Physiological and molecular mechanisms of ZnO quantum dots mitigating cadmium stress in Salvia miltiorrhiza.

This study delved into the physiological and molecular mechanisms underlying the mitigation of cadmium (Cd) stress in the model medicinal plant Salvia miltiorrhiza through the application of ZnO quantum dots (ZnO QDs, 3.84 nm). A pot experiment was conducted, wherein S. miltiorrhiza was subjected to Cd stress for six weeks with foliar application of 100 mg/L ZnO QDs. Physiological analyses demonstrated that compared to Cd stress alone, ZnO QDs improved biomass, reduced Cd accumulation, increased the content of photosynthetic pigments (chlorophyll and carotenoids), and enhanced the levels of essential nutrient elements (Ca, Mn, and Cu) under Cd stress. Furthermore, ZnO QDs significantly lowered Cd-induced reactive oxygen species (ROS) content, including H2O2, O2-, and MDA, while enhancing the activity of antioxidant enzymes (SOD, POD, APX, and GSH-PX). Additionally, ZnO QDs promoted the biosynthesis of primary and secondary metabolites, such as total protein, soluble sugars, terpenoids, and phenols, thereby mitigating Cd stress in S. miltiorrhiza. At the molecular level, ZnO QDs were found to activate the expression of stress signal transduction-related genes, subsequently regulating the expression of downstream target genes associated with metal transport, cell wall synthesis, and secondary metabolite synthesis via transcription factors. This activation mechanism contributed to enhancing Cd tolerance in S. miltiorrhiza. In summary, these findings shed light on the mechanisms underlying the mitigation of Cd stress by ZnO QDs, offering a potential nanomaterial-based strategy for enhancing Cd tolerance in medicinal plants.

Lipopolysaccharide Enhances Tanshinone Biosynthesis via a Ca2+-Dependent Manner in Salvia miltiorrhiza Hairy Roots.

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.

Application of 1H-NMR combined with qRT-PCR technology in the exploration of rosmarinic acid biosynthesis in hair roots of Salvia miltiorrhiza Bunge and Salvia castanea f. tomentosa Stib.

MAIN CONCLUSION: Methyl jasmonate promotes the synthesis of rosmarinic acid in Salvia miltiorrhiza Bunge and Salvia castanea f. tomentosa Stib, and it promotes the latter more strongly. Salvia miltiorrhiza Bunge (SMB) is a traditional Chinese medicinal material, its water-soluble phenolic acid component rosmarinic acid has very important medicinal value. Salvia castanea f. tomentosa Stib (SCT) mainly distributed in Nyingchi, Tibet. Its pharmacological effects are similar to SMB, but its rosmarinic acid is significantly higher than the former. Methyl jasmonate (MJ) as an inducer can induce the synthesis of phenolic acids in SMB and SCT. However, the role of MJ on rosmarinic acid in SMB is controversial. Therefore, this study used SMB and SCT hair root as an experimental material and MJ as a variable. On one hand, exploring the controversial reports in SMB; on the other hand, comparing the differences in the mechanism of action of MJ on the phenolic acids in SMB and SCT. The content of related metabolites and the expression of key genes in the synthesis pathway of rosmarinic acid was analyzed by 1H-NMR combined with qRT-PCR technology. Our research has reached the following conclusions: first of all, MJ promotes the accumulation of rosmarinic acid and related phenolic acids in the metabolic pathways of SMB and SCT. After MJ treatment, the content of related components and gene expression are increased. Second, compared to SMB, SCT has a stronger response to MJ. It is speculated that the different responses of secondary metabolism-related genes to MJ may lead to different metabolic responses of salvianolic acid between the two.

