Promotion of phenolic compounds production in Salvia miltiorrhiza hairy roots by six strains of rhizosphere bacteria

Salvia miltiorrhiza Bunge is an important herb for the treatment of cerebrovascular and cardiovascular diseases with bioactive compounds (phenolic acids and tanshinones). Abundant studies showed that tanshinones could be stimulated by biotic and abiotic stresses, but limited information is available on biosynthesis of phenolic acids promoted by biotic stresses. The aim of the present work was to isolate and identify rhizosphere bacteria which stimulated phenolic compound in Salvia miltiorrhiza hairy roots and investigated the internal mechanism, providing a potential means to enhance content of pharmaceuticals in S. miltiorrhiza. The results showed that six bacteria, namely, HYR1, HYR26, SCR22, 14DSR23, DS6, and LNHR13, belonging to the genus Pseudomonas and Pantoea, significantly promoted the growth and content of major phenolic acids, RA and SAB. Bacteria LNHR13 was the most effective one, with the contents of RA and SAB reaching ～2.5‐fold (30.1 mg/g DW) and ～2.3‐fold (48.3 mg/g DW) as those of the control, respectively. Phytohormones and polysaccharides produced by bacteria showed potential responsibility for the growth and biosynthesis of secondary metabolites of S. miltiorrhiza. Meanwhile, we found that the more abundant the types and contents of phytohormones, the stronger their stimulating effect on the content of salvianolic acids.     

The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza transformed with Agrobacterium rhizogenes ATCC 15834 produced a tiny amount of tanshinones and a constituent level of phenolic acids under normal growth conditions. Upon elicitation with yeast elicitor, the production of both phenolic acids and tanshinones was enhanced. For example, the contents of two phenolic acids, rosmarinic acid and lithospermic acid B were elevated from 1.24% and 2.59% to 2.89% and 2.98% of dry wt, respectively while the intracellular content of cryptotanshinone increased from 0.001% to as much as 0.096% of dry wt. Yeast elicitor also improved the growth of hairy roots (from 3.9 g/l to 7.3 g/l on a dry wt basis). Liquid chromatography-mass spectrometry (LC-MS) was developed for simultaneous detection and identification of phenolic acids and tanshinones in the extracts of S. miltiorrhiza. Rosmarinic acid, lithospermic acid B, cryptotanshinone, tanshinone I, tanshinone IIA and tanshinone IIB were identified by comparison with standards available. Dihydrotanshinone I and methylenetanshiquinone were tentatively identified by the molecular weights and the elution comparable with the literature. An unknown compound with a molecular weight of 280 was found in yeast-elicitor treated hairy root cultures, which was one of the major tanshinones induced.     

SmMYB98b positive regulation to tanshinones in Salvia miltiorrhiza Bunge hairy roots

Plant Cell, Tissue and Organ Culture (PCTOC) - Tanshinones are major secondary metabolites in Salvia miltiorrhiza Bunge, the traditional Chinese medicinal plant Danshen. Increasing the production...     

一氧化氮信号在脱落酸诱导丹参酚酸类成分积累中的作用

为探讨一氧化氮(Nitric oxide,NO)信号在脱落酸(Abscisic acid,ABA)诱导丹参酚酸类成分积累中的作用,采用不同浓度一氧化氮外源供体硝普钠(Sodium nitroprusside,SNP)处理丹参毛状根,6 d后采收,测定酚酸类成分含量;ABA联合一氧化氮清除剂(2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,c-PTIO)或一氧化氮合酶抑制剂(N~G-nitro-L-arginine methyl ester,L-NAME)对丹参毛状根进行处理,测定酚酸类成分含量和关键基因表达量。结果表明,100μmol/L SNP对丹参毛状根中迷迭香酸与丹酚酸B积累的诱导效果最显著,迷迭香酸和丹酚酸B含量分别增加了3倍和4倍。ABA处理能显著促进PAL(Phenylalanine ammonia lyase)、TAT(Tyrosine aminotransferase)和RAS(Rosmarinic acid synthase)基因的表达,促进丹参毛状根中酚酸类成分的积累,而联合c-PTIO或L-NAME共同处理后,3种关键基因表达下调,并显著抑制了酚酸类成分的积累。研究证明NO和ABA均能够促进丹酚酸类成分的积累,NO信号可能介导了ABA对丹酚酸生物合成的诱导作用。     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca(2+) and H(2)O(2) in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10-500 microM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10-100 microM but decreased from 100-200 muM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPgammaS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca(2+) antagonists, but not affected by a protein kinase inhibitor. ATP also induced H(2)O(2) production, which was dependent on both Ca(2+) and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca(2+) level, which was dependent on NO but not H(2)O(2). The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca(2+) and ROS signalling.     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.     

