Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag + and yeast elicitor

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1 Plant Science 168 (2005) Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag + and yeast elicitor Xiuchun Ge a,b, Jianyong Wu a, * a Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China b Department of Plant Protection, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China Received 23 June 2004; received in revised form 4 September 2004; accepted 7 September 2004 Available online 29 September 2004 Abstract This work examined the accumulation of diterpenoid tanshinones and related secondary metabolism pathways in hairy root cultures of Salvia miltiorrhiza Bunge (Lamiacae) induced by a biotic elicitor, the carbohydrate fraction of yeast extract (YE, 100 mgml 1 ), and an abiotic elicitor, Ag + (30 mm). The activity of 3-hydroxy-3-methylglutaryl CoA redutase (HMGR) was only stimulated by Ag +, and 1-deoxy- D-xylulose 5-phosphate synthase (DXS) was stimulated by both, but more strongly by YE. While the non-mva pathway inhibitor fosmidomycin inhibited the tanshinone accumulation induced by both elicitors, the MVA-pathway inhibitor mevinolin only suppressed the Ag-induced tanshinone accumulation. The results suggest that the tanshinone accumulation induced by the two elicitors was mainly synthesized via the non-mva pathway (DXS activity), but could depend on crosstalk between the MVA and non-mva pathways. Furthermore, the pretreatment of hairy roots with Ag + for h potentiated the YE-induced tanshinone production and DXS activity. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Tanshinones; Isoprenoid pathways; Elicitors; Salvia miltiorrhiza; Hairy root culture 1. Introduction Plant hairy roots are transformed roots formed after the infection of wounded plant tissue by the soil bacterium, Agrobaterium rhizogenes. Hairy root culture represents a convenient experimental system for plant science research and a promising bioprocess for the production of medicinal plants and their active constituents, which are mostly plant secondary metabolites [1]. Salvia miltiorrhiza Bunge (Lamiaceae) is a well-known Chinese herbal plant, and its roots, known as Danshen in Chinese, are widely used for the treatment of menstrual disorders and blood circulation diseases, and for the prevention of inflammation. The chief bioactive ingredients of S. miltiorrhiza roots are the diterpenoid pigments, particularly the phenanthrofurane quinone derivatives generally known as tanshinones [2]. Hairy root culture of S. miltiorrhiza has been established as a potential means for tanshinone production [3]. * Corresponding author. Tel.: ; fax: address: bcjywu@polyu.edu.hk (J. Wu). In higher plants, terpenes or isoprenoids are synthesized via at least two different pathways, the mevalonate (MVA) pathway occurring in the cytosol and the non-mva, 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in the plastids of cell [4,5] (Fig. 1). HMGR (3-hydroxy-3- methylglutaryl CoA reductase) catalyzes the formation of mevalonate from 3-hydroxy-3-methylglutaryl CoA, an initial and important step in the MVA pathway, and 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) are the first two enzymes in the DXP pathway. Although diterpenes are generally believed to be synthesized via the non-mva pathway, there has been growing evidence for the involvement of the cytosolic MVA pathway in diterpene biosynthesis and the existence of crosstalk between the two pathways for isoprenoid biosynthesis in some plants [5]. The specific biosynthetic pathways for tanshinone production in the S. miltiorrhiza roots are still not clear. This work aims to further investigate the separate and combined effects of biotic and abiotic elicitors on the secondary metabolism activities of S. miltiorrhiza hairy /$ see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi: /j.plantsci

