Search Results (10)

Phosphorus (P), as one of the essential bulk elements for plant growth and development, plays an important role in root growth, accumulation of secondary metabolites, and regulation of gene expression. In Salvia miltiorrhiza Bunge (S. miltiorrhiza), an important medicinal plant, the accumulation of its active components is closely related to the level of phosphorus supply, but the molecular regulatory mechanism of phosphorus treatment in the growth and secondary metabolism of S. miltiorrhiza is not clear. In this study, we investigated the effects of low phosphorus (P2), moderate phosphorus (P4), and high phosphorus (P6) treatment on the growth and development of S. miltiorrhiza. seedlings, the accumulation of bioactive compounds, and their transcriptional regulation using transcriptomic and metabolomic analyses, and identified the key regulatory genes in the biosynthesis pathways of tanshinone and salvianolic acid. The findings revealed that S. miltiorrhiza biomass exhibited a “peaked” response to phosphorus concentration, peaking at 0.625 mmol·L⁻¹. At this optimal concentration, all four batches achieved maximum root length, root weight, and leaf weight: Batch I (11.3 cm, 2.34 g, 1.62 g), Batch II (12.7 cm, 2.67 g, 1.89 g), Batch III (13.8 cm, 2.85 g, 2.04 g), and Batch IV (15.6 cm, 3.51 g, 2.44 g). Both lower and higher concentrations resulted in growth inhibition and reduced bioactive compound accumulation. Transcription factors associated with root growth and development included bHLH, MYB, and WRKY; in particular, the bZIP23 transcription factor was highly expressed under abnormal phosphorus supply conditions. In addition, the biosynthetic pathways of tanshinone and salvianolic acid were elucidated, and key genes related to the synthesis pathways (CPS, KSL, CYP, PAL, HPPR, and RAS) were identified. The expression of several TFs (such as SmCPS1, SmCYP76AH3, SmCYP76AH1, SmGGPPS1, and SmRAS1) was found to be correlated with tanshinone and salvianolic acid synthesis. The present study provides a theoretical basis for further revealing the molecular mechanism of phosphorus regulation of growth, development, and secondary metabolism of S. miltiorrhiza and provides potential targets for efficient cultivation and molecular breeding of S. miltiorrhiza. Full article

Plant natural products are important sources of innovative drugs, but the extraction and isolation of medicinal natural products from plants is challenging as these compounds have complex structures that are difficult to synthesize chemically. Therefore, utilizing heterologous expression systems to produce medicinal natural products in plants is a novel, environmentally friendly, and sustainable method. In this study, Nicotiana benthamiana was used as the plant platform to successfully produce miltiradiene, the key intermediate of tanshinones, which are the bioactive constituents of the Chinese medicinal plant Salvia miltiorrhiza. The yield of miltiradiene was increased through cytoplasmic engineering strategies combined with the enhancement of isoprenoid precursors. Additionally, we discovered that overexpressing SmHMGR alone accelerated apoptosis in tobacco leaves. Due to the richer membrane systems and cofactors in tobacco compared to yeast, tobacco is more conducive to the expression of plant enzymes. Therefore, this study lays the foundation for dissecting the tanshinone biosynthetic pathway in tobacco, which is essential for subsequent research. Additionally, it highlights the potential of N. benthamiana as an alternative platform for the production of natural products in plants. Full article

The red root of Salvia miltiorrhiza Bunge, a famous traditional Chinese medicine (TCM), was caused by tanshinone in epidermis cells. In order to study the biological function of ncRNAs in the tanshinone synthesis, the expression patterns of mRNA and ncRNAs were comprehensively analyzed in red (high tanshinone content) and white root (low tanshinone content) tissues derived from the same plant. A total of 731 differentially expressed genes (DEGs) were mainly enriched in primary metabolic pathways such as galactose and nitrogen, and some secondary metabolic pathways such as phenylpropanoid and terpenoids. A total of 70 miRNAs, 48 lncRNAs, and 26 circRNAs were identified as differentially expressed (DE) ones. The enrichment pathway of the targets of DE-lncRNA were mainly in ribosome, carbon metabolism, plant hormone signal transduction, and glycerophospholipid metabolism. The function of the targets genes of 59 miRNAs combined with DE-circRNAs was mainly involved in plant–pathogen interaction, endocytosis, phenylpropanoid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis pathways. Most genes of the tanshinone synthesis pathway had a higher expression. Some ncRNAs were predicted to regulate several key enzyme genes of the tanshinone synthesis pathway, such as SmDXS2, SmGGPPS1, SmKSL. Furthermore, most target genes were related to the resistance of pathogens. The present study exhibited the tissue-specific expression patterns of ncRNAs, which would provide a basis for further research into the regulation mechanism of ncRNAs in the tanshinone synthesis process. Full article