smi-miR396b targeted SmGRFs, SmHDT1, and SmMYB37/4 synergistically regulates cell growth and active ingredient accumulation in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: MIR396b had been cloned and overexpressed in Salvia miltiorrhiza hairy roots. MiR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to regulate cell growth and secondary metabolism in S. miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a valuable medicinal herb with two kinds of clinically used natural products, salvianolic acids and tanshinones. miR396 is a conserved microRNA and plays extensive roles in plants. However, it is still unclear how miR396 works in S. miltiorrhiza. In this study, an smi-MIR396b has been cloned from S. miltiorrhiza. Overexpression of miR396b in danshen hairy roots inhibited hairy root growth, reduced salvianolic acid concentration, but enhanced tanshinone accumulation, resulting in the biomass and total salvianolic acids respectively reduced to 55.5 and 72.1% of the control and total tanshinones increased up to 1.91-fold of the control. Applied degradome sequencing, 5'RLM-RACE, and qRT-PCR, 13 targets for miR396b were identified including seven conserved SmGRF1-7 and six novel ones. Comparative transcriptomics and microRNomics analysis together with qRT-PCR results confirmed that miR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to mediate the phytohormone, especially gibberellin signaling pathways and consequentially resulted in the phenotype variation of miR396b-OE hairy roots. Furthermore, miR396b could be activated by methyl jasmonate, abscisic acid, gibberellin, salt, and drought stresses. The findings in this study indicated that smi-miR396b acts as an upstream and central regulator in cell growth and the biosynthesis of tanshinones and salvianolic acids, shedding light on the coordinated regulation of plant growth and biosynthesis of active ingredients in S. miltiorrhiza.

Microbial inoculant and garbage enzyme reduced cadmium (Cd) uptake in Salvia miltiorrhiza (Bge.) under Cd stress.

The uptake and accumulation of cadmium (Cd) in Salvia miltiorrhiza (Bge.) negatively affects the quality of its harvested roots, and seriously threatens human health. This study investigates the effect of a microbial inoculant (MI) and garbage enzyme (GE) on Cd uptake, the accumulation of bioactive compounds, and the community composition of microbes in the rhizosphere soil of S. miltiorrhiza under Cd stress. S. miltiorrhiza seedlings were transplanted to Cd-contaminated pots and irrigated with an MI, GE, a combination of an MI and GE (MIGE) or water (control). The results indicated that treatments with an MI, GE or MIGE can reduce Cd uptake in S. miltiorrhiza. The MIGE treatment had greater efficiency in reducing Cd uptake than the control (reduction by 37.90%), followed by the GE (25.31%) and MI (5.84%) treatments. Treatments with an MI, GE and MIGE had no significant impact on fresh and dry root biomass. Relative to the control, the MI treatment had the highest efficiency in increasing the accumulation of total tanshinones (an increase of 40.45%), followed by the GE treatment (40.08%), with the MIGE treatment (9.90%) treatment not having a more favorable effect than the separate application of an MI or GE. The salvianolic acid content for all groups was higher than the standard prescribed by Chinese pharmacopoeia, notwithstanding a slightly lower level in the treated groups relative to the control. In addition, metagenomic analysis indicated changes in the relative abundance of soil microbes associated with the bioremediation of heavy metals. The relative abundances of Brevundimonas, Microbacterium, Cupriavidus and Aspergillus were significantly greater in the treated groups than in the Control. These results suggest that using MI and GE, either separately or together, may not only improve the quality of S. miltiorrhiza but may also facilitate the microbial remediation of soil contaminated with Cd.

SmPPT, a 4-hydroxybenzoate polyprenyl diphosphate transferase gene involved in ubiquinone biosynthesis, confers salt tolerance in Salvia miltiorrhiza.

