Transcriptomic Analyses Shed Light on Critical Genes Associated with Bibenzyl Biosynthesis in Dendrobium officinale

Abstract

The Dendrobium plants (members of the Orchidaceae family) are used as traditional Chinese medicinal herbs. Bibenzyl, one of the active compounds in Dendrobium officinale, occurs in low amounts among different tissues. However, market demands require a higher content of thes compounds to meet the threshold for drug production. There is, therefore, an immediate need to dissect the physiological and molecular mechanisms underlying how bibenzyl compounds are biosynthesized in D. officinale tissues. In this study, the accumulation of erianin and gigantol in tissues were studied as representative compounds of bibenzyl. Exogenous application of Methyl-Jasmonate (MeJA) promotes the biosynthesis of bibenzyl compounds; therefore, transcriptomic analyses were conducted between D. officinale-treated root tissues and a control. Our results show that the root tissues contained the highest content of bibenzyl (erianin and gigantol). We identified 1342 differentially expressed genes (DEGs) with 912 up-regulated and 430 down-regulated genes in our transcriptome dataset. Most of the identified DEGs are functionally involved in the JA signaling pathway and the biosynthesis of secondary metabolites. We also identified two candidate cytochrome P450 genes and nine other enzymatic genes functionally involved in bibenzyl biosynthesis. Our study provides insights on the identification of critical genes associated with bibenzyl biosynthesis and accumulation in Dendrobium plants, paving the way for future research on dissecting the physiological and molecular mechanisms of bibenzyl synthesis in plants as well as guide genetic engineering for the improvement of Dendrobium varieties through increasing bibenzyl content for drug production and industrialization.

Keywords: Dendrobium officinale; bibenzyl; erianin; gigantol; transcriptome.

Figure 1

(A) Changes of Erianin contents under different exogenous MeJA concentrations treatment using 0.2 mM, 0.5 mM, and 1.5 mM from 0 h to 48 h. (B) Changes of Gigantol contents under different exogenous MeJA concentrations treatment using 0.2 mM, 0.5 mM, and 1.5 mM from 0 h to 48 h. Each bar shows the mean ± SE of triplicate assays. The asterisk (*) at the top of the bar indicate significance differences (p < 0.05) according to the ANOVA and Tukey tests.

Figure 2

(A): Functional GO enrichment analysis for the identified DEGs. GO enrichment analysis of DEGs identified from RNA-Seq analysis. The red, green, and blue bars represent the biological process, cellular component, and molecular function. (B): The top 20 pathways within KEGG analysis. KEGG pathway enrichment analysis of DEGs identified from RNA-Seq analysis. The different colors represent the p-value, ranging from 0.001 to 0.005, while the black circle represents gene number. GO: Gene Ontology; DEG: differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 3

Identification of differentially expressed genes involved in (A) the JA signal pathway and (B–E) TFs involved in bibenzyl compounds biosynthesis. CT signifies the control group, while MJ signifies the treated group. Yellow or blue represents up-regulation or down-regulation, respectively, and black represents the genes at background levels. Scale bar represents fold changes of DEGs expression.

Figure 4

(A): Putative bibenzyl biosynthesis pathways and identification of differentially expressed genes involved in bibenzyl biosynthesis. PAL: phenylalanine ammonia-lyase, C4H: trans-cinnamate 4-monooxygenase, P450: cytochrome P450s, 4CL: 4-coumarate-CoA ligase 1,2-like, and BBS: bibenzyl synthase (or bibenzyl synthase-like). (B): Changes in the expression of the identified DEGs involved in bibenzyl biosynthesis. CT signifies the control group, while MJ signifies the treated group. Yellow or blue represents up-regulation or down-regulation, respectively, and black represents the genes at background levels. Scale bar represents fold changes of DEGs expression.

Figure 5

(A) Phylogenetic analysis and representative members of the CYP450s genes in D. officinale. Tree was constructed using Neighbor-joining with 1000 bootstrap replicates. The code used for different plant CYP450 sequences is AT-Arabidopsis thaliana, LOC-Dendrobium officinale, ‘LOC OS-Oryza sativa. (B) Gene ontology annotation of D. officinale CYP450 A-type and Non A-type genes. The GO annotation allows genes to be classified into three functional groups, including I. Biological processes, II. Molecular function, and III. Cellular components.

Figure 6

Quantitative real-time PCR analysis of four unigenes associated with the bibenzyl bioScheme 1. Each bar shows the mean ± SE of triplicate assays. The asterisk (*) above the columns indicates a significant change of expression level between the control and treatment at p < 0.05 according to the ANOVA tests.