Comparative Transcriptome Analysis Provides Insights into the Molecular Mechanism Underlying the Effect of MeJA Treatment on the Biosynthesis of Saikosaponins in Bupleurum chinense DC.

Bupleurum chinense DC. is a well-known traditional Chinese medicinal plant that produces saikosaponins (SSs), which possess hepatoprotective, antipyretic, and anti-inflammatory activities. Methyl jasmonate (MeJA) is a signalling phytohormone that can increase the accumulation of SSs in the root of Bupleurum plants. However, the molecular understanding of MeJA-mediated SS biosynthesis is not clear. Therefore, it is necessary to explore the molecular mechanism underlying the response of B. chinense DC. to MeJA in roots. In this study, we performed comparative transcriptome analysis of B. chinense DC. roots with different MeJA treatment times. In total, 104,057 unigenes were identified, of which 4053 were differentially expressed genes (DEGs). Most of the DEGs were downregulated after MeJA treatment, and GO enrichment analysis showed that they were mainly related to biological processes involved in stress responses and development. A total of 88 DEGs encoding enzymes known to be involved in the SS synthesis pathway were found, and most were significantly downregulated within 24 h. Based on the DEGs, 99 transcription factors (TFs) belonging to the AP2/ERF, WRKY, bZIP, ZFP, and bHLH families with different expression patterns were also identified. Further integrated analysis indicated that 20 DEGs involved in the SS synthesis pathway and 12 DEGs encoding TFs presented strong correlations with the SS contents, and these DEGs may be critical for the biosynthesis and regulation of SSs. These findings will be critical for further study of the response of B. chinense DC. to MeJA for SS biosynthesis.

Molecular cloning, functional characterization and expression of the ß-amyrin synthase gene involved in saikosaponin biosynthesis in Bupleurum chinense DC.

Bupleurum chinense DC. is a commonly used plant in traditional Chinese medicine, and saikosaponins(SSs) are the main active oleanane-typetriterpene saponins in B. chinense. ß-Amyrin synthase (ß-AS) is an important enzyme in oleanane-type triterpenoid saponin synthesis, but its role in saikosaponin synthesis has rarely been studied. Here, the putative ß-AS gene BcBAS1(Accession No.ON890382) selected according to metabolomic and transcriptomic analyses was cloned and functionally characterized by heterologous expression in Escherichia coli and Pichia pastoris, and its subcellular localization and expression patterns were examined. The molecular weight of the BcBAS1 recombinant protein was approximately 87 kDa, and this protein could catalyse the production of ß-amyrin, the precursor of SSs. Furthermore, BcBAS1 was located in the cytosol, and relative expression in four tissues of the four genotypes was positively correlated with SSa and SSd contents. Our results indicate that BcBAS1 is a ß-AS gene and may play an important role in saikosaponin biosynthesis and regulation. This study sheds light on the role of ß-AS genes in the synthesis of SSs and provides insights for the metabolic engineering of SSs.

Chaetomadramines A-E, a class of siderophores with potent neuroprotective activity from the fungus Chaetomium madrasense cib-1.

Five hydroxamate siderophores, chaetomadramines A-E (1-5), along with seven known compounds were isolated from the fermented rice culture of the fungus Chaetomium madrasense cib-1. Compounds 1-5 were structurally elucidated on the basis of spectroscopic data, which were a group of unusual hydroxamate siderophores, bearing a long fatty acyl on the α-NH2 of the Nδ-hydroxylated ornithine. Compounds 2-5 were new. The structural elucidation and spectroscopic data of 1 were reported for the first time. Compounds 2-4 significantly improved the survival rates of PC12 cells in the neuroprotective activity assay at the concentration of 40 µM.

Plant regeneration via callus-mediated organogenesis in commercial variety of Chuanbeichai No. 1 in Bupleurum chinense DC.

Bupleurum chinense DC is an important medicinal plant with many active ingredients that are used for the treatment of different types of diseases and valued in pharmaceutical markets. In vitro shoot regeneration can efficiently contribute to the improvement of B. chinense. In the present study, we investigated the effects of the explant type and plant growth regulators (PGRs) on embryogenic callus induction and plant regeneration in B. chinense. Our investigation demonstrated that 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 1 mg/L thidiazuron (TDZ) played a major role in promoting callus induction from leaf, hypocotyl and stem 2 explants, whereas the most effective treatment for stem 1 callus formation was Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-D, 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kin). The highest shoot regeneration rate (57.14%) was obtained from hypocotyl-induced calli in MS medium with 0.5 mg/L Kin after 12 weeks of cultivation. This regeneration protocol can be used in large-scale cultivation and may be useful for future genetic modifications of B. chinense .

Transcriptome Level Reveals the Triterpenoid Saponin Biosynthesis Pathway of Bupleurum falcatum L.

Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most "hubness" within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.

Transcriptome and metabolome analysis to reveal major genes of saikosaponin biosynthesis in Bupleurum chinense.

