ORIGINAL PAPER

Molecular mechanism of elicitor-induced tanshinoneaccumulation in Salvia miltiorrhiza hairy root cultures

Guoyin Kai • Pan Liao • Hui Xu • Jing Wang •

Congcong Zhou • Wei Zhou • Yaping Qi •

Jianbo Xiao • Yuliang Wang • Lin Zhang

Received: 28 June 2011 / Revised: 13 January 2012 / Accepted: 17 January 2012 / Published online: 3 February 2012

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012

Abstract To develop an optimal bioprocess for the pro-

duction of tanshinone which is mainly used for the treat-

ment of cardiocerebral vascular disease, the tanshinone

biosynthetic pathway regulation must be better understood.

In this paper, expression of tanshinone biosynthetic path-

way related genes as well as tanshinone accumulation in

Salvia miltiorrhiza hairy root cultures were investigated, in

response to biotic and abiotic elicitors, respectively. Our

results showed tanshinone accumulation in S. miltiorrhiza

hairy roots was highly regulated by the coordination of the

expression of several genes involved in tanshinone

biosynthesis pathway. Our results showed a positive cor-

relation between gene expression and tanshinone accumu-

lation, suggesting that tanshinone accumulation may be the

result of the coexpression up-regulation of several genes

involved in tanshinone biosynthesis under the treatment of

various elicitors. Meantime, SmHMGR, SmDXS2,

SmFPPS, SmGGPPS and SmCPS were identified as the

potential key enzymes in the pathway for targeted meta-

bolic engineering to increase accumulation of tanshinone in

S. miltiorrhiza hairy roots. This is the first report inte-

grating comprehensively the transcript and metabolite

biosynthesis of tanshinone in S. miltiorrhiza hairy roots.

Keywords Dan Shen � Bioactive compounds � Gene

expression � Metabolic engineering � Elicitors

Abbreviations

T2A Tanshinone IIA

CT Cryptotanshinone

BABA b-Aminobutyric acid

MJ Methyl jasmonate

YE Yeast extract

fw Fresh weight

SA Salicylic acid

DW Dry weight

RT Reverse transcriptase

AACT Acetyl-CoA C-acetyltransferase

HMGS 3-Hydroxy-3-methylglutaryl-CoA synthase

HMGR 3-Hydroxy-3-methylglutaryl-CoA reductase

MK Mevalonate kinase

PMK 5-Phosphomevalonate kinase

MDC Mevalonate 5-diphosphate decarboxylase

DXS 1-Deoxy-D-xylulose-5-phosphate synthase

DXR 1-Deoxy-D-xylulose-5-phosphate

reductoisomerase

MCT 2-C-methyl-D-erythritol-4-phosphate cytidyl

transferase

CMK 4-(cytidine 5-diphospho)-2-C-methyl-D-

erythritol kinase

Communicated by B. Borkowska.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11738-012-0940-z) contains supplementarymaterial, which is available to authorized users.

G. Kai (&) � P. Liao � H. Xu � J. Wang � C. Zhou � W. Zhou �Y. Qi � J. Xiao

Laboratory of Plant Biotechnology,

College of Life and Environment Sciences,

Shanghai Normal University, Shanghai 200234,

People’s Republic of China

e-mail: gykai@shnu.edu.cn

Y. Wang

Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,

School of Agriculture and Biology, Shanghai Jiaotong

University, Shanghai 200030, People’s Republic of China

L. Zhang

Department of Pharmacy,

Shaoxing People’s Hospital, Shaoxing 312000, People’s

Republic of China

123

Acta Physiol Plant (2012) 34:1421–1433

DOI 10.1007/s11738-012-0940-z

MECPS 2-C-methylerythritol 2,4-cyclodiphosphate

synthase

HDS 1-Hydroxy-2-methyl-2-(E)-butenyl-4-

diphosphate synthase

HDR 1-Hydroxy-2-methyl-2-(E)-butenyl-4-

diphosphate reductase

IPPI Isopentenyl-diphosphate delta-isomerase

GPPS Geranyl diphosphate synthase

FPPS Farnesyl diphosphate synthase

GGPPS Geranylgeranyl diphosphate synthase

CPS Copalyl diphosphate synthase

KSL Kaurene synthase-like

Introduction

Salvia miltiorrhiza Bunge (Fam. Lamiaceae) (also known

as Dan Shen in China) is a very important and well-known

traditional Chinese medicinal herb for treating many car-

diovascular diseases including inflammation, blood circu-

lation disturbance and menstrual disorders (Chen et al.

2005; Liao et al. 2009; Wu and Shi 2008). Tanshinones, as

one of the major bioactive components mainly in the root

of Dan Shen, are a group of abietane-type diterpenes

including tanshinone IIA (T2A) and cryptotanshinone

(CT), which share many clinical efficacy including antis-

chaemics, antioxidant, antiinflammation, antibacterial and

even antitumor properties (Chang and Chen 1991; Hu et al.

2005; Ji et al. 2008; Xu et al. 2010). Therefore, S. mil-

tiorrhiza has been widely used in clinical practice. For

example, Dan Shen products have been commercially sold

in Japan, the United States and European countries Fur-

thermore, China had the greatest use of Dan Shen, which

had a market that exceeded US$120 million in 2002 (Wu

and Shi 2008; Zhou et al. 2005). However, due to the low

content of tanshinone, huge consumption and long-growth

cycle of cultivated S. miltiorrhiza, tanshinone obtained

from traditional agricultural cultures can not meet the

rapidly increasing market need (Hu et al. 2005; Liao et al.

2009; Zhou et al. 2007). Therefore, it is clear that in the

foreseeable future, the supply of tanshinone will depend on

biotechnological methods.

Currently, as most of tanshinones are biosynthesized

from the root of Dan Shen, in vitro hairy root cultures

induced by Agrobacterium rhizogenes have been suggested

as a promising tool and an alternative way for large-scale

production of useful secondary metabolites. Furthermore,

the use of hairy root culture of S. miltiorrhiza to improve

tanshinone yield had been investigated by various kinds of

treatments such as biotic elicitor (yeast extract, YE) and

abiotic elicitors such as methyl jasmonate (MJ), Ag?, Co2?,

a-amino isobutyric acid, and b-aminobutyric acid (BABA),

and their results had been well documented recently (Ge

and Wu 2005a, b; Wang et al. 2007a, b; Yan et al. 2005,

2006; Zhao et al. 2010). Most of the previous works have

focused on the induction effect to the end product tanshi-

none under various elicitors in S. miltiorrhiza hairy roots,

while the molecular mechanism of tanshinone accumula-

tion induced by various elicitors is still unclear (Ge and Wu

2005a; Gao et al. 2009; Wang et al. 2008; Wu et al. 2009).

This, however, significantly relies on a comprehensive

understanding of the pathway for tanshinone biosynthesis,

the enzymes catalyzing the reaction chain, especially the

rate-limiting steps, and the genes encoding these proteins.

The tanshinone biosynthesis process is complex and not

fully characterized up to now. As a kind of diterpene,

tanshinone is biosynthesized from the central five-carbon

intermediate isopentenyl diphosphate (IPP) which is from

the mevalonate (MVA) pathway in cytoplasm, and

1-deoxy-D-xylulose phosphate (DXP) pathway in plastids,

for that there is crosstalk between the two pathways in the

S. miltiorrhiza (Ge and Wu 2005a; Jiang et al. 2006; Liao

et al. 2009). Several genes such as SmAACT, SmCMK,

SmIPPI, SmFPPS, SmCPS, SmKSL involved in biosyn-

thesis of tanshinone have been isolated by the team led by

Professor Luqi Huang (Gao et al. 2008, 2009; Wang et al.

