Dongfeng Yang

Effects of abscisic acid, gibberellin, ethylene and their interactions on production of phenolic acids in salvia miltiorrhiza bunge hairy roots.

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Phenolic acids mainly including caffeic acid, rosmarinic acid and salvianolic acid B are a group of active ingredients in S. miltiorrhiza. Abscisic acid (ABA), gibberellin (GA) and ethylene are three important phytohormones. In this study, effects of the three phytohormones and their interactions on phenolic production in S. miltiorrhiza hairy roots were investigated. The results showed that ABA, GA and ethylene were all effective to induce production of phenolic acids and increase activities of PAL and TAT in S. miltiorrhiza hairy roots. Effects of phytohormones were reversed by their biosynthetic inhibitors. Antagonistic actions between the three phytohormones played important roles in the biosynthesis of phenolic acids. GA signaling is necessary for ABA and ethylene-induced phenolic production. Yet, ABA and ethylene signaling is probably not necessary for GA3-induced phenolic production. The complex interactions of phytohormones help us reveal regulation mechanism of secondary metabolism and scale-up production of active ingredients in plants.

Ag+ as a More Effective Elicitor for Production of Tanshinones than Phenolic Acids in Salvia miltiorrhiza Hairy Roots

Phenolic acids and tanshinones are two groups of bioactive ingredients in Salvia miltiorrhiza Bunge. As a heavy metal elicitor, it has been reported that Ag+ can induce accumulations of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. In this study, the effects of Ag+ treatment on accumulations of six phenolic acids and four tanshinones in S. miltiorrhiza hairy roots were investigated. To further elucidate the molecular mechanism, expressions of key genes involved in the biosynthesis of these ingredients were also detected. The results showed that although the total phenolic acids content was almost not affected by Ag+, accumulations of rosmarinic acid (RA), caffeic acid and ferulic acid were significantly increased, while accumulations of salvianolic acid B (LAB), danshensu (DSU) and cinnamic acid were decreased. We speculate that LAB probably derived from the branch pathway of DSU biosynthesis. Contents of four tanshinones were enhanced by Ag+ and their accumulations were more sensitive to Ag+ than phenolic acids. Genes in the upstream biosynthetic pathways of these ingredients responded to Ag+ earlier than those in the downstream biosynthetic pathways. Ag+ probably induced the whole pathways, upregulated gene expressions from the upstream pathways to the downstream pathways, and finally resulted in the enhancement of ingredient production. Compared with phenolic acids, tanshinone production was more sensitive to Ag+ treatments. This study will help us understand how secondary metabolism in S. miltiorrhiza responds to elicitors and provide a reference for the improvement of the production of targeted compounds in the near future.

Cloning and Characterization of a Putative R2R3 MYB Transcriptional Repressor of the Rosmarinic Acid Biosynthetic Pathway from Salvia miltiorrhiza

Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plants in China. Phenolic acids that are derived from the rosmarinic acid pathway, such as rosmarinic acid and salvianolic acid B, are important bioactive components in S. miltiorrhiza. Accumulations of these compounds have been reported to be induced by various elicitors, while little is known about transcription factors that function in their biosynthetic pathways. We cloned a subgroup 4 R2R3 MYB transcription factor gene (SmMYB39) from S. miltiorrhiza and characterized its roles through overexpression and RNAi-mediated silencing. As the results showed, the content of 4-coumaric acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and total phenolics was dramatically decreased in SmMYB39-overexpressing S. miltiorrhiza lines while being enhanced by folds in SmMYB39-RNAi lines. Quantitative real-time PCR and enzyme activities analyses showed that SmMYB39 negatively regulated transcripts and enzyme activities of 4-hydroxylase (C4H) and tyrosine aminotransferase (TAT). These data suggest that SmMYB39 is involved in regulation of rosmarinic acid pathway and acts as a repressor through suppressing transcripts of key enzyme genes.

Transcriptomics, proteomics, and metabolomics to reveal mechanisms underlying plant secondary metabolism

Plant secondary metabolites play an important role in the fields of food, medicine, agriculture, and biofuels. Secondary metabolites are an important focus of crop breeding and metabolic engineering research. However, our understanding of secondary metabolism is far from complete, particularly in nonmodel plants. Functional genomics, which includes transcriptomics, proteomics, and metabolomics, opens a new avenue for deciphering secondary metabolism. Here we review the applications of functional genomics in secondary metabolism, including the discovery of novel genes, the identification of gene function, and the detection of novel pathways of the metabolic network. Results from these studies will accelerate our understanding of elicitation mechanism of secondary metabolism and how plants interact with their environment, ultimately improving the production of secondary metabolites by means of metabolic engineering.

DsTRD: Danshen Transcriptional Resource Database.

Salvia miltiorrhiza has been comprehensively studied as a medicinal model plant. However, research progress on this species is significantly hindered by its unavailable genome sequences and limited number of expressed sequence tags in the National Center for Biotechnology Information database. Thus, a transcript database must be developed to assist researchers to browse, search, and align sequences for gene cloning and functional analysis in S. miltiorrhiza. In this study, the Danshen Transcriptional Resource Database (DsTRD) was built using 76,531 transcribed sequences assembled from 12 RNA-Seq transcriptomes. Among these 12 RNA-seq data, ten were downloaded from NCBI database. The remaining two were enced on the Hiseq2000 platform using the stem and hairy-root of S. miltiorrhiza. The transcripts were annotated as protein-coding RNAs, long non-coding RNAs, microRNA precursors, and phased secondary small-interfering RNA genes through several bioinformatics methods. The tissue expression levels for each transcript were also calculated and presented in terms of RNA-Seq data. Overall, DsTRD facilitates browsing and searching for sequences and functional annotations of S. miltiorrhiza. DsTRD is freely available at http://bi.sky.zstu.edu.cn/DsTRD/home.php.

