Molecular mechanism of elicitor-induced tanshinone ... - Springer Link

Acta Physiol Plant (2012) 34:1421–1433 DOI 10.1007/s11738-012-0940-z

ORIGINAL PAPER

Molecular mechanism of elicitor-induced tanshinone accumulation 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 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2012

Abstract To develop an optimal bioprocess for the production of tanshinone which is mainly used for the treatment of cardiocerebral vascular disease, the tanshinone biosynthetic pathway regulation must be better understood. In this paper, expression of tanshinone biosynthetic pathway 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 correlation between gene expression and tanshinone accumulation, suggesting that tanshinone accumulation may be the result of the coexpression up-regulation of several genes Communicated by B. Borkowska.

Electronic supplementary material The online version of this article (doi:10.1007/s11738-012-0940-z) contains supplementary material, 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: [email protected] 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

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 metabolic engineering to increase accumulation of tanshinone in S. miltiorrhiza hairy roots. This is the first report integrating 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-Derythritol kinase

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MECPS HDS HDR IPPI GPPS FPPS GGPPS CPS KSL

Acta Physiol Plant (2012) 34:1421–1433

2-C-methylerythritol 2,4-cyclodiphosphate synthase 1-Hydroxy-2-methyl-2-(E)-butenyl-4diphosphate synthase 1-Hydroxy-2-methyl-2-(E)-butenyl-4diphosphate reductase Isopentenyl-diphosphate delta-isomerase Geranyl diphosphate synthase Farnesyl diphosphate synthase Geranylgeranyl diphosphate synthase Copalyl diphosphate synthase 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 cardiovascular diseases including inflammation, blood circulation 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 antischaemics, 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. miltiorrhiza has been widely used in clinical practice. For example, Dan Shen products have been commercially sold in Japan, the United States and European countries Furthermore, 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?, Co? 2,

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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 tanshinone under various elicitors in S. miltiorrhiza hairy roots, while the molecular mechanism of tanshinone accumulation 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 biosynthesis 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 biosynthesis 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 biosynthesis pathway in S. miltiorrhiza hairy roots. The production changes of tanshinone IIA and cryptotanshinone and the expression profiles of all the known tanshinone biosynthesis 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 biosynthesis and enhance tanshinones’ production in S. miltiorrhiza hairy roots by metabolic engineering in the near future.

Acta Physiol Plant (2012) 34:1421–1433 Fig. 1 The metabolic pathway leading to tanshinone. AACT, Acetyl-CoA C-acetyltransferase; HMGS, 3-hydroxy-3-methylglutarylCoA synthase; HMGR, 3-hydroxy-3-methylglutarylCoA reductase; MK, Mevalonate kinase; PMK, 5-phosphomevalonate kinase; MDC, mevalonate 5-diphosphate decarboxylase; DXS, 1-deoxy-D-xylulose-5phosphate synthase; DXR, 1-deoxy-D-xylulose-5phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol4-phosphate cytidyl transferase; CMK, 4-(cytidine 5-diphospho)-2-C-methyl-Derythritol kinase; MECPS, 2-Cmethylerythritol 2,4cyclodiphosphate synthase; HDS, 1-hydroxy-2-methyl-2(E)-butenyl-4-diphosphate synthase; HDR, 1-hydroxy-2methyl-2-(E)-butenyl-4diphosphate reductase; IPPI, Isopentenyl-diphosphate deltaisomerase; 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

1423 Plastidial DXP pathway Pyruvate + Glyceraldehyde 3-phosphate

Cytosol MVA pathway

DXS

2

Acetyl-CoA

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

AACT

DXR

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

HMGS

MCT

3S-Hydroxy-3-methylglutary-CoA (HMGS-CoA) 4-(Cytidine 5’-diphospho)-2-C-methyl-D-erythritol (CDP-ME)

HMGR

CMK

3R-Mevalonate acid (MVA)

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

MK

MECPS

Mevalonate acid-5-diphosphate 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate (cMEPP)

PMK

HDS

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

HDR

Isopentencyl diphosphate (IPP)

HDR

IPPI

Dimethylally diphosphate (DMAPP)

GPPS

Geranyl diphosphate (GPP)

Monoterpene

FPPS

Farnesyl diphosphate (FPP)