2009). Recently, we cloned other genes including

SmHMGS, SmHMGR, SmDXR, SmDXS1, SmDXS2,

SmGGPPS (Kai et al. 2010; Liao et al. 2009; Yan et al.

2009) (Fig. 1). Although much progress has been made in

elicitor induction effect and gene cloning, there is limited

information about which genes are the key targets and

molecular regulation mechanism in the tanshinone bio-

synthesis pathway of S. miltiorrhiza under treatment of

different elicitors. Once the key targets are identified, the

targeted metabolic engineering strategies could be used to

improve tanshinone yield by transferring the key genes

involved in the tanshinone biosynthetic pathway into

S. miltiorrhiza alone or in combination with effective elicitors

in the near future (Nims et al. 2006; Wang and Wu 2010).

Biotic (YE) and abiotic elicitors (MJ and Ag?) were

used to examine the regulation of the tanshinone biosyn-

thesis pathway in S. miltiorrhiza hairy roots. The produc-

tion changes of tanshinone IIA and cryptotanshinone and

the expression profiles of all the known tanshinone bio-

synthesis related genes including SmAACT, SmHMGS,

SmHMGR, SmDXR, SmDXS1, SmDXS2, SmCMK, SmIPPI,

SmFPPS, SmGGPPS, SmCPS and SmKSL were evaluated

over time. This work should be useful for our further

understanding of molecular regulation mechanism of genes

encoding related enzymes involved in tanshinones bio-

synthesis and enhance tanshinones’ production in S. mil-

tiorrhiza hairy roots by metabolic engineering in the near

future.

1422 Acta Physiol Plant (2012) 34:1421–1433

123

Materials and methods

Hairy root culture

The S. miltiorrhiza hairy root culture was derived after

infection of S. miltiorrhiza leaves with the disarmed

A. tumefaciens strain C58C1 (received from Prof. Kexuan-

Tang’s laboratory, Shanghai Jiao Tong University, China)

which carries A. rhizogenes Ri plasmid pRiA4 in our lab-

oratory (Zhou et al. 2007). Stock culture of hairy roots was

maintained on 1/2 MS solid medium with 8 g l-1 agar and

30 g l-1 sucrose, at 25�C in the dark, details for getting

stock culture of hairy roots have been given in another

paper of our lab (Kai et al. 2011). The whole stages for

getting S. miltiorrhiza hairy root from S. miltiorrhiza leaves

can also be seen in Supplementary material 2. The pH of

the medium was adjusted to 5.8 prior to autoclaving at

121�C for 20 min. The experiments on the effects of

elicitors were all carried out in shake-flask cultures, with

250-ml Erlenmeyer flasks each containing 200 ml 1/2 MS

liquid medium on an orbital shaker controlled at 100 rpm

in darkness at 25�C. Each flask was inoculated with 3 g of

Diterpene tanshinones

Geranyl diphosphate (GPP)

Farnesyl diphosphate (FPP)

Geranylgeranyl diphosphate (GGPP)

Abietane-type diterpene (Miltiradiene)

Copalyl diphosphate (CPP)

GPPS

HDR

HDS

MECPS

MCT

Acetoacetyl-CoA

3R-Mevalonate acid (MVA)

Mevalonate acid-5-diphosphate

Mevalonate diphosphate

Dimethylally diphosphate (DMAPP)

AACT

HMGS

HMGR

1-Deoxy-D-xylulose 5-phosphate (DXP)

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate (cMEPP)

1-Hydroxy-2-methyl-2-(E)-buteny 4-diphosphate (HMBPP)

Isopentencyl diphosphate (IPP)

Monoterpene

Sesquiterpene

Polyterpenes

DXS

DXR

CMK

IPPI

GGPPS

CPS

FPPS

KSL

HDR

PMK

MDC

MK

Cytosol MVA pathway

Plastidial DXP pathway

2 Acetyl-CoA

Pyruvate + Glyceraldehyde 3-phosphate

3S-Hydroxy-3-methylglutary-CoA (HMGS-CoA)

2-C-Methyl-D-erythritol 4-phosphate (MEP)

4-(Cytidine 5’-diphospho)-2-C-methyl-D-erythritol (CDP-ME)

4-(Cytidine 5’-diphospho)-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P)

Fig. 1 The metabolic pathway

leading to tanshinone. AACT,

Acetyl-CoA

C-acetyltransferase; HMGS,

3-hydroxy-3-methylglutaryl-

CoA synthase; HMGR,

3-hydroxy-3-methylglutaryl-

CoA reductase; MK,

Mevalonate kinase; PMK,

5-phosphomevalonate kinase;

MDC, mevalonate

5-diphosphate decarboxylase;

DXS, 1-deoxy-D-xylulose-5-

phosphate synthase; DXR,

1-deoxy-D-xylulose-5-

phosphate reductoisomerase;

MCT, 2-C-methyl-D-erythritol-

4-phosphate cytidyl transferase;

CMK, 4-(cytidine

5-diphospho)-2-C-methyl-D-

erythritol kinase; MECPS, 2-C-

methylerythritol 2,4-

cyclodiphosphate synthase;

HDS, 1-hydroxy-2-methyl-2-

(E)-butenyl-4-diphosphate

synthase; HDR, 1-hydroxy-2-

methyl-2-(E)-butenyl-4-

diphosphate reductase; IPPI,

Isopentenyl-diphosphate delta-

isomerase; GPPS, Geranyl

diphosphate synthase; FPPS,

Farnesyl diphosphate synthase;

GGPPS, Geranylgeranyl

diphosphate synthase; CPS,

Copalyl diphosphate synthase;

KSL, Kaurene synthase-like. UP

to now, MK, PMK, MDC,

MCT, MECPS, HDS, HDR and

GPPS still have not been

isolated from S. miltiorrhiza

Acta Physiol Plant (2012) 34:1421–1433 1423

123

fresh weight (fw) of roots from 2-month-old shake-flask

cultures which were sub-cultured for three times (the liquid

1/2 MS media were changed every 20 days). Elicitor

treatments of the hairy root cultures were performed on day

18 after inoculation, and the hairy roots were harvested

from the culture medium at selected times [0 (before

treatment), 3, 6, 9 days after stress treatment for tanshinone

extraction and RNA isolation. All treatments were per-

formed in triplicate and the results of tanshinone contents

were represented by the mean ± standard deviation (SD).

Elicitors preparation

MJ was first dissolved in a small volume of DMSO, and

then in distilled water at 50 mM (Ge and Wu 2005b). Ag?

was supplied to the culture with a concentrated silver

thiosulfate (Ag2S2O3) solution prepared by mixing AgNO3

and Na2S2O3 at 1:4 molar ratio (Zhang et al. 2004). All the

solutions were filter sterilized through 0.22-lm filters, and

added to cultures to the desired final concentrations (Ge

and Wu 2005b). Yeast elicitors (YE) were purchased from

Bio Basic Inc. The yeast extract (25 g) was dissolved in

125 ml distilled water, and then 100 ml ethanol was added.

The solution was allowed to precipitate for 4 days at 4�C,

and the supernatant was decanted. The remaining gummy

precipitate was redissolved in 125 ml distilled water and

subjected to another round of ethanol precipitation. The

precipitate was dissolved in 100 ml distilled water and then

sterilized by autoclaving at 121�C for 20 min (Ge and Wu

2005b). After cooled at room temperature, the prepared YE

solution was stored at 4�C prior to use (Wang et al. 2007b).

Elicitor treatments were chosen at following concentrations

(MJ 0.1 mM, YE 100 mg l-1, Ag? 0.03 mM) as effective

points based on previous studies to produce the maximum

responses (Ge and Wu 2005a, b).