High-performance liquid chromatography based chemical fingerprint analysis and chemometric approaches for the identification and distinction of three endangered Panax plants in Southeast Asia

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high-performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.

Effects of cerous nitrate on growth and tanshinone production in salvia miltiorrhiza hairy roots

Abstract Salvia miltiorrhiza, a popular traditional Chinese medicine, is widely used for treatments in cardiotonic disease. Tanshinones are a group of bioactive ingredients in S. miltiorrhiza. In this study, Ce3+ was used as an elicitor to enhance tanshinones production in S. miltiorrhiza hairy roots. The results showed that contents of dihydrotanshinone I (DTI) and cryptotanshinone (CT) were significantly enhanced by 50 μmol/L Ce3+, and reached to 0.875 mg/g and 0.271 mg/g, respectively. However, tanshinone II A (TIIA) and tanshinone I (TI) contents were significantly decreased to 59% and 62% of the control. Simultaneously, expressions of genes (HMGR, DXR, DXS1, DXS2 and GGPPS) involved in tanshinone biosynthesis were upregulated by Ce3+. Responses of DXS1, DXS2 and GGPPS to Ce3+ treatments were later than HMGR and DXR. We speculated that branch pathways of DTI and CT biosynthesis were probably different from TIIA and TI. This work will help us understand biosynthetic mechanism of tanshinones in plants.

Microarray and Degradome Sequencing Reveal MicroRNA Differential Expression Profiles and Their Targets in Pinellia pedatisecta

MicroRNAs (miRNAs) are endogenous small non-coding RNAs which play a critical role in gene regulation in plants. Pinellia pedatisecta is one of the most important herbs in traditional Chinese medicine, but there are no microRNAs of Pinellia pedatisecta were deposited in miRBase and the research of the related miRNA biological functions is still insufficient. To detect Pinellia pedatisecta miRNAs and discover their expression difference with Pinellia ternata , we carried out a microarray profiling. A total of 101 miRNAs belonging to 22 miRNA families were detected both in Pinellia pedatisecta and Pinellia ternata respectively, among them 21 miRNAs showed their differentially expression. GO (gene ontology) term enrichment analysis of the target genes of differential expression miRNAs reveal that these miRNAs mainly affect the reproduction, transcription factor activity and plant developmental process. To elucidate the target function of miRNAs, we constructed a degradome library from Pinellia pedatisecta leaf. The result showed that a total of 18 transcript were identified as targets of miRNAs and further analysis indicated that miR156 and miR529 may function together to repress SPL14.

Nitric Oxide Plays a Central Role in Water Stress-Induced Tanshinone Production in Salvia miltiorrhiza Hairy Roots.

Nitric oxide (NO), a well-known signaling molecule plays an important role in abiotic and biotic stress-induced production of plant secondary metabolites. In this study, roles of NO in water stress-induced tanshinone production in Salvia miltiorrhiza hairy roots were investigated. The results showed that accumulations of four tanshinone compounds in S. miltiorrhiza hairy roots were significantly stimulated by sodium nitroprusside (SNP, a NO donor) at 100 μM. Effects of SNP were just partially arrested by the mevalonate (MVA) pathway inhibitor (mevinolin), but were completely inhibited by the 2-C-methyl-d-erythritol-4-phosphate pathway (MEP) inhibitor (fosmidomycin). The increase of tanshinone accumulation and the up-regulation of HMGR and DXR expression by PEG and ABA treatments were partially inhibited by an inhibitor of NO biosynthesis (Nω-nitro-L-arginine methyl ester (L-NAME)) and a NO scavenger (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)). Simultaneously, NO generation in the hairy roots was triggered by PEG and ABA, and the effects were also arrested by c-PTIO and L-NAME. These results indicated that NO signaling probably plays a central role in water stress-induced tanshinone production in S. miltiorrhiza hairy roots. SNP mainly stimulated the MEP pathway to increase tanshinone accumulation.

A correlation model of UPLC fingerprints and anticoagulant activity for quality assessment of Panax notoginseng by hierarchical clustering analysis and multiple linear regression analysis

Panax notoginseng (Burk.) F.H. Chen (commonly known as Tianqi or Sanqi) is a famous traditional Chinese medicine and has been widely used in the treatment of cardio- and cerebro-vascular diseases. However, the quality assessment of Sanqi is quite difficult because of the ingredient complexity. In this work, the chemical fingerprints of 15 batches of P. notoginseng from different sources were investigated by ultra-performance liquid chromatography (UPLC). Fourteen common peaks in these samples were detected including 20(S)-protopanaxatriol saponins (PTS) and 20(S)-protopanaxadiol saponins (PDS). These samples were divided into three clusters by hierarchical clustering analysis (HCA). Clusters I and III possessed stronger anti-coagulation effects than Cluster II. Multiple linear regression analysis (MLRA) showed that notoginsenoside R1, ginsenoside Rb1, ginsenoside Rd and an unknown compound might be the major effective compounds for the quality control of Sanqi. PDS (Rb1, Rd, and the unknown compound) possesses a stronger anticoagulant activity than PTS (R1 only). This conjecture was confirmed by the dose–effect relationship evaluation of PDS and PTS. This study provides a general correlation model of the fingerprint and efficacy relationship for the quality control of Sanqi and other TCMs.