KEY MESSAGE: SmPPT, which encodes 4-hydroxybenzoate polyprenyl diphosphate transferase involved in ubiquinone biosynthesis, confers salt tolerance to S. miltiorrhiza through enhancing the activities of POD and CAT to scavenge ROS. Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a key electron transporter in the mitochondrial respiratory system. UQ is composed of a benzene quinone ring and a polyisoprenoid side chain. Attachment of polyisoprenoid side chain to the benzene quinone ring is a rate-limiting step catalyzed by 4-hydroxybenzoate polyprenyl diphosphate transferase (PPT). So far, only a few plant PPT-encoding genes have been functionally analyzed. Through genome-wide analysis and subsequent molecular cloning, a PPT-encoding gene, termed SmPPT, was identified from an economically and academically important medicinal model plant, Salvia miltiorrhiza. SmPPT contained many putative cis-elements associated with abiotic stresses in the promoter region and were responsive to PEG-6000 and methyl jasmonate treatments. The deduced SmPPT protein contains the PT_UbiA conserved domain of polyprenyl diphosphate transferase and an N-terminal mitochondria transit peptide. Transient expression assay of SmPPT-GFP fusion protein showed that SmPPT was mainly localized in the mitochondria. SmPPT could functionally complement coq2 mutation and catalyzed UQ6 production in yeast cells. Overexpression of SmPPT increased UQ production and enhanced salt tolerance in S. miltiorrhiza. Under salinity stress conditions, transgenic plants accumulated less H2O2 and malondialdehyde and exhibited higher peroxidase (POD) and catalase (CAT) activities compared with wild-type plants. It indicates that SmPPT confers salt tolerance to S. miltiorrhiza at least partially through enhancing the activities of POD and CAT to scavenge ROS.

Smoke-Isolated Karrikins Stimulated Tanshinones Biosynthesis in Salvia miltiorrhiza through Endogenous Nitric Oxide and Jasmonic Acid.

Although smoke-isolated karrikins (KAR1) could regulate secondary metabolism in medicinal plants, the signal transduction mechanism has not been reported. This study highlights the influence of KAR1 on tanshinone I (T-I) production in Salvia miltiorrhiza and the involved signal molecules. Results showed KAR1-induced generation of nitric oxide (NO), jasmonic acid (JA) and T-I in S. miltiorrhiza hairy root. KAR1-induced increase of T-I was suppressed by NO-specific scavenger (cPTIO) and NOS inhibitors (PBITU); JA synthesis inhibitor (SHAM) and JA synthesis inhibitor (PrGall), which indicated that NO and JA play essential roles in KAR1-induced T-I. NO inhibitors inhibited KAR1-induced generation of NO and JA, suggesting NO was located upstream of JA signal pathway. NO-induced T-I production was inhibited by SHAM and PrGall, implying JA participated in transmitting signal NO to T-I accumulation. In other words, NO mediated the KAR1-induced T-I production through a JA-dependent signaling pathway. The results helped us understand the signal transduction mechanism involved in KAR1-induced T-I production and provided helpful information for the production of S. miltiorrhiza hairy root.

Heterologous Expression of Salvia miltiorrhiza MicroRNA408 Enhances Tolerance to Salt Stress in Nicotiana benthamiana.

MicroRNAs (miRNAs) are a class of endogenous small RNAs that regulate the expression of target genes post-transcriptionally; they are known to play major roles in development and responses to abiotic stress. MicroRNA408 (miR408) is a conserved small RNA in plants; it was reported that miR408 genes were involved in abiotic stress in Arabidopsis. However, miR408 in Salvia miltiorrhiza has been rarely investigated. In this study, we cloned Sm-MIR408, the miR408 precursor sequence, and its promoter sequence from S. miltiorrhiza and the role in tolerance to salt stress is described. The effects of salt stress on miR408 expression were studied by using ß-glucuronidase (GUS) staining. Our data indicated that transgenic tobacco overexpressing Sm-MIR408 promoted seed germination and reduced the accumulation of reactive oxygen species under salt stress. Transcript levels of antioxidative genes, i.e., NbSOD, NbPOD, and NbCAT, and their enzyme activities increased in salinity-stressed transgenic tobacco plants, suggesting a better antioxidant system to cope the oxidative damage caused by salinity stress. Taken together, these findings indicated that miR408 functions in positive responses to salt tolerance in tobacco.

Transcriptome Analyses from Mutant Salvia miltiorrhiza Reveals Important Roles for SmGASA4 during Plant Development.