Extraction and determination of tanshinones

Hairy roots were harvested from shake-flask cultures,

washed three times with distilled water, blotted dry on a

paper towel, then dried at 45�C in an oven until reaching a

constant dry weight (DW). Tanshinones were extracted

according to the protocol of previous report (Ge and Wu

2005b) with some modification. Dry hairy roots (200 mg)

were ground with a pestle and mortar, soaked in 16 ml

100% methanol, powder of dry hairy roots in 100%

methanol was sonicated for 1 h, and then kept at room

temperature for 24 h. The extraction solvent (methanol)

was then evaporated under vacuum and the residue was

redissolved in 1.0 ml methanol. The solution was filtered

through a 0.22-lm filter followed by HPLC analysis.

HPLC analyses were performed on a Waters 600 liquid

chromatography (USA). Tanshinones were separated on a

C18 column (150 mm 9 4.6 mm; 5 lm; Waters, USA) at

30�C. The mobile phase consisted of methanol/water at

75:25 (v/v) and UV detection wavelength was 270 nm. The

flow rate was 1.0 ml min-1 and the injection volume was

10 ll (Lv et al. 2008; Yan et al. 2006). Two tanshinone

species, cryptotanshinone (CT) and tanshinone IIA (T2A),

were detected and quantified with authentic standards.

Tanshinone content shown in the results is the sum of CT

and T2A contents. Because tanshinones are lipophilic and

barely soluble in an aqueous solution, tanshinone content

(dissolved) in the medium was negligible and not deter-

mined (Wu and Shi 2008).

RNA preparation

Salvia miltiorrhiza hairy roots under various treatments at

selected points in time were used for RNA isolation. Total

RNA was isolated from the hairy root samples using RNA

prep pure plant kit (Tiangen Biotech Co., Ltd). DNase I

(Tiangen Biotech Co., Ltd) was used to remove all DNA

from the samples according to the manufacture’s instruc-

tions. The quality and concentration of the extracted RNA

were checked and stored as described before (Liao et al.

2009).

cDNA synthesis and RT-PCR

First strand cDNA was synthesized using reverse trans-

criptase (RT) (Takara, Japan) from 2 lg total RNA. For

each reaction, 1/50th of the RT reaction was used as the

template for PCR. Table 1 contains a list of primers of

S. miltiorrhiza genes used. Amplifications were performed

under the following condition: 94�C for 5 min followed by

35 cycles of amplification (94�C for 45 s, 58�C for 60 s

and 72�C for 2 min 30 s). While plant 18S rRNA gene

with the specific primers 18SF (50-CCAGGTCCAGACAT

AGTAAG-30) and 18SR (50-GTACAAAGGGCAGGGAC

GTA-30) was used to estimate whether equal amounts of

RNA among samples were used in semi-quantitative

RT-PCR under the following condition: 94�C for 5 min

followed by 22 cycles of amplification (94�C for 45 s,

58�C for 60 s and 72�C for 40 s).

Apparatus and reagents

Methyl jasmonate (MJ, 98%) was obtained from Sigma Co.

(Mo, USA). Silver nitrate (AgNO3, 99.9%) was purchased

from Shanghai Research Institute of Fine Chemical Tech-

nology (Shanghai, China). Sodium thiosulfate (Na2S2O3,

98%) was obtained from Shanghai Putuo Chemical

Industry Research Institute (Shanghai, China). Cryptotan-

shinone (CT) and tanshinone IIA (T2A) standards were

obtained from the Institute for Identification of

1424 Acta Physiol Plant (2012) 34:1421–1433

123

Pharmaceutical and Biological Products (Beijing, China).

All other reagents and solvents were of analytical grade

and used without further purification unless otherwise

noted. All aqueous solutions were prepared using newly

double-distilled water.

Experimental design and statistical analysis

Previous studies (Ge and Wu 2005a, b; Wang et al. 2007a;

Yan et al. 2006) showed that tanshinone accumulation in

S. miltiorrhiza hairy roots can be stimulated to a different

extent by MJ, YE, Ag? and YE–Ag? respectively. It is

believed that the elicitor inducing secondary metabolite

biosynthesis in plant cells or tissues requires the activation

of genes coded for the enzymes involved in secondary

metabolite biosynthetic pathways (Gao et al. 2009; Wu

et al. 2009). Therefore, in this study, what we are more

concerned with the expression profiles of genes in tanshi-

none biosynthetic pathway in hairy roots of S. miltiorrhiza

and the relationship between these genes and tanshinones

production under treatment of different elicitors.

All the experiments including semi-quantitative RT-

PCR, HPLC analysis were repeated three times. Results of

tanshinone content were presented as mean values ± SD.

Data were analyzed by the Students’s t test.

Results

Effect of elicitors on tanshinones (CT ? T2A)

production

In this work, the effects of the chemical elicitor MJ, biotic

elicitor YE and heavy metal ion (Ag?) on the yield of

tanshinones were investigated. CT and T2A were evaluated

on 0, 3, 6 and 9 days after the cultures were treated with

each elicitor and tanshinone content shown in the results is

the sum of CT and T2A in S. miltiorrhiza hairy roots

(Fig. 2; Table 2, Supplementary material 1).

Under MJ treatment, the accumulation of CT ? T2A

was gradually increased. As shown in Fig. 2a, tanshinone

accumulation enhanced after 3 days, with a maximum

value of 0.931 mg g-1 DW measured on day 9, about 5.78

times that of the control, 0.161 mg g-1 DW.

Under the treatment of Ag?, the accumulation of

CT ? T2A was also gradually increased but with lower

Table 1 List of S. miltiorrhiza genes and primer pairs used for RT-PCR

Gene GeneBank accession Primer name Primer sequence PCR product (bp)

AACT F635969 SmAACTKF 50-ATGGCACCAGAAGCTGCTTC-30 1,200

SmAACTKR 50-TCAAGACAAGGGCCGAGGCG-30

HMGS FJ785326 SmHMGSKF 50-AGATCTATGGCCAAGAATGTCGGGATCCT-30 1,396

SmHMGSKR 50-GGTCACCTCAGTGGCCGTTCGCAACTGTGC-30

HMGR EU680958 SmHMGRKF 50-AGATCTATGGATATCCGCCGGAGGC-30 1,711

SmHMGRKR 50-GGTGACCTCAGGAGCCAATCTTCGTG-30

DXS1 EU670744 SmDXS1KF 50-GGATCCATGGCTTTATGCCCATTTGCATT-30 2,157

SmDXS1KR 50-GAGCTCCTATGACATAATTTCCAGAGCCT-30

DXS2 FJ643618 SmDXS2KF 50-AGATCTATGGCGTCGTCTTGTGGAGTTAT-30 2,188

SmDXS2KR 50-GGTCACCTTACAAGTTGTTGATGAGATGAA-30

DXR DQ991431 SmDXRKF 50-ATGGCTCTAAACTTGATGTC-30 1,425

SmDXRKR 50-TCATACAAGAGCAGGACTCG-30

CMK EF534309 SmCMKKF 50-ATGGCTTCCTCTTCCTCCCA-30 1,191

SmCMKKR 50-CTATTCAACATCGCACGTCG-30

IPPI EF635967 SmIPPIKF 50-ATGTCGTCCTTGACCAGCAT-30 918

SmIPPIKR 50-CTAAGTCAGCTTGTGAATTG-30

FPPS EF635968 SmFPPSKF 50-ATGGCGAATCTGAACGGAGA-30 1,050

SmFPPSKR 50-TTATTTCTGCCTCTTGTATA-30

GGPPS FJ643617 SmGGPPSKF 50-AAGGATCCATGAGATCTATGAATCTGGT-30 1,111

SmGGPPSKR 50-CCGAGCTCTTAGTTCTGCCTATGTGCAA-30

CPS EU003997 SmCPSKF 50-ATGGCCTCCTTATCCTCTAC-30 2,382

SmCPSKR 50-TCACGCGACTGGCTCGAAAAG-3

KSL EF635966 SmKSLKF 50-ATGTCGCTCGCCTTCAACCC-30 1,788

SmKSLKR 50-TCATTTCCCTCTCACATTAT-30

Acta Physiol Plant (2012) 34:1421–1433 1425

123

levels than MJ cultures. Tanshinone accumulation enhanced

after 3 days, with a maximum value of 0.354 mg g-1 DW

measured on day 9, about 2.2 times that of the control,

0.161 mg g-1 DW (Fig. 2b).