BACKGROUND: Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. In the present study, we performed an integrated analysis of metabolic composition and the expressed genes involved in saikosaponin biosynthetic pathways among four organs (the root, flower, stem, and leaf) of B. chinense to discover the genes related to the saikosaponin biosynthetic pathway. RESULTS: Transcript and metabolite profiles were generated through high-throughput RNA-sequencing (RNA-seq) data analysis and liquid chromatography tandem mass spectrometry, respectively. Evaluation of saikosaponin contents and transcriptional changes showed 152 strong correlations (P < 0.05) over 3 compounds and 77 unigenes. These unigenes belonged to eight gene families: the acetoacetyl CoA transferase (AACT) (6), HMG-CoA synthase (HMGS) (2), HMG-CoA reductase (HMGR) (2), mevalonate diphosphate decarboxylase (MVD) (1), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) (3), farnesyl diphosphate synthase (FPPS) (11), ß-amyrin synthase (ß-AS) (13) and cytochrome P450 enzymes (P450s) (39) families. CONCLUSIONS: Our results investigated the diversity of the saikosaponin triterpene biosynthetic pathway in the roots, stems, leaves and flowers of B. chinese by integrated transcriptomic and metabolomic analysis, implying that manipulation of P450s genes such as Bc95697 and Bc35434 might improve saikosaponin biosynthesis. This is a good candidate for the genetic improvement of this important medicinal plant.

Analysis of unigenes involved in lateral root development in Bupleurum chinense and B. scorzonerifolium.

MAIN CONCLUSION: We identified IAA13 negatively associated with lateral root number by comparing the differential expressed genes between Bupleurum chinense and B. scorzonerifolium. Dried roots of the genus Bupleurum L. are used as a herbal medicine for diseases in Asia. Bupleurum chinense has a greater number of lateral roots than B. scorzonerifolium, but the genetic mechanisms for such differences are largely unknown. We (a) compared the transcriptome profiles of the two species and (b) identified a subset of candidate genes involved in auxin signal transduction and explored their functions in lateral root development. By isoform sequencing (Iso-Seq) analyses of the whole plant, more unigenes were found in B. scorzonerifolium (118,868) than in B. chinense (93,485). Given the overarching role of indole-3-acetic acid (IAA) as one of the major regulators of lateral root development, we identified 539 unigenes associated with auxin signal transduction. Fourteen and 44 unigenes in the pathway were differentially expressed in B. chinense and B. scorzonerifolium, respectively, and 3 unigenes (LAX2, LAX4, and IAA13) were expressed in both species. The number of lateral root primordia increased after exogenous auxin application at 8 h and 12 h in B. scorzonerifolium and B. chinense, respectively. Since overexpression of IAA13 in Arabidopsis reduced the number of lateral roots, we hypothesized that IAA13 is involved in the reduction of the number of lateral roots in B. scorzonerifolium.

Differential Expression of Genes Involved in Saikosaponin Biosynthesis Between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd.

Radix Bupleuri (roots of Bupleurum spp.) is an important medicinal herb. Triterpenoid saponins of saikosaponins generally constitute the main class of secondary metabolites of plants in the Bupleurum genus. However, the molecular regulatory mechanism underlying their biosynthesis remains elusive. In this study, we observed significantly different saikosaponin biosynthesis between Bupleurum chinense and Bupleurum scorzonerifolium at the seedling stage. The sequential and expression characterization of 232 genes in the triterpenoid saponin biosynthetic pathway, which includes the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway, between B. chinense and B. scorzonerifolium was also investigated. Sixty of these genes may be involved in saikosaponin biosynthesis. Manipulation of these genes, especially those of the ß-AS, P450, and UGT families, may improve saikosaponin production.

[Extraction technology of diester-type alkaloids from Radix Aconiti lateralis].

OBJECTIVE: To determine the suitable extraction technology of diester-type alkaloids, such as mesaconitine, aconitine and hypaconitine from the roots of Aconitum carmichaeli (Radix Aconiti lateralis). METHODS: The contents of mesaconitine, aconitine and hypaconitine were determined by HPLC. Single-factor experiment was used to study the extraction factors. RESULTS: The suitable extraction technology for diester-type alkaloids was as follows: coarse sizings of Radix Aconiti lateralis was extracted by 10 times ethanol for 3 times (each time for 1 day) at 15 degrees C. The total yield of diester-type alkaloids was 0.57%, including 0.16% aconitine, 0.032% mesaconitine and 0.38% hypaconitine, and the purity was 18.81%. CONCLUSION: The extraction technology is efficient, harmfulless, economical, convenient and can be used for industrial production.

[Study on quality changes of Radix Aconiti Lateralis Praeparata under different storage conditions].

OBJECTIVE: To analyze the changes of the content of water, soluble protein, total alkaloids, polysaccharides of Radix Aconiti Lateralis Praeparata under different storage treatments. METHODS: The experiment was conducted in 4 kinds of storage methods, such as sack storage, sand storage, cold storage, and the natural heap as the control. RESULTS: During storage, the content of water, total alkaloids and polysaccharides tended to decline, while the content of soluble protein increased. CONCLUSION: Compared with the others, the cold storage is most suitable for keeping good quality of Radix Aconiti Lateralis Praeparata.