Salvia miltiorrhiza (S. miltiorrhiza) is an important Chinese herb that is derived from the perennial plant of Lamiaceae, which has been used to treat neurasthenic insomnia and cardiovascular disease. We produced a mutant S. miltiorrhiza (MT), from breeding experiments, that possessed a large taproot, reduced lateral roots, and defective flowering. We performed transcriptome profiling of wild type (WT) and MT S. miltiorrhiza using second-generation Illumina sequencing to identify differentially expressed genes (DEGs) that could account for these phenotypical differences. Of the DEGs identified, we investigated the role of SmGASA4, the expression of which was down-regulated in MT plants. SmGASA4 was introduced into Arobidopsis and S. militiorrhiza under the control of a CaMV35S promoter to verify its influence on abiotic stress and S. miltiorrhiza secondary metabolism biosynthesis. SmGASA4 was found to promote flower and root development in Arobidopsis. SmGASA4 was also found to be positively regulated by Gibberellin (GA) and significantly enhanced plant resistance to salt, drought, and paclobutrazol (PBZ) stress. SmGASA4 also led to the up-regulation of the genes involved in salvianolic acid biosynthesis, but inhibited the expression of the genes involved in tanshinone biosynthesis. Taken together, our results reveal SmGASA4 as a promising candidate gene to promote S. miltiorrhiza development.

Identification and Characterization of Flavonoid Biosynthetic Enzyme Genes in Salvia miltiorrhiza (Lamiaceae).

Flavonoids are a class of important secondary metabolites with a broad spectrum of pharmacological functions. Salviamiltiorrhiza Bunge (Danshen) is a well-known traditional Chinese medicinal herb with a broad diversity of flavonoids. However, flavonoid biosynthetic enzyme genes have not been systematically and comprehensively analyzed in S.miltiorrhiza. Through genome-wide prediction and molecular cloning, twenty six flavonoid biosynthesis-related gene candidates were identified, of which twenty are novel. They belong to nine families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), respectively. Analysis of intron/exon structures, features of deduced proteins and phylogenetic relationships revealed the conservation and divergence of S.miltiorrhiza flavonoid biosynthesis-related proteins and their homologs from other plant species. These genes showed tissue-specific expression patterns and differentially responded to MeJA treatment. Through comprehensive and systematic analysis, fourteen genes most likely to encode flavonoid biosynthetic enzymes were identified. The results provide valuable information for understanding the biosynthetic pathway of flavonoids in medicinal plants.

Characterization of the polyphenol oxidase gene family reveals a novel microRNA involved in posttranscriptional regulation of PPOs in Salvia miltiorrhiza.

Salvia miltiorrhiza is a well-known material of traditional Chinese medicine. Understanding the regulatory mechanisms of phenolic acid biosynthesis and metabolism are important for S. miltiorrhiza quality improvement. We report here that S. miltiorrhiza contains 19 polyphenol oxidases (PPOs), forming the largest PPO gene family in plant species to our knowledge. Analysis of gene structures and sequence features revealed the conservation and divergence of SmPPOs. SmPPOs were differentially expressed in plant tissues and eight of them were predominantly expressed in phloem and xylem, indicating that some SmPPOs are functionally redundant, whereas the others are associated with different physiological processes. Expression patterns of eighteen SmPPOs were significantly altered under MeJA treatment, and twelve were yeast extract and Ag+-responsive, suggesting the majority of SmPPOs are stress-responsive. Analysis of high-throughput small RNA sequences and degradome data showed that miR1444-mediated regulation of PPOs existing in P. trichocarpa is absent from S. miltiorrhiza. Instead, a subset of SmPPOs was posttranscriptionally regulated by a novel miRNA, termed Smi-miR12112. It indicates the specificity and significance of miRNA-mediated regulation of PPOs. The results shed light on the regulation of SmPPO expression and suggest the complexity of SmPPO-associated phenolic acid biosynthesis and metabolism.

SmLEA2, a gene for late embryogenesis abundant protein isolated from Salvia miltiorrhiza, confers tolerance to drought and salt stress in Escherichia coli and S. miltiorrhiza.