The accumulation or content of CT ? T2A was also

induced by YE. Results showed that tanshinone accumu-

lation enhanced after 3 days, with a maximum value of

0.643 mg g-1 DW measured on day 6, about 3.99 times

that of the control, 0.161 mg g-1 DW, but then decreased

to 0.338 mg g-1 DW on day 9 (Fig. 2c).

The combination of YE and Ag? cultures showed the

greatest accumulation of CT ? T2A. As shown in

Figs. 2d, 3, tanshinone accumulation enhanced after

3 days, with a maximum value of 2.08 mg g-1 DW mea-

sured on day 9, which was about 12.92-fold over the

control.

Effects of MJ elicitor on the tanshinone biosynthetic

pathway

In order to investigate the expression profiles of genes

in tanshinone biosynthetic pathway in hairy roots of

S. miltiorrhiza under MJ treatment, a variety of gene

transcripts were analyzed by RT-PCR from the tanshinone

biosynthetic pathway (Table 1). The results showed that

the expressions of most investigated genes were up-reg-

ulated by MJ. As shown in Fig. 4, mRNA levels of

SmAACT, SmHMGS, SmHMGR, SmDXR, SmDXS2,

SmGGPPS, SmIPPI and SmCPS, reached the highest level

on day 3, then showed different expression profiles in

varying degrees on days 6 and 9; mRNA levels of

SmCMK reached the highest level on day 6, and then

decreased on day 9; mRNA levels of SmFPPS on day 3

and 6 were steady similar to the control (day 0), and

increased on day 9; but transcript expressions of SmDXS1

could not be detected at all the tested points in time;

mRNA levels of SmKSL could not be detected on days 0,

and 3, but dramatically increased on day 6 and reached

the highest level on day 9. These results revealed that

most of investigated genes in the tanshinone biosynthetic

pathway in hairy roots of S. miltiorrhiza were responsive

to MJ elicitor and could be effectively elicited at least at

transcription level, coinciding with the MJ induction

effect for improving the tanshinones’ production on day

3. It is interesting to note that the expression of tanshi-

none biosynthesis related genes (SmHMGR-M6, SmDXR-

M9, SmIPPI-M9, SmCPS-M9) is down-regulated (Fig. 4),

even though tanshinone accumulation is gradually

increased after 3, 6 and 9 days under the treatment of MJ

(Fig. 2a), suggesting that enzyme activities of SmHMGR,

SmDXR, SmIPPI and SmCPS may persist long after their

cognate mRNAs are decrease or almost absent from the

hairy roots, similar results were also reported by Gao

et al. (2009) and Nims et al. (2006).

0

0.5

1

1.5

2

mg·

g-1

(DW

)

MJ treatment time (days)

Tanshinone content

0

0.5

1

1.5

2

mg·

g-1

(DW

)

Ag+ treatment time (days)

Tanshinone content

0

0.5

1

1.5

2

mg·

g-1

(DW

)

YE treatment time (days)

Tanshinone content

0

0.5

1

1.5

2

0 3 6 9 0 3 6 9

0 3 6 9 0 3 6 9

mg·

g-1

(DW

)

YE-Ag treatment time (days)

Tanshinone content

+

a b

c d

Fig. 2 Time courses of

tanshinone (CT ? T2A) content

in S. miltiorrhiza hairy roots

after treatment with a MJ,

b Ag?, c YE, d YE–Ag?

respectively. Values are means

of triplicate results and errorbars represent standard

deviation, n = 3. *P \ 0.05;

**P \ 0.01

1426 Acta Physiol Plant (2012) 34:1421–1433

123

Effects of Ag? elicitor on the tanshinone biosynthetic

pathway

As shown in Fig. 5, under Ag? treatment, mRNA levels of

SmAACT gradually decreased on days 3, and 6, and slightly

increased on day 9 compared with the control (0 day);

mRNA levels of SmHMGS gradually decreased on days 3,

6, 9; mRNA levels of SmHMGR, SmDXS2, SmIPPI,

SmGGPPS reached the highest level on day 6, and then

decreased on day 9; mRNA levels of SmDXR, SmCMK did

not change on days 3 and 6 compared with the control, and

then decreased on day 9; mRNA levels of SmFPPS grad-

ually increased and reached the highest level on day 9;

transcript expressions of SmDXS1 and SmKSL could not be

detected at all the tested points in time; mRNA levels of

SmCPS reached the highest level on day 3, and decreased

thereafter. These results showed that the expressions of

SmHMGR, SmDXS2, SmIPPI, SmFPPS, SmGGPPS,

SmCPS were induced by Ag?, coincided with their

induction effects for improving the tanshinones’ produc-

tion, implying that the activation of these six genes coding

for the respective six enzymes contribute to the improve-

ment of the tanshinones’ production under the treatment of

Ag?. These six genes may play important roles in the

tanshinones’ biosynthetic pathway under the treatment of

Ag?.

0.00

0.05

0.10

0.15

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

c

Elution time (min)

AU

1

2

0.00

0.02

0.04

0.06

0.08

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

10.4

76

17.0

81

Elution time (min)

a

AU

2

1

0.00

0.02

0.04

0.06

0.08

0.10

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

b

Elution time (min)

AU

12

Fig. 3 HPLC chromatograms

of tanshinones from a mixture

of authentic standards of CT (1)

and T2A (2), b control hairy

root cultures, and c YE–Ag?

treated hairy root cultures on

day 9 (tanshinones peaks, 1 for

CT, 2 for T2A)

Acta Physiol Plant (2012) 34:1421–1433 1427

123

Effects of YE elicitor on the tanshinone biosynthetic

pathway

In this work, YE elicitor was also used to investigate

expression profiles of genes in the tanshinone biosynthetic

pathway in hairy roots of S. miltiorrhiza. As shown in Fig. 6,

mRNA levels of SmAACT, SmHMGS, SmDXR, SmDXS2,

SmCMK, SmIPPI, SmFPPS, SmGGPPS and SmCPS were

stimulated by YE at different points in time and in varying

degrees respectively. And mRNA levels of SmHMGR

declined on day 3, but increased to the similar level of the

control on day 6, and then declined to undetectable levels by

day 9; transcript expressions of SmDXS1 and SmKSL also

could not be detected at all the tested points in time. These

results showed that the expressions of SmAACT, SmHMGS,

SmDXR, SmDXS2, SmCMK, SmIPPI, SmFPPS, SmGGPPS,

SmCPS were responsive to YE, and the expression profiles of

some genes such as SmHMGS, SmDXR, SmDXS2, SmCMK,

SmIPPI, SmCPS under YE treatment were consistent with the

YE induction effects for stimulating the tanshinones’ pro-

duction. Hence, co-activation of these six genes (SmHMGS,

SmDXR, SmDXS2, SmCMK, SmIPPI, SmCPS) coding for the

respective six enzymes dramatically improved the tanshi-

nones’ production under the treatment of YE.

