Elicitor-induced phenolic acids accumulation in Salvia virgata Jacq. hairy root cultures

Plant Cell, Tissue and Organ Culture (PCTOC) - Phenolic acids, as the predominant secondary metabolites of Salvia plants, are largely used in pharmaceutical industries. The main aim of the study...     

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。     

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.     

Effect of methyl jasmonate on phenolic acids accumulation and the expression profile of their biosynthesis-related genes in Mentha spicata hairy root cultures

Plant Cell, Tissue and Organ Culture (PCTOC) - Mentha spicata L., as a rich source of phenolic acids, is known for its therapeutic importance. Elicitors play a crucial role in biosynthetic pathways...     

Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures

Hairy roots were induced by infecting stems and leaves of Glycyrrhiza inflata with Agrobacterium rhizogenes ATCC 15834. The optimization of growth and glycyrrhizin accumulation of G. inflata hairy roots was studied. Sucrose (6%, w/v) was optimal for growth and glycyrrhizin accumulation in G. inflata hairy roots. Effects of elicitors like chitosan, methyl jasmonate, and yeast extract on glycyrrhizin production were studied. Methyl jasmonate (100 microM) was most efficient in enhancing glycyrrhizin production up to almost 109 microg/g dry weight on day 5 of elicitation. These results indicate that application of elicitors can enhance the capacity of G. inflata hairy roots to produce glycyrrhizin.     

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     

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.     

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.     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

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.     

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.     

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.     

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.     

The signaling role of extracellular ATP and its dependence on Ca2+ flux in elicitation of Salvia miltiorrhiza hairy root cultures

The application of a polysaccharide elicitor from yeast extract,YE, to Salvia miltiorrhiza hairy root cultures induced transientrelease of ATP from the roots to the medium, leading to a dose-dependentincrease in the extracellular ATP (eATP) level. The eATP levelrose to a peak (about 6.5 nM with 100 mg l^–1 YE) at about10 h after YE treatment, but dropped to the control level 6h later. The elicitor-induced ATP release was dependent on membraneCa²⁺ influx, and abolished by the Ca²⁺ chelator EGTA or thechannel blocker La³⁺. The YE-induced H₂O₂ production was stronglyinhibited by reactive blue (RB), a specific inhibitor of membranepurinoceptors. On the other hand, the application of exogenousATP at 10–100 µM to the cultures also induced rapidand dose-dependent increases in H₂O₂ production and medium pH,both of which were effectively blocked by RB and EGTA. The non-hydrolyzableATP analog ATPS was as effective as ATP, but the hydrolyzedderivatives ADP or AMP were not so effective in inducing thepH and H₂O₂ increases. Our results suggest that ATP releaseis an early event and that eATP plays a signaling role in theelicitation of plant cell responses; Ca²⁺ is required for activationof the elicitor-induced ATP release and the eATP signal transduction.This is the first report on ATP release induced by a fungalelicitor and its involvement in the elicitor-induced responsesin plant cells.     

Methyl jasmonate increases the production of valepotriates by transformed root cultures of Valerianella locusta

Transformed roots of V. locusta (Valerianaceae) were obtained through transformation with Agrobacterium rhizogenes strains A4 and ATCC 15834. Six known valepotriates, including diavaltrate, acevaltrate, didrovaltrate, IVHD-valtrate, isovaltrate, and valtrate were the major components detected. An LC/PDA method was used in the quantitation of these compounds in the transformed root extracts. The treatment of transformed roots with biotic (methyl jasmonate, salicylic acid, yeast extract) and abiotic elicitors (CuSO₄, HgCl₂, CaCl₂) was used as a strategy to improve the production of valepotriates. Methyl jasmonate appeared to be the best elicitor for valepotriate production, yielding up to a 7-fold increase in total valepotriate content, while HgCl₂ had the most deteriorating effect on the production of valepotriates. Salicylic acid-, CuSO₄- and CaCl₂-treated roots showed significant increases in the production at a short duration of exposure; the production decreased as the time of elicitation increased. The highest total valepotriate content achieved in this study was 139 mg g^–1 DW (13.9%) from transformed roots treated for 10 days with 100 M methyl jasmonate. This amount was >50- and 12-fold higher than the values reported from the cultivated plants and callus culture, respectively, and was comparable to the amount reported from the high valepotriate-producing species Valeriana thalictroides Graebn. The production of diavaltrate, acevaltrate, didrovaltrate, and isovaltrate were significantly higher, while the production of IVHD-valtrate was lower and that of valtrate was similar to that of the control. The IVAL/VAL production ratio was affected by the treatment with methyl jasmonate but not by other elicitors. The use of transformed root cultures in combination with the treatment with biotic and abiotic elicitors offer a new route for high valepotriate production.     

Growth patterns and alkaloid accumulation in hairy root and untransformed root cultures of Datura stramonium

The aim of this work was to study the possible relationship between alkaloid production and growth measured as: biomass increase and cellular division frequency, in Datura stramonium in vitro root cultures (hairy root and normal cultures). A comparison of growth values on a fresh and dry weight basis showed that there were differences between transformed and non-transformed lines. The differential growth between lines occurred due to a real biomass increase and not because of water accumulation. On the other hand, the rate of cell division showed a similar pattern for all lines studied. Therefore, the differences in growth are not due to different cell division rates, nor to the presence of larger meristems, but to the development and growth of lateral roots and the presence of active intercalary meristematic zones in each line. The maximum alkaloid production occurred when the cultures were not growing. This suggests an inverse relationship. Finally, the data support a specific model of growth at the level of cell division in root cultures which has not been described before in the literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of phytoalexin formation in crown gall and hairy root culture of Salvia miltiorrhiza by methyl viologen

Methyl viologen (MV) (20–150 M), a generator of superoxide anion (O₂ ^–), but not hydrogen peroxide (H₂O₂) (10 M–2 mM) triggered the formation of cryptotanshinone (a phytoalexin) in cultures of both crown galls and hairy roots of Salvia miltiorrhiza. MV also inhibited the biomass formation and decreased the contents of phenolic acids in both cultures whereas H₂O₂ did not. In addition, MV and yeast elicitor induced cryptotanshinone formation synergistically only in crown gall cultures. Treatment of the cultures with 3.3 M diphenylene iodonium, an inhibitor of NAD(P)H oxidase, did not exhibit any detrimental effect on the yeast elicitor-induced cryptotanshinone formation in hairy root cultures whereas 1 M diphenylene iodonium was inhibitory on yeast elicitor-induced cryptotanshinone formation in crown gall cultures.