一株促进丹参生长和提高丹酚酸含量的活性内生真菌

利用菌根接种技术,丹参幼苗接种固体培养的SMF34菌株后,在温室和试验田条件下栽培,比较处理组和对照组在表观性状、生物量、总酚酸和丹酚酸B含量间的差异,结果处理组丹参均优于对照组。该活性菌株经形态和分子鉴定为拟青霉属Paecilomyces sp.真菌。提示该活性菌对丹参生长发育和丹酚酸含量的提高具有促进作用,是一株具有很好应用和研究价值的真菌。     

Diterpenoids and triterpenoids in hairy roots of Salvia sclarea

Hairy root culture of Salvia sclarea L. was established following infection with Agrobacterium rhizogenes LBA 9402. The culture was grown in growth regulator-free half-strength B5 Gamborg medium with 30 g l⁻¹ sucrose and was investigated with respect to its capability of producing diterpenoids and triterpenoids. Four diterpenoids (ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) and two ursene-type triterpenoids (2α,3α-dihydroxy-urs-12-en-28-oic acid and 2α,3α,24-trihydroxy-urs-12-en-28-oic acid) were isolated from the hairy roots. The presence of three sterols (β-sitosterol, stigmasterol and campesterol), as well as oleanolic and ursolic acids was also shown by GC–MS analysis. The quantitative and qualitative differences in diterpenoid and triterpenoid production patterns between hairy roots grown in the light and in the dark were described.     

蔗糖浓度对三裂叶野葛毛状根生长及其次生物质产生的影响

本文研究了蔗糖浓度对发根农杆菌ATCC15834诱导产生的三裂叶野葛毛状根生长及其葛根素和异黄酮类化合物产生的影响以及液体培养基中蔗糖的消耗变化。结果表明：毛状根在含5％、4％、3％和2％蔗糖的MS培养基中培养16天后的干重增殖倍数分别为11．7、11．9、10．1和5．9;其中尤以3％的蔗糖浓度最有利于毛状根中异黄酮类化合物及葛根素的积累;培养12天后,毛状根的葛根素含量达到最高,约5．147mg／gDW;而其异黄酮类化合物的含量则在培养16天后达到最高,约27．76mg／gDW。在毛状根液体培养过程中培养基的蔗糖浓度随着毛状根的生长而降低,其消耗速率与毛状根的生长速度及其可溶性总糖含量成正比。毛状根的可溶性总糖含量在培养12天时达到最高,而培养16天后培养基中的蔗糖消耗完毕。     

Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures

Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.     

内生真菌枝孢属Cladosporium sp.对丹参生长和丹酚酸含量的影响

本文介绍从药用植物丹参内生真菌中筛选新获得的一株对丹参生长和丹酚酸含量具有显著促进作用的菌株,Cladosporium sp. SM58。运用菌根生物技术,于离体、盆栽和田栽条件下,分别将丹参组培苗或幼苗接种SM58固体菌种后,观察测定菌株SM58对丹参组培苗生长的影响,以及丹参幼苗接菌培植6个月后菌株SM58对丹参丹酚酸有效成分含量和生物量的影响;同时进行显微观察,对菌株SM58侵入田栽丹参根组织的状况进行分析。结果表明,SM58菌株不仅对丹参组培苗的株高、根长和生物量均有显著促生长作用（P<0.01）,而且对盆栽和田栽丹参的丹酚酸有效成分含量和根干重的提高也均有显著效果（P<0.01）。与对照相比较,接菌组田栽丹参的根干重、总酚酸含量、丹酚酸A的含量分别提高68%、47%、11%。显微观察显示SM58菌丝主要通过侵染丹参的根表皮细胞和皮层细胞而对丹参产生影响。本研究表明,SM58菌株能对丹参品质和产量产生有益影响,是一株具有很好研究价值和开发应用的活性菌。     

Isolation and identification of endophytic bacteria from root tissues of Salvia miltiorrhiza Bge. and determination of their bioactivities

Endophytic bacteria are microorganisms that live in host plants, but do not cause diseases to the hosts. This study examined the occurrence, distribution, growth-promoting and antifungal activities of endophytes in the root of Salvia miltiorrhiza Bge. Six endophytic bacterial strains, which belong to genera of Pseudomonas, Rhizobium, Bacillus and Novosphingobium, were isolated from the root of healthy S. miltiorrhiza. Cell suspension (approx. 10⁹ cell?·?ml^?1) of two isolates and cell-free fermentation filtrate of four isolates substantially promoted the growth of hypocotyl and radicle of muskmelon seeds. The cell-free fermentation filtrate of six isolates had no inhibiting effect on tested pathogenic fungi, namely Fusarium solani, F. oxysporum f. sp. vasinfectum and F. oxysporum. Six compounds were isolated from one of the six endophytic bacteria, namely, Bacillus aryabhattai, and two of these compounds displayed certain antifungal activity against three tested S. miltiorrhiza pathogens. Our work indicates that endophytic bacteria occur in the root of S. miltiorrhiza, and that associated bacterial isolates have growth-promoting effect on muskmelon seeds and are expected to be a potential source for bioactive metabolites.     

PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots

Tanshinones, a group of active ingredients in Salvia miltiorrhiza, are derived from at least two biosynthetic pathways, which are the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastids. Abscisic acid (ABA) and methyl jasmonate (MJ) are two well-known plant hormones induced by water stress. In this study, effects of polyethylene glycol (PEG), ABA and MJ on tanshinone production in S. miltiorrhiza hairy roots were investigated, and the role of MJ in PEG- and ABA-induced tanshinone production was further elucidated. The results showed that tanshinone production was significantly enhanced by treatments with PEG, ABA and MJ. The mRNA levels of 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS), as well as the enzyme activities of HMGR and DXS were stimulated by all three treatments. PEG and ABA triggered MJ accumulation. Effects of PEG and ABA on tanshinone production were completely abolished by the ABA biosynthesis inhibitor [tungstate (TUN)] and the MJ biosynthesis inhibitor [ibuprofen (IBU)], while effects of MJ were almost unaffected by TUN. In addition, MJ-induced tanshinone production was completely abolished by the MEP pathway inhibitor [fosmidomycin (FOS)], but was just partially arrested by the MVA pathway inhibitor [mevinolin (MEV)]. In conclusion, a signal transduction model was proposed that exogenous applications of PEG and ABA triggered endogenous MJ accumulation by activating ABA signaling pathway to stimulate tanshinone production, while exogenous MJ could directly induce tanshinone production mainly via the MEP pathway in S. miltiorrhiza hairy roots.     

Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures

 Salvia miltiorrhiza contains two groups of biologically active secondary metabolites termed phenolic compounds (e.g. rosmarinic acid) and tanshinones (e.g. cryptotanshinone). Their roles in plant defense responses were examined using a simplified system consisting of a yeast elicitor and a Ti C58 transformed S. miltiorrhiza cell line. Both dosage and time course studies were carried out on the effects of yeast elicitor on the formation of rosmarinic acid and cryptotanshinone. It was found that the yeast elicitor reduced the constituent level of rosmarinic acid (from ca. 5% to ca. 3.0% of dry cell weight) whereas the level of cryptotanshinone was enhanced greatly (from a negligible amount to ca. 20 mg/l). These results suggest that in S. miltiorrhiza, rosmarinic acid and cryptotanshinone may take part in plant passive and active defense responses, respectively, against pathogen attack. Cryptotanshinone was identified as a phytoalexin in S. miltiorrhiza for the first time. Results of the treatment of cell cultures with 2-aminoindan-2-phosphonic acid, a highly specific and potent inhibitor of phenylalanine ammonia-lyase (EC 4.3.1.5), indicated that this compound did not inhibit yeast elicitor induced tanshinone formation, but did inhibit rosmarinic acid biosynthesis. Received: 22 May 1999 / Revision received: 27 August / Accepted: 1 September 1999     

Production of lithospermic acid B and rosmarinic acid in hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium rhizogenes ATCC 15834, and the transformation was proved by direct detection of the inserted T-DNA by the polymerase chain reaction. As determined by HPLC, these hairy root cultures had the ability to produce lithospermic acid B (LAB), rosmarinic acid (RA) and other related phenolic compounds, the water-soluble active components of the plant. The effect of five different basal media, MS, MS-NH<INF>4</INF> (MS without ammonium nitrate), B5, WPM and 6,7-V on the root growth and phenolic compound production was studied. It was found that MS-NH<INF>4</INF> and 6,7-V media were superior to MS, B5 and WPM media in terms of both root growth and phenolic compound production. The time course of biomass accumulation and phenolic compound formation was also examined in the culture using MS-NH<INF>4</INF>medium. During cultivation, the content of RA in the roots was stable being approximately 0.48% of dry weight while the content of LAB fluctuated between 0.73% and 1.61% of dry weight, and decreased gradually at the stationary phase of growth. The highest production of LAB and RA was about 64 mg L⁻¹ and 23 mg L⁻¹, respectively. Received 05 November 1998/ Accepted in revised form 06 February 1999     

Overexpression of Ath-MIR160b increased the biomass while reduced the content of tanshinones in Salvia miltiorrhiza hairy roots by targeting ARFs genes

Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root...     

Modeling of tanshinone synthesis and phase distribution under the combined effect of elicitation and in situ adsorption in Salvia miltiorrhiza hairy root cultures

Modeling of tanshinone synthesis and phase distribution under the combined effect of elicitation and adsorption was studied. The simulated results showed that enhancement of tanshinone production was mainly due to the effect of the elicitor and that resin addition resulted in adsorbance of the tanshinones from the root and alteration of tanshinone distribution. Furthermore, parameter sensitivities analysis showed that the rate of transport of tanshinones from the root to the medium was the important factor that influenced tanshinone accumulation in the resin. In conclusion, the modeling of tanshinone synthesis and phase distribution identified the process mechanism under the combined effect of elicitation and adsorption and this modeling can be used in similar plant tissue culture systems.