Combined effects of YE elicitor and Ag? elicitor

on the tanshinone biosynthetic pathway

This work and previous studies all came to the conclusion

that the elicitation by combination of a biotic elicitor (YE)

and an abiotic elicitor (Ag?) can generate a synergistic

effect, which is more effective than single elicitors to pro-

mote tanshinone production in S. miltiorrhiza hairy root

cultures (Wang et al. 2007b; Yan et al. 2006). But its

functional mechanism is still unknown. Therefore, it’s

worth investigating the expression profiles of genes

involved in tanshinone biosynthesis induced by YE–Ag?,

which will be helpful to uncover molecular induction

mechanisms treated by YE–Ag?. Here, YE–Ag? were also

used to further investigate the expression profiles of genes

in the tanshinone biosynthetic pathway in hairy roots of

S. miltiorrhiza. As shown in Fig. 7, under YE–Ag? treatment,

mRNA levels of SmAACT, SmHMGS, SmDXR, SmDXS2,

SmCMK, SmFPPS, SmGGPPS, and SmCPS were stimu-

lated (most of them reached the highest level on day 6) in

varying degrees respectively; mRNA levels of SmHMGR

dramatically decreased on day 3, but rapidly increased to

the similar levels as the control on days 6, and 9; transcript

Fig. 4 Effects of MJ on the expression of related genes in the

tanshinone biosynthetic pathway during S.miltiorrhiza hairy roots

culture period. 18S rRNA gene was used as the control to show the

normalization of the templates in PCR. The experiment was repeated

three times. M represents for MJ

Fig. 5 Effects of Ag? on the expression of related genes in the

tanshinone biosynthetic pathway during S. miltiorrhiza hairy roots

culture period. 18S rRNA gene was used as the control to show the

normalization of the templates in PCR. The experiment was repeated

three times. A represents for Ag?

1428 Acta Physiol Plant (2012) 34:1421–1433

123

expressions of SmDXS1 and SmKSL were also could not be

detected at all the tested points in time; mRNA levels of

SmIPPI slightly decreased on days 3, and 6, but rapidly

increased to the similar level as the control. These results

showed that the expressions of SmAACT, SmHMGS,

SmDXR, SmDXS2, SmCMK, SmFPPS, SmGGPPS and

SmCPS were induced by YE–Ag?. This coincided with

their induction effects for improving the tanshinones’ pro-

duction. At the same time, the mRNA of SmHMGR, SmIPPI

on days 6, and 9 constantly maintained as high of levels as

the control and/or even higher than the control also con-

tribute to the improvement in the production of tanshinones.

Therefore, the co-activation of eight genes (SmAACT,

SmHMGS, SmDXR, SmDXS2, SmCMK, SmFPPS,

SmGGPPS, SmCPS) coding for the respective eight

enzymes together with constantly high mRNA levels of

SmHMGR and SmIPPI further dramatically improved the

tanshinones’ production under the treatment of YE–Ag?.

Discussion

In a word, both of our experimental results and other pre-

vious studies (Ge and Wu 2005a, b; Wang et al. 2007a;

Yan et al. 2006) showed that tanshinone accumulation in

S. miltiorrhiza hairy roots can be stimulated in different

degrees by MJ, YE, Ag? and YE–Ag?, respectively. At the

same time, our experimental results comprehensively

revealed that tanshinone accumulation in S. miltiorrhiza

hairy roots under various elicitors may be the result of

expressions increased in many genes involving tanshinone

biosynthesis under treatment of various elicitors for the

first time. Under the treatment of MJ, tanshinone accu-

mulation reached a maximum value day 9 (Fig. 2a), it may

result from MJ induction of most of the test genes

including SmAACT, SmHMGS, SmHMGR, SmDXR,

SmDXS2, SmCMK, SmIPPI, SmGGPPS, SmCPS (Fig. 4).

Under the treatment of Ag?, tanshinone accumulation

enhanced after 3 days (Fig. 2b). Accordingly, mRNA

expressions of six genes in tanshinone biosynthetic path-

way including SmHMGR, SmDXS2, SmIPPI, SmFPPS,

SmGGPPS and SmCPS were induced by Ag? (Fig. 5). This

implies that these six genes may play more important roles

in improving tanshinones’ production under the treatment

of Ag?. Under the treatment of YE, tanshinone accumu-

lation enhanced after 3 days (Fig. 2c), and mRNA

expressions of six genes in the tanshinone biosynthetic

pathway including SmHMGS, SmDXR, SmDXS2, SmCMK,

Fig. 6 Effects of YE on the expression of related genes in the

tanshinone biosynthetic pathway during S. miltiorrhiza hairy roots

culture period. 18S rRNA gene was used as the control to show the

normalization of the templates in PCR. The experiment was repeated

three times. Y represents for YE

Fig. 7 Effects of combination of YE and Ag? on the expression of

related genes in the tanshinone biosynthetic pathway during

S. miltiorrhiza hairy roots culture period. 18S rRNA gene was used

as the control to show the normalization of the templates in PCR. The

experiment was repeated three times. YA represents for YE–Ag?

Acta Physiol Plant (2012) 34:1421–1433 1429

123

SmIPPI and SmCPS were induced by YE at corresponding

points in time (Fig. 6), implying that these six genes may

play more important roles in improving the production of

tanshinones under the treatment of YE. Under the com-

bined treatment of YE and Ag?, tanshinone accumulation

enhanced after 3 days, with a maximum value on day 9

(Fig. 2d), showed the best induced effect to improve tan-

shinone production, and mRNA expressions of eight genes

in the tanshinone biosynthetic pathway including SmAACT,

SmHMGS, SmDXR, SmDXS2, SmCMK, SmFPPS,

SmGGPPS and SmCPS were induced by YE–Ag? (Fig. 7),

implying that there exists a direct relationship between

expressions of these eight genes and tanshinone accumu-

lation stimulated by YE–Ag?. These eight genes may play

more important roles in improving tanshinones’ production

under the treatment of YE–Ag?.

Tanshinone accumulation was stimulated in different

degrees under treatment of MJ, Ag?, YE, YE–Ag? in

S. miltiorrhiza hairy roots. At the same time, the results of

RT-PCR (Figs. 4 MO, 5 A0, 6 Y0, 7 YA0) showed that

mRNA expression levels of different genes in the tanshi-

none biosynthetic pathway are various. In un-induced

S. miltiorrhiza hairy roots, transcript expressions of SmDXS1

and SmKSL could not be detected, and mRNA expression

levels of SmHMGR, SmDXS2, SmFPPS, SmGGPPS and

SmCPS were much lower than that of SmAACT, SmHMGS,

SmDXR, SmCMK and SmIPPI. These results implied that

SmHMGR, SmDXS2, SmFPPS, SmGGPPS and SmCPS

may serve as rate -limiting genes in tanshinone biosyn-

thesis because of their low mRNA expression in un-

induced S. miltiorrhiza hairy roots. This perception was

also supported by the effects of induction by both biotic

and abiotic elicitors. As shown in Figs. 4, 5 mRNA

expression levels of SmHMGR, SmDXS2, SmFPPS,

SmGGPPS and SmCPS were dramatically increased under

the treatment of MJ and Ag? respectively. The mRNA

expression levels of SmDXS2, SmFPPS, and SmCPS were

also dramatically increased under the treatment of YE, and

mRNA expression levels of SmHMGR and SmGGPPS on

days 3 and 6 still remain steady, similar to the control

under the treatment of YE (Fig. 6). What’s more, under the

treatment of YE–Ag?, mRNA expression levels of

SmDXS2, SmFPPS, SmGGPPS and SmCPS were also

dramatically increased (Fig. 7). But the timing and extent

of this up-regulation varies for each individual gene.

Therefore, these results demonstrated that SmHMGR,

SmDXS2, SmFPPS, SmGGPPS and SmCPS may be more

effective or play more important roles in enhancing tan-

shinone accumulation in S. miltiorrhiza hairy roots, and

these five genes may function as key rate-limiting genes in

the tanshinone biosynthetic pathway.

HMGR, which catalyzes the conversion of 3-hydroxy-

methylglutaryl-CoA (HMG-CoA) to mevalonate, has been

considered as the first key step in the MVA pathway in

plants (Chapell 1995; Ha et al. 2003). Previous study also

reported that there is crosstalk between MVA and DXP

pathways for tanshinone biosynthesis in S. miltiorrhiza

hairy roots (Ge and Wu 2005a). Our previous study

revealed that SmHMGR was expressed highest in the roots,

followed by stems and leaves and the expression of

SmHMGR could be up-regulated by MJ in different tissues

of S. miltiorrhiza including roots, stems and leaves (Liao

et al. 2009). Our results here demonstrated that there exists

a tight relationship between expressions of SmHMGR

(Figs. 4, 5) and tanshinone accumulation (Fig. 2a, b) not

only under the treatment of MJ, but also under the treat-

ment of Ag? in S. miltiorrhiza hairy roots. Therefore,

SmHMGR is most likely one of the key genes in the tan-

shinone biosynthetic pathway in S. miltiorrhiza hairy roots.

DXS catalyze the first-limiting step in the DXP bio-

synthetic pathway (Estevez et al. 2001). Over-expression of

DXS in Arabidopsis thaliana and Lavandula latifolia dra-

matically enhanced terpenoid production respectively

(Estevez et al. 2001; Lois et al. 2000; Munoz-Bertomeu

et al. 2006), confirming that DXS is one of the key regu-

latory targets for terpenoid metabolism. Here, it’s worth

noting that transcript expressions of SmDXS1 could not be

detected during all the test points in time under the treat-

ment of any elicitors including MJ, Ag?, YE and YE–Ag?

(Figs. 4, 5, 6, 7). However, mRNA expression of SmDXS2

could be detected in untreated S. miltiorrhiza hairy roots

and could be dramatically induced by MJ, Ag?, YE and

YE–Ag? respectively. Both situations of the mRNA

expression of SmDXS2 in untreated S. miltiorrhiza hairy

roots and mRNA expression changes of SmDXS2 in

S. miltiorrhiza hairy roots under treatment of MJ, Ag?, YE

and YE–Ag?, respectively, showed a direct relationship

between the content of tanshinone and SmDXS2 expression

(Figs. 2, 3, 4, 5, 6, 7). These results suggest that SmDXS1,

SmDXS2 may play different roles in S. miltiorrhiza.

SmDXS2 may play an important role for the secondary

metabolism production of diterpenoid tanshinones in

S. miltiorrhiza hairy roots, while SmDXS1 may be involved

in other processes like photosynthesis and/or primary

metabolism. Results of sequence alignment, evolutional

analysis and expression pattern analysis in different

S. miltiorrhiza tissues including roots, stems and leaves of

SmDXS1/SmDXS1 and SmDXS2/SmDXS2 in our experi-

ments could also come to the same conclusion (unpub-

lished data). Similar results were also found in Medicago

truncatula DXSs (named as MtDXS1, MtDXS2) (Floß et al.

2008). Therefore, based on our results and other previous

reports, SmDXS1 may functions as a housekeeping gene

in S.miltiorrhiza and SmDXS2 may play an important role

in the biosynthesis of secondary diterpenoid tanshinones in

S. miltiorrhiza hairy roots.

1430 Acta Physiol Plant (2012) 34:1421–1433

123

DXR catalyzes the second step in the DXP biosynthetic

pathway, which converts DXP to MEP. This has been

noted to play an important role in regulating the MEP

pathway (Lois et al. 2000). DXR-over-expressing lines of

Arabidopsis showed an increased accumulation of MEP-

derived plastid isoprenoids such as chlorophylls and

carotenoids (Carretero-Paulet et al. 2006). The function of

SmDXR was also complemented in Escherichia coli by our

previous study and another study (Wu et al. 2009; Yan

et al. 2009). What’s more, the positive relationship between

mRNA expression of SmDXR and tanshinone accumulation

after exposure to hyperosmotic stress and YE was con-

firmed in S. miltiorrhiza hairy roots (Wu et al. 2009). In

this work, transcription levels of SmDXR were also

observed to be up-regulated after exposure to YE in par-

allel with increased tanshinone accumulation in S. mil-

tiorrhiza hairy roots (Figs. 2c, 6). The above studies

suggest that SmDXR may also be a target for the exploi-

tation of a metabolic engineering approach to manipulating

tanshinone biosynthesis in S. miltiorrhiza hairy roots

(Carretero-Paulet et al. 2006; Wu et al. 2009; Yan et al.

2009). To our surprise, transcription levels of SmDXR were

just slightly induced under the treatment of YE and YE–

Ag? respectively, expression of SmDXR were not induced

by Ag?, while tanshinone accumulation was dramatically

stimulated under the treatment of YE, Ag? and YE–Ag?

respectively. Furthermore, mRNA expression levels of

SmDXR were very high and were much higher than that of

SmHMGR, SmDXS2, SmFPPS, SmGGPPS and SmCPS in

un-induced S. miltiorrhiza hairy roots (Figs. 4 MO, 5 A0, 6

Y0, 7 YA0), implying that there is a sufficient quantity of

SmDXR in un-induced S. miltiorrhiza hairy roots for the

synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP),

while enough production of MEP is necessary for the

production of diterpenoid tanshinones in S. miltiorrhiza

hairy root cultures. Therefore, these results suggest that

SmDXR was involved in the tanshinone biosynthesis but

may not be a suitable target for higher production of tan-

shinone in S. miltiorrhiza hairy roots.

The condensation reaction catalyzed by GGPPS is an

important branch point for the transformation from basic

terpenoid precursor to universal diterpenoid precursor

(Engprasert et al. 2004). There exists a positive relationship

between expression levels of Taxus canadensis GGPPS and

diterpenoid taxol accumulation in T. canadensis suggests

that GGPPS is an important target for metabolic regulation

in diterpenoid biosynthesis (Hefner et al. 1998). Our pre-

vious work demonstrated that SmGGPPS encoded a func-

tional protein and played an important role in promoting

carotenoid pathway flux by color complementation assay in

E. coli (Kai et al. 2010). Furthermore, results here show

that mRNA expression of SmGGPPS was dramatically

induced by MJ, Ag? and YE–Ag? respectively (Figs. 4, 5,

7), suggesting there exists a tight relationship between

expressions of SmGGPPS and tanshinone accumulation in

S. miltiorrhiza hairy roots (Fig. 2a, b, d. So SmGGPPS was

also identified as one of the key enzymes in biosynthesis of

tanshinone.

Recently, SmCPS has been isolated and confirmed to be

the first enzyme in the late specific tanshinone biosynthesis

pathway, which cyclization of GGPP to form normal

copalyl diphosphate (CPP) (Gao et al. 2009). Transcrip-

tional expression of SmCPS was found to have a positive

relationship with tanshinone accumulation not only under

the treatment of MJ, but also under the treatment of YE–

Ag? in S. miltiorrhiza hairy roots (Gao et al. 2009). Our

work here also shows the similar results under the treat-

ment of MJ and YE–Ag? respectively (Figs. 2a, d, 4, 7).

Moreover, transcription levels of SmCPS were also

observed to be dramatically up-regulated after exposure to

Ag? and YE respectively (Figs. 2b, c, 5, 6) in parallel with

increased tanshinone accumulation in S. miltiorrhiza hairy

roots in this work. Importantly, mRNA levels of SmCPS in

un-induced S. miltiorrhiza hairy roots were very low

among all the tested genes here (Figs. 4 MO, 5 A0, 6 Y0, 7

YA0). Therefore, SmCPS was also considered as a key

rate-limiting enzyme involved in tanshinone biosynthesis

in S. miltiorrhiza hairy roots.

