Accumulation differences of high-value ingredients in different phenotype Lonicera macranthoides: insights from integrative metabolome and transcriptome analyses

Abstract

Background: Lonicera macranthoides Hand.-Mazz., the primary sources of Lonicerae Flos(Shanyinhua), brings great medicinal and economic value as an invaluable source of natural bioactive compounds. Nutrient and metabolites accumulation generally changed accompany with its floral development and opening. While the specific accumulation pattern and the underlying molecular regulatory networks remain unclear.

Methods: The present study intergrated a comparative analysis upon UPLC-MS/MS-based metabolomics and RNA-seq-based transcriptomics to revealed the differences in accumulation of flavonoids, phenolic acids, and terpenoids between the xianglei-type (corolla-closed) and wild-type (corolla-unfolded) of L. macranthoides flowers.

Results and conclusion: 674 differentially accumulated metabolites(DAMs) were identified in WT and XL, with 5,776 common differentially expressed genes(DEGs), revealing a significant differences in accumulation of flavonoids, phenolic acids, and terpenoids during the late stage of flower development between the xianglei-type and wild-type of L. macranthoides flowers. Combined analysis further identified 36 hub genes, major transcription factors and hormone-related genes, which play key roles in the differential accumulation of the abovementioned metabolites. These lines of evidences provide a molecular basis for the metabolic changes occurring during growth and can be significantly implicated in further research on the biosynthetic pathways associated with high-value potent active components in woody plants.

Keywords: Lonicera macranthoides Hand.-Mazz.; active ingredients; metabolome; regulatory network; transcriptome.

Figure 1

Analysis of metabolite contents in the flowers of WF and XF at different developmental stages. WF1-WF7 represent the seven developmental stages of Wild type LM, and XF1-XF7 represent the seven developmental stages of XiangLei type LM. (A) Morphological comparison of WT and XL LM. (B) Classes of the metabolites with annotated structures. Metabolites are divided into twenty-five categories. (C) Metabolite variation tendencies among the twenty cluster profiles. (D) KEGG pathway assignment of metabolites identified as flavonoids, phenolic acids and terpenoids of LM.

Figure 2

Functional analysis of the DAMs involved in WF and XF flower development. (A) DAMs number in all compare groups. (B) Venn analysis of differently accumulate flavonoids, phenolic acids and terpenoids in WF, XF, and XF-WF compare groups. (C) KEGG Ontology enrichment circle diagram and (D) KEGG enrichment analysis of DAMs for pre-floral development stage XF123 vs. WF123. (E) KEGG Ontology enrichment circle diagram and (F) KEGG enrichment analysis of DAMs for mid-floral development stage XF45 vs. WF45. (G) KEGG Ontology enrichment circle diagram and (H) KEGG enrichment analysis of DAMs for late-floral development stage XF67 vs. WF67.

Figure 3

Transcriptome sequencing of WF and XF samples at seven stages. (A) Number of DEGs in all compared groups. (B) Venn diagram of DEGs associated with WF, XF, and XF-WF. (C) Correlation heatmap of 5,776 common DEGs. (D) Heatmap of the transcriptome expression of 5,776 common DEGs. (E) KEGG pathway assignment of all DEGs among WF and XF. (F) GO classification of all DEGs among WF and XF.

Figure 4

Integration of related genes and metabolites involved in flavonoid, phenolic acid and terpenoid biosynthesis The left column represents the proposed pathways for flavonoid (A), phenolic acid (B), and terpenoid (C) biosynthesis in LM. The right column represents the correlation network between structural genes and metabolites.

Figure 5

Hormone-related genes involved in the biosynthesis of active ingredients. (A) Cluster analysis of 200 hormone-related genes. (B) Bar graph of the distribution of genes in the eight clusters.

Figure 6

Weighted gene coexpression network analysis (WGCNA) of the relative DEGs. (A) Construction of the coexpression modules of genes. 15 modules labeled different colors represent gene set with different expression patterns. The correlation coefficient between the module and the flower stage displayed in the cell at the row−column intersection according to the color scale on the right. (B) Correlation analysis between 20 key flavoids and gene modules. (C) Correlation analysis between 20 phenolic acids and gene modules. (D) Correlation analysis between 20 terpenolids and gene modules.

Figure 7

Quantitative real-time(RT−qPCR) and RNA−seq(TPM) analysis of genes involved in metabolite pathways. Relative expression levels of transcription factors and structural genes are shown as the means ± standard deviations of three biological replicates.

Figure 8

Transcriptional regulatory network of active ingredients in LM. The red star represent key structural genes, and the yellow triangle represent key TFs. The connecting lines with arrows and dashed line represent positive and negative regulation, respectively. Different colored lines were used to distinguish the regulation of structural genes by different TF. (A) Regulatory network of phenolic acids biosynthesis in LM. (B) Regulatory network of flavonoids biosynthesis in LM. (C) Regulatory network of terpenoids biosynthesis in LM. (D) The network diagram of several hormones involved in the transcription factor regulation of downstream active component structural genes.