Transcriptomic and metabolomic analyses provide insights into the biosynthesis of chlorogenic acids in Lonicera macranthoides Hand.-Mazz

Abstract

Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in South China. The developmental stage and corolla dehiscence of the flower are the important factors affecting the quality of medicinal ingredients. However, neither the regulatory mechanism controlling chlorogenic acids biosynthesis in L. macranthoides nor the molecular basis of effect of corolla dehiscence on the quality of medicinal materials is fully understood. In this study, metabolomics and transcriptomics were used to analyze the metabolic and transcriptional differences of two different cultivars closed bud type (Bt), and flowering type (Ft), as well as the effect of jasmonic acid methyl ester (MeJA) on chlorogenic acids (CGAs) biosynthesis. In total, large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were filtered among three lines of samples. Gene metabolite correlation analyses revealed a 'core set' of 30 genes and 54 genes that were strongly correlated with CGAs biosynthesis and regulating the flowering, respectively. Quantitative real-time polymerase chain reaction results proved the alterations in the expression levels of genes encoding the pathways involved in CGAs biosynthesis. The ion abundances of CGAs were most significantly increased, while some of the CGAs derived and Caffeoyl-CoA-derived substances showed the most largely reduced abundances in the closed bud type (Bt) compared to the flowering type (Ft). MeJA may leads to the activation of downstream genes in CGAs biosynthesis pathway. Overall, there were significant differences in the transcriptional and metabolic levels of CGAs biosynthesis pathway in flower buds of different flowering cultivars. The redirection of metabolic flux may contribute to increased accumulation of CGAs. However, whether MeJA and flowering have direct effects on the accumulation of CGAs needs further studied. These researches effectively expanded the functional genomic library and provide new insights into CGAs biosynthesis in L. macranthoides.

Fig 1. Image shows the different types of Lonicera. macranthoides Hand.-Mazz (L. macranthoides).

(a): The flowering type (Ft). (b): The closed bud type (Bt). (c): The MeJA-treated closed bud type (Bt1000).

Fig 2. Number and heat map of metabolite profiles of different varieties.

(a) Numbers of different types of metabolites in all samples. (b) Clustering heat map of the metabolites detected in the total samples. Each example is visualized in a single column, and each metabolite is represented by a single row. Red indicates high abundance, whereas low relative metabolites are shown in green. Ft indicates the flowering type, Bt indicates closed bud type and Bt1000 indicates MeJA-treated closed bud type (Bt1000).

Fig 3. Multivariate analysis of metabolic profiling.

(a): principal components analysis (PCA) of metabolites. (b): Venn diagram representing the numbers of differentially accumulated metabolites (DAMs). Ft indicates the flowering type, Bt indicates closed bud type and Bt1000 indicates MeJA-treated closed bud type (Bt1000).

Fig 4. Differential metabolite analysis.

(a): Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot. Blue dots indicate Bt, and red dots indicate Bt1000. (b): OPLS-DA score plot. Blue dots indicate Bt, and red dots indicate Ft. (c): Volcano plot of differential metabolites between Bt and Bt1000. (d): Volcano plot of differential metabolites between Bt and Ft. Each point in the volcanic plot represents a metabolite, the abscissa represents the logarithm of the quantitative difference multiples of a metabolite in two samples, and the ordinate represents the variable importance in project (VIP) value. The green dots in the figure represent downregulated differentially expressed metabolites, the red dots represent upregulated differentially expressed metabolites, and the black dots represent detected metabolites but that are not significantly different. Ft indicates the flowering type, Bt indicates closed bud type and Bt1000 indicates MeJA-treated closed bud type (Bt1000).

Fig 5. Identification and functional characterization of differentially accumulated metabolites (DAMs).

Kyoto Encyclopedia of Genes and Genomes Ortholog database (KEGG) enrichment analysis of the DAMs between (a) Ft and Bt and between (b) Bt1000 and Bt. Top 20 metabolite changes between (c) Ft and Bt and between (d) Bt1000 and Bt. Ft indicates the flowering type, Bt indicates closed bud type and Bt1000 indicates MeJA-treated closed bud type.

Fig 6. Transcription profiles of Lonicera macranthoides Hand.-Mazz.

(a): The cluster heat map for differentially expressed genes (DEGs). (b): Venn diagram showing the distribution of DEGs from Bt, Ft and Bt1000. (c): Volcano plot of differentially expressed genes between Ft and Bt. (d): Volcano plot of differentially expressed genes between Bt1000 and Bt. (e): KEGG enrichment analysis of the differentially accumulated metabolites (DAMs) between Ft and Bt. (e): Kyoto Encyclopedia of Genes and Genomes Ortholog database (KEGG) enrichment analysis of the DAMs between Bt1000 and Bt. Ft indicates the flowering type, Bt indicates closed bud type and Bt1000 indicates MeJA-treated closed bud type.

Fig 7. Biosynthetic pathway of chlorogenic acids.

(a): Metabolic pathway map of integrative chlorogenic acids biosynthesis in L. macranthoides. Red indicates an increase in content, and the blue indicates a decrease in content. (b): Heat map of changes in transcripts in chlorogenic acids biosynthesis. (c): Heat map of changes in metabolites in chlorogenic acids biosynthesis. Upregulated (red) and downregulated (green) genes and metabolites are indicated. Ft indicates the flowering type, Bt indicates closed bud type and Bt1000 indicates MeJA-treated closed bud type.