At the same time, SmKSL (kaurene synthase-like) has

also been isolated from S. miltiorrhiza hairy roots by

functional genomics-based approach, which identified as

another diterpene synthase further cyclization and rear-

rangement of normal CPP to form an abietane-type diter-

pene named miltiradiene (Gao et al. 2009). Furthermore,

MJ and YE–Ag? were found to increase both the mRNA

levels of SmKSL and, subsequently, tanshinone IIA in

S. miltiorrhiza hairy roots (Gao et al. 2009). Our results here

also showed that SmKSL can be induced by MJ, mRNA

levels of SmKSL could not be detected on days 0, and 3, but

dramatically increased on day 6 and reached the highest

level on day 9. However, the expression of SmKSL could not

be detected at all the test points in time under the treatment

of other elicitors including YE, Ag? and YE–Ag? respec-

tively, suggesting that SmKSL may not induced by YE, Ag?

and YE–Ag? respectively or the mRNA levels of SmKSL

were too low to be detected. The expression profile of

SmKSL under the treatment of YE–Ag? is inconsistent with

that in previous report (Gao et al. 2009). This inconsistency

may be because of a difference of the bacterial strain used

for transformation and the status of analyzed root line.

Further experiments need to be carried out to further

understand the role of SmKSL in the late specific tanshinone

biosynthetic pathway.

According to the results in this work, tanshinone accu-

mulation was dramatically stimulated by MJ (Fig. 2a), and

almost all the tested genes encoding related enzymes

Acta Physiol Plant (2012) 34:1421–1433 1431

123

involved in tanshinone biosynthesis were coordinately

induced by MJ (Fig. 4), providing direct molecular evi-

dence for improving tanshinone content by MJ. ORCA-

similar transcript factors may exist in S. miltiorrhiza for

global control of several tanshinone biosynthetic genes, as

illustrated for terpenoid indole alkaloids (TIA) in Catha-

ranthus roseus (Van der Fits and Memelink 2000). This

current work leads us in a direction where it will be pos-

sible to clone upstream regulatory regions of the MJ-

induced genes. Then we will be able to analyze, identify

the cis-acting elements and trans-acting factors of pro-

moters respectively, and find the transcription factors

which may play important roles (global control several

tanshinones’ biosynthetic genes) in promoting tanshinone

accumulation induced by MJ.

It’s necessary to note that most of the genes tested in this

work are involved in the early tanshinone biosynthesis

pathway except SmCPS and SmKSL. Unfortunately, the late

specific tanshinone biosynthetic pathway after SmKSL is

still unknown. It’s certain that there are several unknown

genes involved in the late specific tanshinone biosynthetic

pathway also play more important roles in tanshinone

biosynthesis and may also be induced by biotic (YE) and

abiotic elicitors (MJ and Ag?). Additionally, there may be

more unknown genes involved in the late specific tanshi-

none biosynthetic pathway be induced by YE–Ag? than by

single elicitors like YE or Ag? respectively. So it is

important to clone and identify the unknown genes

involved in the late specific tanshinone biosynthesis path-

way for better understanding the whole tanshinone bio-

synthetic pathway and further uncover the functional

mechanism why combination of a biotic elicitor (YE) and

an abiotic elicitor (Ag?) can generate a synergistic effect to

promote tanshinone production in S. miltiorrhiza hairy root

cultures. More work needs to be done to find the unknown

genes involved in the late specific tanshinone biosynthesis

pathway with the technologies, such as mRNA differential

display, 2D-electrophoresis, cDNA microarray (Cui et al.

2011) etc.

Conclusion

In conclusion, this current work demonstrated that regu-

lation of the tanshinone biosynthetic pathway occurs at the

level of mRNA and that there is a tight correlation between

steady-state transcript abundance and respective tanshinone

accumulation. This comprehensively revealed that the

enhancement of tanshinone accumulation under various

elicitors may be the result of co-expressions up-regulation

of several genes involving tanshinone biosynthesis under

the treatment of various elicitors. Several pathway bottle-

necks to be targeted for metabolic engineering that can

potentially increase tanshinone accumulation in the near

future have been identified. Additionally, this work shows

the need to discover the unknown MJ-induced regulatory

elements and unknown genes involved in the late specific

tanshinone biosynthesis pathway for further understanding

the whole tanshinone biosynthesis pathway and further

increasing tanshinone content in S. miltiorrhiza in the near

future.

Author contribution GYK designed the study, analyzed

data and revised the manuscript. PL performed the exper-

iments, analyzed the data and drafts the manuscript. HX

and JW participated in RNA extracting and gene expres-

sion analysis. CCZ and WZ helped to culture hairy roots.

YPQ drafted part of the manuscript and revised the man-

uscript. YLW, JBX and LZ helped extracting tanshinone

and analyzing the content of tanshinone. All authors have

read and approved the final manuscript.

Acknowledgments This work was supported by National Natural

Science Fund (30900110), Shanghai Science and Technology Com-

mittee Project (10JC1412000, 09QH1401900, 06QA14038,

08391911800, 073158202, 075405117, 065458022, 05ZR14093),

Project from Ministry of Science and Technology of China

(NC2010AE0075, NC2010AE0372), Zhejiang Provincial Natural

Science Fund (Y2080621), Shanghai Education Committee Fund

(09ZZ138, 06DZ015, J50401), Fujian Science and Technology

Committee Key Special Project (2008NZ0001-4), National Trans-

genic Organism New Variety Culture Key Project (2009ZX08012-

002B), Project from Shanghai Normal University (SK201230,

SK201236, SK200830). We gratefully thank Kyle Andrew Schneider

(University of Dayton, Ohio, USA) for grammatical correction of the

manuscript.

References

Carretero-Paulet L, Cairo A, Botella-Pavıa P, Besumbes O, Campos

N, Boronat A, Rodrıguez-Concepcion M (2006) Enhanced flux

through the methylerythritol 4-phosphate pathway in Arabidop-sis plants overexpressing deoxyxylulose 5-phosphate reducto-

isomerase. Plant Mol Biol 62:683–695

Chang WL, Chen CF (1991) Cytotoxic activities of tanshinone

against human carcinoma cell line. Am J China Med 19:207

Chapell J (1995) Biochemistry and molecular biology of the

isoprenoid biosynthesis pathway in plants. Annu Rev Plant

Physiol Plant Mol 46:521–547

Chen AJ, Li CH, Gao WH, Hua ZD, Chen XG (2005) Application of

non-aqueous micellar electrokinetic chromatography to the

analysis of active components in Radix Salvia miltiorrhiza and

its medicinal preparations. J Pharm Biomed 37:811–814

Cui GH, Huang LQ, Tang XJ, Zhao JX (2011) Candidate genes

involved in tanshinone biosynthesis in hairy roots of Salviamiltiorrhiza revealed by cDNA microarray. Mol Biol Rep

38:2471–2478

Engprasert S, Taura F, Kawamukai M, Shoyama Y (2004) Molecular

cloning and functional expression of geranylgeranyl pyrophos-

phate synthase from Coleus forskohlii Briq. BMC Plant Biol

4:1–8

Estevez JM, Cantero A, Reindl A, Reichler S, Leon P (2001) 1-deoxy-

D-xylulose-5-phosphate synthase, a limiting enzyme for

1432 Acta Physiol Plant (2012) 34:1421–1433

123

plastidic isoprenoid biosynthesis in plants. J Biol Chem

276:22901–22909

Floß DS, Hause B, Lange PR, Kuster H, Strack D, Walter MH (2008)