Abiotic stresses, such as drought and high salinity, are major factors that limit plant growth and productivity. Late embryogenesis abundant (LEA) proteins are members of a diverse, multigene family closely associated with tolerance to abiotic stresses in numerous organisms. We examined the function of SmLEA2, previously isolated from Salvia miltiorrhiza, in defense responses to drought and high salinity. Phylogenetic analysis indicated that SmLEA2 belongs to the LEA_2 subfamily. Its overexpression in Escherichia coli improved growth performance when compared with the control under salt and drought stresses. We further characterized its roles in S. miltiorrhiza through overexpression and RNAi-mediated silencing. In response to drought and salinity treatments, transgenic plants overexpressing SmLEA2 exhibited significantly increased superoxide dismutase activity, reduced levels of lipid peroxidation, and more vigorous growth than empty-vector control plants did. However, transgenic lines in which expression was suppressed showed the opposite results. Our data demonstrate that SmLEA2 plays an important role in the abiotic stress response and its overexpression in transgenic S. miltiorrhiza improves tolerance to excess salt and drought conditions.

Comparative proteomic analysis of the response to silver ions and yeast extract in Salvia miltiorrhiza hairy root cultures.

Biotic and abiotic stresses can inhibit plant growth, resulting in losses of crop productivity. However, moderate adverse stress can promote the accumulation of valuable natural products in medicinal plants. Elucidating the underlying molecular mechanisms thus might help optimize the variety of available plant medicinal materials and improve their quality. In this study, Salvia miltiorrhiza hairy root cultures were employed as an in vitro model of the Chinese herb Danshen. A comparative proteomic analysis using 2-dimensional gel electrophoresis and MALDI-TOF-MS was performed. By comparing the gel images of groups exposed to the stress of yeast extract (YE) combined with Ag(+) and controls, 64 proteins were identified that showed significant changes in protein abundance for at least one time point after treatment. According to analysis based on the KEGG and related physiological experimental verification, it was found that YE and Ag(+) stress induced a burst of reactive oxygen species and activated the Ca(2+)/calmodulin signaling pathway. Expression of immune-suppressive proteins increased. Epidermal cells underwent programmed cell death. Energy metabolism was enhanced and carbon metabolism shifted to favor the production of secondary metabolites such as lignin, tanshinone and salvianolic acids. The tanshinone and salvianolic acids were deposited on the collapsed epidermal cells forming a physicochemical barrier. The defense proteins and these natural products together enhanced the stress resistance of the plants. Since higher levels of natural products represent good quality in medicinal materials, this study sheds new light on quality formation mechanisms of medicinal plants and will hopefully encourage further research on how the planting environment affects the efficacy of herbal medicines.

Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process. Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

Synergistic effects of ultraviolet-B and methyl jasmonate on tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.

Tanshinones are major bioactive diterpenoids of Salvia miltiorrhiza roots used for the treatment of cardiocerebral diseases. To develop effective elicitation and bioprocess strategies for the enhanced production of tanshinones, ultraviolet-B (UV-B) irradiation and methyl jasmonate (MeJA) elicitation were applied alone or in combination respectively in S. miltiorrhiza hairy root cultures. Our results showed 40-min UV-B irradiation at 40µW/cm(2) stimulated tanshinone production without any suppression of root growth, suggesting a new effective elicitor to S. miltiorrhiza hairy root cultures for tanshinone production. Moreover, the combined treatment of UV-B irradiation and MeJA exhibited synergistic effects on the expression levels of 3-hydroxy-3-methylglutaryl-CoA reductase (SmHMGR) and geranylgeranyl diphosphate synthase (SmGGPPS) genes in the tanshinone biosynthetic pathway. When hairy roots of 18-day-old cultures were exposed to the combined elicitation for 9days, the maximum production of tanshinone reached to 28.21mg/L, a 4.9-fold increase over the control. The combined elicitation of UV-B and MeJA was firstly used to stimulate the production of biologically important secondary metabolites in hairy root cultures.

[Effects of plant growth regulator uniconazole on plant morphology and biomass allocation of Salvia miltiorrhiza].

In this study, we use pot experiment to evaluate the effect of plant growth regulator on plant morphology and biomass allocation of Salvia miltiorrhiza. Different concentrations of uniconazole were supplied to S. miltioohiza by means of foliar spray. Height, breadth and stem diameter were measured dynamically, the biomass of leaf, stem, flower and fruit, root biomass and biomass ratio were also examined at the harvest time. Owing to the treatment, plant morphology showed significant changes, the height had been greatly reduced and the breadth decreased largely. Meanwhile, the biomass allocation changed too. The biomass ratio of leaf and stem had been notably reduced while the biomass ratio of root had been increased remarkably. It appears that foliar application of uniconazole during vigorous growth period in S. miltioohiza has dramatic effect on dwarfing plant and improving resistant to lodging. This measure could also be applied to condensed cultivation of S. miltioohiza to increase production.