2 488 X. Ge, J. Wu / Plant Science 168 (2005) Fig. 1. An overview of the cytosolic mevalonate pathway and plastidial mevalonate-independent pathway for the biosynthesis of terpenoids (isoprenoids) in plant cells. (DMAPP, dimethylallyl diphosphate; DXP, 1- deoxy-d-xylulose-5-phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; GA-3P, glyceraldehyde-3-phosphate; HMG-CoA, 3-hydroxy-3- methylglutaryl-coa; HMGR, HMG-CoA reductase; IPP, isopentenyl diphosphate. Mevinolin and fosmidomycin are inhibitors of HMGR and DXR, respectively.) roots in suspension cultures, and to identify the specific metabolic pathways responsible for the biosynthesis of tanshinones. The carbohydrate (polysaccharide) fraction of yeast extract was used as a biotic elicitor and Ag + as an abiotic elicitor. 2. Materials and methods 2.1. Hairy root culture The S. miltiorrhiza hairy root culture was derived after the infection of plantlets with a Ri T-DNA bearing Agrobacterium rhizogenes bacterium (ATCC15834) in the laboratory of Dr. Steven F. Chen at The University of Hong Kong [6]. Stock cultures of the hairy roots were maintained on solid, hormone-free MS medium [7] with 8 g L 1 agar and 30 g L 1 sucrose but without ammonium nitrate, at 25 8C in the dark. Experiments in this study on the effects of elicitors were all carried out in shake-flask suspension cultures, with 125-mL Erlenmeyer flasks each containing 25 ml medium on an orbital shaker set at rpm and 25 8C, as described elsewhere [8]. Hairy roots were harvested from the culture medium by filtration and blotted dry with paper towels (yielding the fresh weight, fw), and then dried at 45 8C in an oven until constant weight (dry weight, dw). Briefly, 25 g of yeast extract (Sigma, St. Louis, MO, Cat. Y4250) was dissolved in 125 ml of distilled water, followed by the addition of 100 ml of ethanol. The solution was allowed to precipitate for 4 days at 4 8C in a refrigerator, and the supernatant was decanted. The remaining gummy precipitate was redissolved in 125 ml of distilled water and subjected to another round of ethanol precipitation. The final precipitate was dissolved in 100 ml of distilled water, sterilized by autoclaving at 121 8C for 2 h and stored at 4 8C in a refrigerator before use. The elicitor dose was expressed by the total carbohydrate content, which was determined by the phenol sulfuric acid method [10] using sucrose as a standard. Silver ion Ag + was supplied to the culture with a concentrated silver thiosulfate (Ag 2 S 2 O 3 ) solution prepared by mixing AgNO 3 and Na 2 S 2 O 3 at 1:4 molar ratio as described previously [8]. Mevinolin (Sigma, Cat. M2147) was used as an inhibitor of the mevalonate pathway [11] and fosmidomycin (sodium salt FR-31564, Molecular Probes Inc., Eugene, OR) as an inhibitor of the non-mva pathway [4]. Both have been proven specific and effective for the respective pathways, mevinolin for HMGR and fosmidomycin for DXR (Fig. 1). Stock solutions of mevinolin (Mev, 5 mm) and fosmidomycin (Fos, 100 mm) were prepared in distilled water and sterilized by filtration (0.22 mm membrane). Elicitor and inhibitor treatments were applied to the hairy root culture on day 18 post inoculation, at concentrations (Ag + 30 mm, YE 100 mg ml 1, Mev 5 mm and Fos 100 mm) chosen based on previous studies to produce the maximum responses. All treatments were performed in triplicate, and the results were represented by the mean standard error (S.E.) Analysis of tanshinones Tanshinones were extracted from the roots with methanol (MeOH) (ca. 100 mg/ml). Tanshinones in the culture medium were extracted with ethyl acetate (1:1, v/v), which was then evaporated to dryness and redissolved in methanol. Tanshinone content in the MeOH extract solution was determined by the HPLC method and instruments as described previously [8] with a 25 cm 0.46 cm Alltech Econosphere 5 m C-l8 column. Three tanshinone species, cryptotanshinone (CT), tanshinone I (T1) and tanshinone IIA (T2A) (Fig. 2), were detected and quantified with authentic standards obtained from the Institute for Identi Preparation of elicitors and metabolic inhibitors The yeast elicitor (YE) was the carbohydrate (polysaccharide) fraction of yeast extract prepared by ethanol precipitation, as described by Hahn and Albersheim [9]. Fig. 2. Molecular structures of three major tanshinones in Salvia miltiorrhiza roots.