Knock-down of the MEP pathway isogene 1-deoxy-D-xylulose

5-phosphate synthase 2 inhibits formation of arbuscular mycor-

rhiza-induced apocarotenoids, and abolishes normal expression

of mycorrhiza-specific plant marker genes. Plant J 56:86–100

Gao W, Cui GH, Kong JQ, Cheng KD, Wang W, Yuan Y, Huang LQ

(2008) Optimizing expression and purification of recombinant

Salvia miltiorrhiza copalyl diphosphate synthase protein in

E. coli and preparation of rabbit antiserum against SmCPS. Acta

Pharm Sinica 43:766–772

Gao W, Hillwig ML, Huang LQ, Cui GH, Wang XY, Kong JQ, Yang

B, Peters RJ (2009) A functional genomics approach to

tanshinone biosynthesis provides stereochemical insights. Org

Lett 11:5170–5173

Ge XC, Wu JY (2005a) Tanshinone production and isoprenoid

pathways in Salvia miltiorrhiza hairy roots induced by Ag? and

yeast elicitor. Plant Sci 168:487–491

Ge XC, Wu JY (2005b) Induction and potentiation of diterpenoid

tanshinone accumulation in Salvia miltiorrhiza hairy roots by b-

aminobutyric acid. Appl Microbiol Biotechnol 68:183–188

Ha SH, Kim JB, Hwang YS, Lee SW (2003) Molecular character-

ization of three 3-hydroxy-3-methylglutaryl-CoA reductase

genes including pathogen-induced Hmg2 from pepper (Capsi-cum annuum). Biochim Biophys Acta 1625:253–260

Hefner J, Ketchum REB, Croteau R (1998) Cloning and functional

expression of a cDNA encoding geranylgeranyl diphosphate

synthase from Taxus canadensis and assessment of the role of

this prenyltransferase in cells induced for taxol production. Arch

Biochem Biophys 360:62–74

Hu P, Luo GA, Zhao ZZ, Jiang ZH (2005) Quality Assessment of

Radix Salvia Miltiorrhiza. Chem Pharm Bull 53:481–484

Ji YL, He JT, Zhou QH, Chen J, Li Q, Zhu J, Liu HY (2008) The

inhibiting effect and its molecular mechanism of Tanshinone on

human lung cancer cell line in vitro. Chin J Lung Cancer

11:202–205

Jiang JH, Kai GY, Cao XY, Chen FM, He DN, Liu Q (2006)

Molecular cloning of a HMG-CoA reductase gene from

Eucommia ulmoides Oliver. Biosci Rep 26:171–181

Kai GY, Liao P, Zhang T, Zhou W, Wang J, Xu H, Liu YY, Zhang L

(2010) Characterization, expression profiling and functional

identification of a gene encoding geranylgeranyl diphosphate

synthase from Salvia miltiorrhiza. Biotechnol Bioproc E

15:236–245

Kai GY, Xu H, Zhou C, Liao P, Xiao JB, Luo XQ, You LJ, Zhang L

(2011) Metabolic engineering tanshinone biosynthetic pathway

in Salvia miltiorrhiza hairy root cultures. Metab Eng 13:319–327

Liao P, Zhou W, Zhang L, Wang J, Yan XM, Zhang Y, Zhang R, Li

L, Zhou GY, Kai GY (2009) Molecular cloning, characterization

and expression analysis of a new gene encoding 3-hydroxy-3-

methylglutaryl coenzyme A reductase from Salvia miltiorrhiza.

Acta Physiol Plant 31:565–572

Lois LM, Rodriguez-Concepcion M, Gallego F, Campos N, Boronat

A (2000) Carotenoid biosynthesis during tomato fruit develop-

ment: regulatory role of 1-deoxy-D-xylulose 5-phosphate syn-

thase. Plant J 22:503–513

Lv DM, Yuan Y, Huang LQ, Liang RX, Qiu DY (2008) Studies on

accumulation of effective components of Salvia miltiorrhizahairy roots in different culture periods. China J Chin Materia

Medica 33:1078–1080

Munoz-Bertomeu J, Arrillaga I, Ros R, Segura J (2006) Up-regulation

of 1-deoxy-D-xylulose-5-phosphate synthase enhances

production of essential oils in transgenic spike lavender. Plant

Physiol 142:890–900

Nims E, Dubois CP, Roberts SC, Walker EL (2006) Expression

profiling of genes involved in paclitaxel biosynthesis for targeted

metabolic engineering. Metab Eng 8:385–394

Van der Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive

transcriptional regulator of plant primary and secondary metab-

olism. Science 289:295–297

Wang XY, Cui GH, Huang LQ, Qiu DY (2007a) Effects of methyl

jasmonate on accumulation and release of tanshinones in

suspension cultures of Salvia miltiorrhiza hairy root. China J

Chin Materia Medica 32:300–302

Wang XY, Cui GH, Huang LQ, Qiu DY (2007b) Effects of elicitors

on accumulation of tanshinones in suspension cultures of Salviamiltiorrhiza hairy root. China J Chin Materia Medica

32:976–978

Wang XY, Cui GH, Huang LQ, Gao W, Yuan Y (2008) A full length

cDNA of 4-(cytidine 5-diphospho)-2-C-methyl-D- erythritol

kinase cloning and analysis of introduced gene expression in

Salvia miltiorrhiza. Acta Pharm Sinica 43:1251–1257

Wang XY, Cui GH, Gao W, Huang LQ (2009) New method of

‘‘ingredient difference phonetypical cloning’’ for functional gene

cloning from medicinal plants. China J Chin Materia Medica

34:14–17

Wu JY, Shi M (2008) Ultrahigh diterpenoid tanshinone production

through repeated osmotic stress and elicitor stimulation in fed-

batch culture of Salvia miltiorrhiza hairy roots. Appl Microbiol

Biotechnol 78:441–448

Wu SJ, Shi M, Wu JY (2009) Cloning and characterization of the

1-deoxy-D-xylulose 5-phosphate reductoisomerase gene for

diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza (Chi-

nese sage) hairy roots. Biotechnol Appl Biochem 52:89–95

Wang JW, Wu JY (2010) Tanshinone biosynthesis in Salviamiltiorrhiza and production in plant tissue cultures. Appl

Microbiol Biotechnol 88:437–449

Xu H, Zhang L, Zhou CC, Xiao JB, Liao P, Kai GY (2010) Metabolic

regulation and genetic engineering of pharmaceutical component

tanshinone biosynthesis in Salvia miltiorrhiza. J Med Plants Res

4:2591–2597

Yan Q, Hu ZD, Tan RX, Wu JY (2005) Efficient production and

recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy

root cultures with in situ adsorption, elicitation and semi-

continuous operation. J Biotechnol 119:416–424

Yan Q, Hu ZD, Wu JY (2006) Synergistic effects of biotic and abiotic

elicitors on the production of tanshinones in Salvia miltiorrhizahairy root culture. China J Chin Materia Medica 31:188–190

Yan XM, Zhang L, Wang J, Liao P, Zhang Y, Zhang R, Kai GY

(2009) Molecular characterization and expression of 1-deoxy-D-

xylulose 5-phosphate reductoisomerase (DXR) gene from Salviamiltiorrhiza. Acta Physiol Plant 31:1015–1022

Zhang CH, Yan Q, Cheuk J, Wu JY (2004) Enhancement of

tanshinone production in Salvia miltiorrhiza hairy root culture by

Ag? and nutrient feeding. Planta Med 70:147–151

Zhao JL, Zhou LG, Wu JY (2010) Promotion of Salvia miltiorrhizahairy root growth and tanshinone production by polysaccharide-

protein fractions of plant growth-promoting rhizobacterium

Bacillus cereus. Process Biochem 45:1517–1522

Zhou LM, Zuo Z, Chow MSS (2005) Danshen: an overview of its

chemistry, pharmacology, pharmacokinetics, and clinical use.

J Clin Pharmacol 45:1345–1359

Zhou W, Shen YF, Chen JF, Dai LM, Kai GY (2007) Research and

development of biotechnology of Salvia miltiorrhiza Bunge.

J Biol (in Chinese). 24:48–51

Acta Physiol Plant (2012) 34:1421–1433 1433

123