Study on molecular mechanism of volatiles variation during Bupleurum scorzonerifolium root development based on metabolome and transcriptome analysis

Abstract

Bupleurum scorzonerifolium Willd. is a medicinal herb. Its root has a high content of volatile oil (BSVO), which shows a variety of biological activities. Currently, BSVO in the injectable form is used for treating fever in humans and livestock. The yield and quality of volatile oils depends on the developmental stages of plants. However, the changes in BSVO yield and quality during root development in Bupleurum scorzonerifolium and the underlying molecular regulatory mechanisms remain unclear. This knowledge gap is limiting the improvement in the quality of BSVO. In the present study, B. scorzonerifolium root was collected at germinative, vegetative, florescence, fruiting and defoliating stages. The yield of BSVO, metabolic profile of volatile components and transcriptome of root samples at various developmental stages were comprehensively determined and compared. BSVO continuously accumulated from the germinative to fruiting stages, and its level slightly decreased from the fruiting to defoliating stages. A total of 82 volatile components were detected from B. scorzonerifolium root, of which 22 volatiles were identified as differentially accumulated metabolites (DAMs) during the root development. Of these volatiles, fatty acids and their derivatives accounted for the largest proportion. The contents of most major volatiles were highest at the fruiting stage. A large number of differentially expressed genes (DEGs) were detected during B. scorzonerifolium root development, of which 65 DEGs encoded various enzymes and transcription factors regulating the biosynthesis of fatty acids and their derivatives. In further analysis, 42 DEGs were identified to be significantly correlated with DAMs, and these DEGs may be the key genes for the biosynthesis of volatiles. To the best of our knowledge, this is the first study to comprehensively report the changes in the composition and content of volatiles and underlying mechanism during B. scorzonerifolium root development. This study provided important reference for future studies to determine the harvest time of B. scorzonerifolium roots and improve the quality of BSVO.

Keywords: Bupleurum scorzonerifolium Willd.; fatty acids and their derivatives; metabolome; transcriptome; volatiles.

Figure 1

The yield of volatile oil from Bupleurum scorzonerifolium root at five developmental stages. Samples labeled BS_1, BS_2, BS_3, BS_4, and BS_5 represent the samples of B. scorzonerifolium root collected at germinative, vegetative, florescence, fruiting and defoliating stages, respectively. Different letters (a, b, c) indicate statistical significant differences.

Figure 2

Relative abundance of different classes of volatiles in Bupleurum scorzonerifolium root at five developmental stages. Samples labeled BS_1, BS_2, BS_3, BS_4, and BS_5 are the samples of B. scorzonerifolium root collected at germinative, vegetative, florescence, fruiting and defoliating stages, respectively.

Figure 3

Cluster diagram of volatile metabolites in Bupleurum scorzonerifolium root at five developmental stages. The abscissa represents the sample name, and the ordinate represents the corresponding compound name. The color represents the value of the correlation coefficient. Samples labeled BS_1, BS_2, BS_3, BS_4, and BS_5 are the samples of B. scorzonerifolium root collected at germinative, vegetative, florescence, fruiting and defoliating stages, respectively.

Figure 4

Differentially accumulated metabolites (DAMs) during Bupleurum scorzonerifolium root development. (A) Principal component analysis of the fifteen samples based on the volatile metabolic profiles. (B) The numbers of DAMs in different compared groups. (C) Venn diagram representing the DAMs in different compared groups. Samples labeled BS_1, BS_2, BS_3, BS_4, and BS_5 are the samples of B. scorzonerifolium root collected at germinative, vegetative, florescence, fruiting and defoliating stages, respectively.

Figure 5

Differentially expressed transcripts during Bupleurum scorzonerifolium root development. (A) Total numbers of differentially expressed genes (DEGs), upregulated genes, and downregulated genes in different compared groups. (B) Venn diagram of the DEGs in different compared groups. (C) GO annotation analysis of the upregulated genes and downregulated genes in BS_1 vs BS_4 group. (D) GO enriched analysis of DEGs in BS_1 vs BS_4 group. (E) KEGG annotation analysis of DEGs in BS_1 vs BS_4 group. (F) KEGG enriched analysis of DEGs in BS_1 vs BS_4 group. Samples labeled BS_1, BS_2, BS_3, BS_4, and BS_5 are the samples of B. scorzonerifolium root collected at germinative, vegetative, florescence, fruiting and defoliating stages, respectively.

Figure 6

Pathways and genes involved in biosynthesis of fatty acids and their derivatives in Bupleurum scorzonerifolium root. Acetyl-CoA carboxylase (ACCase), 3-Oxoacyl-ACP synthase (FabF), 3-Oxoacyl-ACP reductase (FabG), 3-Hydroxyacyl-ACP dehydratase (FabZ), Fatty acyl-ACP thioesterase B (FATB), Stearoyl-ACP desaturase (FAB), Fatty acid desaturase (FAD), Lipoxygenase (LOX), Hydroperoxide lyase (HPL), Aldehyde dehydrogenase (ALDH), Alcohol dehydrogenase (ADH), Alcohol acyltransferase (AAT).

Figure 7

Total genes and differentially expressed genes (DEGs) occupied a proportion of the transcription factors in the de novo assembled transcriptome of Bupleurum scorzonerifolium root.

Figure 8

Gene-metabolite Pearson correlation network between differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). Gene-metabolite pairs were connected by edges; light blue and grey lines represent positive and negative correlation respectively; circle and hexagon represent DEGs and DAMs, respectively.

Figure 9

The qRT-PCR and FPKM-value analyses of 11 genes encoding enzymes involved in biosynthesis of fatty acids and their derivatives in Bupleurum scorzonerifolium root. Relative expression levels of 11 genes were analyzed, using the actin gene (GAPDH) as a reference for the normalization, all performed with three technical triplicates. Data represent mean ± SE.