3 fication of Pharmaceutical and Biological Products (Beijing). The tanshinone peaks of samples were further confirmed by co-chromatography with the standards. The total tanshinone content shown in the results is the sum of the three in the roots and the culture medium Enzyme assays X. Ge, J. Wu / Plant Science 168 (2005) All enzymes assayed in this work were extracted from the hairy roots with an extraction buffer of 50 mm Tris HCl, 10 mm b-mercaptoethanol, 1% (w/v) polyvinylpyrrolidone (PVP) and ph 7.5. The roots were ground in the extraction buffer (1 g fw ml 1 ) for 5 min with a pestle and mortar on ice, followed by centrifugation at 14,000 rpm and 4 8C for 30 min to obtain a solids-free extract. The protein content was determined by the Bradford method [12] using bovine serum albumin as a standard. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was determined by the method of Toroser and Huber [13]. The enzyme extract was added (ca. 50 mg protein per ml) to a 50 mm of Tris HCl assay buffer (ph 7.0) containing 0.3 mm of HMG-CoA (Sigma, Cat. H6132), 0.2 mm of NADPH and 4 mm of dithiothreitol. NADPH oxidation in the reaction solution was monitored at 25 8C by the decreasing absorbance at 340 nm, against the solution free of HMG-CoA as a blank. One HMGR enzyme unit is equivalent to the oxidation of 1 mmol of NADPH per minute. The activity of 1-deoxy-D-xylulose 5-phosphate synthase was determined by the fluorometric method of Querol et al. [14], which is based on the reaction of 1-deoxy-D-xylulose 5- phosphate with 3,5-diaminobenzoic acid in an acidic medium to form a highly fluorescing quinaldine derivative. The reaction mixture contained 40 mm of Tris HCl (ph 7.5), 2.5 mm of MgCl 2, 5 mm of b-mercaptoethanol, 1 mm of thiamin diphosphate, 10 mm of sodium pyruvate, and 20 mm of DL-glyceraldehyde 3-phosphate, and the enzyme extract (ca. 50 mg protein per ml reaction solution). The reaction solution was maintained at 37 8C for 1 h, stopped by heating at 80 8C for 5 min, and then spun down at 13,000 rpm for 5 min to remove the denatured proteins. The supernatant was mixed with 1 ml of 10 mm 3,5- diaminobenzoic acid in 5 M phosphoric acid, and then heated in a boiling water bath for 15 min. The fluorescence intensity of the reaction product, which is proportional to DXS activity, was determined with a PerkinElmer LS50B fluorescence spectrophotometer at 396 nm excitation and 510 nm emission. Fig. 3. Effects of Ag + (30 mm) and yeast elicitor (YE, 100 mgml 1 carbohydrate) on hairy root growth and tanshinone accumulation (Ctrl, control). Elicitor was added to culture on day 18 post inoculation; error bars for standard errors (S.E.), n = 3. by YE (Fig. 3). The maximum increment in the total tanshinone content was 1.2-fold by Ag + and 3.1-fold by YE (day 4) over the control Effects of elicitors on key enzymes of terpenoid biosynthetic pathways The activity of HMGR in hairy roots was only stimulated by Ag + but not affected by the yeast elicitor (Fig. 4). The HMGR activity rose rapidly to the peak level (80% higher) in 12 h (0.5 day) and then declined gradually to the control level on day 6 after Ag + treatment. DXS activity was enhanced by both elicitors, but more rapidly and dramatically by YE. The elicitor-induced increase of DXS activity was transient, reaching a peak within h (1 2 days) and coming off the peak in the next 1 2 days. At the peak level, the DXS activities of YE and Ag-treated cultures were respectively 12-fold and 8-fold higher than that of the control Effects of Ag + pretreatment on YE-induced tanshinone production and DXS activity The pretreatment of hairy root cultures with Ag + (30 mm) for 12, 24 or 48 h (0.5 2 days) prior to the addition of YE significantly enhanced YE-induced tanshinone accumulation and DXS activity (Fig. 5). The enhancing effect became more appreciable with the length of pretreatment period 3. Results 3.1. Effect of elicitors on tanshinone production Tanshinone accumulation was stimulated within 2 days after treatment, by both Ag + and YE, but more significantly Fig. 4. Effects of Ag + and yeast elicitor on HMGR and DXS activities of hairy roots. Elicitor dose and day of addition to culture were all the same as in Fig. 3.

4 490 X. Ge, J. Wu / Plant Science 168 (2005) Fig. 5. Effects of Ag + pretreatment on yeast elicitor-induced tanshinone production and DXS activity. Ag + (30 mm) was added to the hairy root cultures 12 h ( 12 h), 24 h ( 24 h) or 48 h ( 48 h) prior to the addition of YE (100 mgml 1 ), and measurements were all taken 2 days after YE addition. before the yeast elicitor treatment, as seen from the increasingly higher tanshinone content and DXS activity from Ag ( 12 h) + YE to Ag ( 48 h) + YE. The increasingly stronger effect with a longer pretreatment period suggests that sufficient time is needed for the cells to be primed for the elicitation of secondary metabolism responses by the yeast elicitor. Fig. 5 also shows the contents of three tanshinone species, T1, T2A and CT, which were all stimulated by various elicitor treatments, with CT being enhanced most dramatically by most treatments. Ag + pretreatment did not have any significant effect on HMGR activity as compared with those attained with YE treatment alone or