Production of herbicide-resistant medicinal plant Salvia miltiorrhiza transformed with the bar gene.

In this study, we successfully performed Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza and produced herbicide-resistant transformants. Leaf discs of S. miltiorrhiza were infected with Agrobacterium tumefaciens EHA105 harboring pCAMBIA 3301. The pCAMBIA 3301 includes an intron-containing gus reporter and a bar selection marker. To increase stable transformation efficiency, a two-step selection was employed which consists of herbicide resistance and gus expression. Here, we put more attention to the screening step of herbicide resistance. The current study provides an efficient screening system for the transformed plant of S. miltiorrhiza harboring bar gene. To determine the most suitable phosphinothricin concentration for plant selection, non-transformed leaf discs were grown on selection media containing six different phosphinothricin concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/l). Based on the above results of non-transformed calluses, the sensitivity of phosphinothricin (0, 0.4, 0.8, 1.2, 1.6 mg/l) was tested in the screening of transgenic S. miltiorrhiza. We identified that 0.6 mg/l phosphinothricin should be suitable for selecting putatively transformed callus because non-transformed callus growth was effectively inhibited under this concentrations. When sprayed with Basta, the transgenic S. miltiorrhiza plants were tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to S. miltiorrhiza.

Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots.

Tanshinone is a group of active diterpenes, which are widely used in the treatment of cardiovascular disease. In this study, methyl jasmonate (MJ) and salicylic acid (SA) were used to investigate their effects on tanshinone accumulation and biosynthetic gene expression in the hairy roots of geranylgeranyl diphosphate synthase (SmGGPPS) overexpression line (G50) in Salvia miltiorrhiza. High-performance liquid chromatography analysis showed that total tanshinone content in G50 was obviously increased by 3.10-fold (11.33 mg/g) with MJ at 36 H and 1.63 times (5.95 mg/g) after SA treatment for 36 H in comparison with their mimic treatment control. Furthermore, quantitative reverse-transcription PCR analysis showed that the expression of isopentenyl-diphosphate delta-isomerase (SmIPPI), SmGGPPS, copalyl diphosphate synthase (SmCPS), and kaurene synthase-like (SmKSL) increased significantly with MJ treatment. However, the expression of SmIPPI reached the highest level at 144 H, whereas those of SmGGPPS, SmCPS, and SmKSL only increased slightly with SA treatment. The two elicitor treatments suggested that tanshinone accumulation positively correlated to the expression of key genes such as SmGGPPS, SmCPS, and SmKSL. Meanwhile, the study also indicated that it was a feasible strategy to combine elicitor treatment with transgenic technology for the enhancement of tanshinone, which paved the way for further metabolic engineering of tanshinone biosynthesis.

[Influence of plant growth regulater on yield and quality of Salvia miltiorrhiza].

The study is aimed to investigate the effect of plant growth regulators on yield and quality of the Salvia miltiorrhiza. The plant growth regulators was spraying on Salvia plants in July or August in field experiment, then the yield, ingredient content and the antioxidant activity were determined. The results showed that plant growth regulator 'Zhuanggenling' could increase the yield of Salvia with root-planting by 38.45%. Plant growth regulator 'Duoxiaozuo' could increase the yield of Salvia with seedling planting by 14.19%. Both plant growth regulator significantly reduced the antioxidant activity of Salvia in vitro, but they had no significant effect on active ingredient contents.

Hydrogen peroxide is involved in salicylic acid-elicited rosmarinic acid production in Salvia miltiorrhiza cell cultures.

Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H2O2) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H2O2 in rosmarinic acid (RA) production in Salvia miltiorrhiza cell cultures were investigated. The results showed that SA significantly enhanced H2O2 production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H2O2 could also promote PAL activity and enhance RA production. If H2O2 production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H2O2 is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation.