylalanine ammonia-lyase (PAL), flavanone synthase FLS, and CHS8] were validated through quantitative real-time PCR (qRT-PCR). Most of these genes showed high expression in leaf and root as compared with other tissue. Therefore, the presence of bibenzyls and flavonoids in different parts of A. graminifolia and their molecular regulators can provide a quick source to decipher the medicinal efficacy of orchids.

Keywords: Arundina graminifolia; HPLC-MS/MS; WGCNA; medicinal constituents; model orchid.

Figure 1

Distribution of secondary metabolite data into enzyme classes (A), major biological process enrichments (B) and up- and downregulated differentially expressed genes (DEGs) for different tissues (C).

Figure 2

The heatmap of major DEGs involved in the biosynthesis of flavonoids (A) and phenylpropanoids (B).

Figure 3

Clustering analysis of DEGs related to secondary metabolites (A) major pathways enriched in DEGs related to secondary metabolites (B) protein–protein interaction among DEGs related to secondary metabolites (C). The circle colors show the enrichment of pathways shown in part (B).

Figure 4

Weighted gene coexpression network analysis (WGCNA); cluster dendrogram (A), eigengene adjacency heatmap of modules (B), key modules with expression intensities (C), and module membership of yellow module for tissue-specific gene significance (D).

Figure 5

(A) Gene regulators of phenylpropanoids, flavonoids, and bibenzyls; (B) biosynthesis route of flavonoids and bibenzyls. Colored in red are the enzymes that regulate different steps of biosynthetic pathways; (C) selection of candidates that involve biosynthesis of flavonoids and bibenzyls through coexpressed modules; (D) concentrations of phenylpropanoids, bibenzyls, and flavonoids through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); (E) quantitative real-time PCR (qRT-PCR) expression of 10 selected DEGs related to bibenzyl and flavonoid biosynthesis. The statistical significance is shown at p ≤ 0.05 (^*) or p ≤ 0.01 (^**).