Ag + YE at the same time (data not shown). In addition, most of the elicitor treatments had a notable negative effect on the growth of hairy root biomass (Fig. 6) Effects of pathway inhibitors on elicitor-induced tanshinone production The non-mva isoprenoid pathway inhibitor, fosmidomycin, nearly arrested tanshinone accumulation induced by both Ag + and YE, while the MVA pathway inhibitor, Fig. 6. Effects of various elicitor treatment schemes on the growth of hairy root biomass. All conditions were same as specified in Fig. 5. Fig. 7. Effects of MVA and non-mva pathway inhibitors, fosmidomycin (Fos, 100 mm) and mevinolin (Mev, 5 mm), on Ag- and YE-induced tanshinone production and root biomass growth (Ctrl, culture not treated by any elicitor). mevinolin, only partially inhibited the Ag- but not YEinduced tanshinone accumulation (Fig. 7). These inhibitors did not significantly depress the tanshinone content of the control culture and the biomass growth of elicitor-treated cultures. 4. Discussion Our experimental results show that tanshinone accumulation and related enzyme activities in S. miltiorrhiza hairy roots can be stimulated by both biotic and abiotic elicitors. The YE-induced tanshinone accumulation in the S. miltiorrhiza hairy roots appeared to be mainly derived from a non-mva pathway involving DXS since YE did not stimulate HMGR activity (Fig. 4). This perception is also supported by the effects of the two pathway inhibitors on YE-induced tanshinone accumulation, which was effectively blocked by the non-mva pathway inhibitor, fosmindomycin, but not by the MVA-pathway inhibitor, mevinolin (Fig. 7). On the other hand, Ag-induced tanshinone accumulation appeared to be dependent on both pathways because it was suppressed by the inhibitors of both pathways (Fig. 7), and Ag + stimulated the key enzymes of both pathways, DXS and HMGR (Fig. 4). Therefore, there may be crosstalk between the MVA and non-mva isoprenoid pathways in the S. miltiorrhiza roots, which influenced tanshinone biosynthesis induced by Ag +. It is worth noting that HMGR was not stimulated by the yeast elicitor. Similarly, the accumulation of sesquitepenes including rishitin and lubimins by Datura stramonium hairy roots was induced only by heavy metal ions Cu 2+ and Cd 2+ but not by cellulase and various fungal extracts [15]. This was in contrast to the induction and stimulation of sesquitepene accumulation in plant hairy root cultures by various biotic elicitors, such as rishitin and lubimin in

5 X. Ge, J. Wu / Plant Science 168 (2005) Hyoscyamus muticus [16] hairy root culture by a fungal elicitor. HMGR is the first key enzyme in the MVA pathway, which is responsible for the synthesis of many terpenoids such as sesquiterpenes and triterpenes (Fig. 1). Perhaps, the induction of HMGR activity by pathogen infection and elicitor treatment mainly occurs in plant species which resort to the MVA pathway for the biosynthesis of phytoalexins such as sesquiterpenes and triterpenes. For example, fungal elicitor-induced HMGR activity has been observed in potato tuber tissues [17] and rice cell cultures [18];inTaxus media cell cultures, however, three abiotic elicitors, methyl jasmonate (MeJA), arachidonic acid and vanadyl sulphate, enhanced the accumulation of diterpenoid taxanes but depressed intracellular HMGR activity [19]. The enhancement of YE-induced tanshinone accumulation as well as the related enzyme activity by Ag + pretreatment (Fig. 5) suggests that Ag + potentiated the biotic elicitor-induced tanshinone production of S. miltiorrhiza hairy roots. Earlier, MeJA and SA pretreatment augmented the defense responses in plant cells induced by fungal elicitors such as the oxidative burst and coumarin production in parsley cells [20 22]. These abiotic agents prime the plant cells to be more sensitive and responsive to elicitation, although the mechanism of priming is still not fully understood. In addition to plant responses to biotic elicitors and pathogens, priming may potentiate the responses of plant cells to abiotic stresses such as osmotic and temperature shocks [23]. However, there is still a lack of information about the priming of plant cells by biotic elicitors to potentiate the responses and secondary metabolism induced by an abiotic elicitor. Moreover, the potentiation has been mostly attained with the signal compounds such as MeJA, SA and their analogues in the elicitation of plant defense responses, and our present study is perhaps the first ever on the effect of a heavy metal ion. Acknowledgements This work was supported financially by the Hong Kong Polytechnic University (G-YD28 and ASD fund) and the Areas of Excellence Scheme established under the University Grants Committee of the Hong Kong SAR, China (AoE/B 10/2001 and AoE/P 10/2001). References [1] A. Giri, L. Narasu, Transformed hairy roots: recent trends and applications, Biotechnol. Adv. 18 (2000) [2] W. Tang, G. Eisenbrand, Chdrugs of Plant Origin: Chemistry, Pharmacology and Use in Traditional and Modern Medicine, Springer- Verlag, Berlin, 1992, pp [3] Z.B. Hu, A.W. Alfermann, Diterpenoid production in hairy root cultures of Salvia miltiorrhiza, Phytochemistry 2 (1993) [4] H.K. 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