Tanshinones are the bioactive constituents of Danshen (Salvia miltiorrhiza Bunge), which is used in Traditional Chinese Medicine to treat cardiovascular and other diseases, and they synthesize and accumulate in the root periderm of S. miltiorrhiza. However, there is no relevant report on the initial stage of tanshinone synthesis, as well as the root structure and gene expression characteristics. The present study aims to provide new insights into how these bioactive principles begin to synthesize by characterizing possible differences in their biosynthesis and accumulation during early root development from both spatial and temporal aspects. The morphological characteristics and the content of tanshinones in roots of S. miltiorrhiza were investigated in detail by monitoring the seedlings within 65 days after germination (DAGs). The ONT transcriptome sequencing was applied to investigate gene expression patterns. The periderm of the S. miltiorrhiza storage taproot initially synthesized tanshinone on about 30 DAGs. Three critical stages of tanshinone synthesis were preliminarily determined: preparation, the initial synthesis, and the continuous rapid synthesis. The difference of taproots in the first two stages was the smallest, and the differentially expressed genes (DEGs) were mainly enriched in terpene synthesis. Most genes involved in tanshinone synthesis were up regulated during the gradual formation of the red taproot. Plant hormone signal transduction and ABC transport pathways were widely involved in S. miltiorrhiza taproot development. Five candidate genes that may participate in or regulate tanshinone synthesis were screened according to the co-expression pattern. Moreover, photosynthetic ferredoxin (FD), cytochrome P450 reductase (CPR), and CCAAT binding transcription factor (CBF) were predicted to interact with the known downstream essential enzyme genes directly. The above results provide a necessary basis for analyzing the initial synthesis and regulation mechanism of Tanshinones. Full article

The pathogenic form of thermophilic Naegleria sp. i.e., Naegleria fowleri, also known as brain eating amoeba, causes primary amoebic encephalitis (PAM) with a >97% fatality rate. To date, there are no specific drugs identified to treat this disease specifically. The present antimicrobial combinatorial chemotherapy is hard on many patients, especially children. Interestingly, Naegleria fowleri has complex lipid biosynthesis pathways like other protists and also has a strong preference to utilize absorbed host lipids for generating energy. The ergosterol biosynthesis pathway provides a unique drug target opportunity, as some of the key enzymes involved in this pathway are absent in humans. Sterol 24-C Methyltransferase (SMT) is one such enzyme that is not found in humans. To select novel inhibitors for this enzyme, alkaloids and terpenoids inhibitors were screened and tested against two isozymes of SMT identified in N. gruberi (non-pathogenic) as well as its homolog found in yeast, i.e., ERG6. Five natural product derived inhibitors i.e., Cyclopamine, Chelerythrine, Berberine, Tanshinone 2A, and Catharanthine have been identified as potential drug candidates based on multiple criteria including binding affinity, ADME scores, absorption, and, most importantly, its ability to cross the blood brain barrier. This study provides multiple leads for future drug exploration against Naegleria fowleri. Full article

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca²⁺ signaling is essential for the LPS-triggered immune response, Ca²⁺ channel blocker LaCl₃ and CaM antagonist W-7 were used to investigate the role of Ca²⁺ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl₃ and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca²⁺ signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca²⁺ signaling. Ca²⁺ signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza. Full article

Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway. Full article

Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza. Full article

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 (N^ω-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. Full article

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. Full article