Sesquiterpene

GGPPS

Geranylgeranyl diphosphate (GGPP)

Polyterpenes

CPS

Copalyl diphosphate (CPP) KSL

Abietane-type diterpene (Miltiradiene)

Diterpene tanshinones

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. KexuanTang’s laboratory, Shanghai Jiao Tong University, China) which carries A. rhizogenes Ri plasmid pRiA4 in our laboratory (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

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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 performed 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

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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 determined (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 instructions. 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 transcriptase (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 Technology (Shanghai, China). Sodium thiosulfate (Na2S2O3, 98%) was obtained from Shanghai Putuo Chemical Industry Research Institute (Shanghai, China). Cryptotanshinone (CT) and tanshinone IIA (T2A) standards were obtained from the Institute for Identification of


1425

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

SmHMGSKF

50 -AGATCTATGGCCAAGAATGTCGGGATCCT-30

HMGS HMGR DXS1 DXS2

FJ785326 EU680958 EU670744 FJ643618

0

SmHMGSKR

5 -GGTCACCTCAGTGGCCGTTCGCAACTGTGC-3

SmHMGRKF

50 -AGATCTATGGATATCCGCCGGAGGC-30

SmHMGRKR

50 -GGTGACCTCAGGAGCCAATCTTCGTG-30

SmDXS1KF

50 -GGATCCATGGCTTTATGCCCATTTGCATT-30

SmDXS1KR

50 -GAGCTCCTATGACATAATTTCCAGAGCCT-30

SmDXS2KF

50 -AGATCTATGGCGTCGTCTTGTGGAGTTAT-30

1,396 0

1,711

0

2,157 2,188 0

SmDXS2KR

5 -GGTCACCTTACAAGTTGTTGATGAGATGAA-3

DXR

DQ991431

SmDXRKF SmDXRKR

50 -ATGGCTCTAAACTTGATGTC-30 50 -TCATACAAGAGCAGGACTCG-30

1,425

CMK

EF534309

SmCMKKF

50 -ATGGCTTCCTCTTCCTCCCA-30

1,191

SmCMKKR

50 -CTATTCAACATCGCACGTCG-30

SmIPPIKF

50 -ATGTCGTCCTTGACCAGCAT-30

SmIPPIKR

50 -CTAAGTCAGCTTGTGAATTG-30

SmFPPSKF

50 -ATGGCGAATCTGAACGGAGA-30

SmFPPSKR

50 -TTATTTCTGCCTCTTGTATA-30

SmGGPPSKF

50 -AAGGATCCATGAGATCTATGAATCTGGT-30

IPPI FPPS GGPPS CPS KSL

EF635967 EF635968 FJ643617 EU003997 EF635966

0

SmGGPPSKR

5 -CCGAGCTCTTAGTTCTGCCTATGTGCAA-3

SmCPSKF

50 -ATGGCCTCCTTATCCTCTAC-30

SmCPSKR

50 -TCACGCGACTGGCTCGAAAAG-3

SmKSLKF

50 -ATGTCGCTCGCCTTCAACCC-30

SmKSLKR

50 -TCATTTCCCTCTCACATTAT-30

918 1,050 1,111

0

2,382 1,788

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.

tanshinone content were presented as mean values ± SD. Data were analyzed by the Students’s t test.

Experimental design and statistical analysis

Effect of elicitors on tanshinones (CT ? T2A) production

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 tanshinone 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 RTPCR, HPLC analysis were repeated three times. Results of

Results

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

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1426

a

b

Tanshinone content 2

mg·g -1 (DW)

mg· g -1 (DW)

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 error bars represent standard deviation, n = 3. *P \ 0.05; **P \ 0.01


1.5 1 0.5

Tanshinone content 2 1.5 1 0.5 0

0 0

3

6

9

MJ treatment time (days)

c

d

2

mg·g -1 (DW)

mg·g -1 (DW)

Tanshinone content

1.5 1 0.5 0

3

6

9

YE treatment time (days)

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 accumulation 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 measured 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-regulated by MJ. As shown in Fig. 4, mRNA levels of

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3

6

9

Tanshinone content 2 1.5 1 0.5 0

0

0

Ag+ treatment time (days)

0

3

6

9

+

YE-Ag treatment time (days)

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 tanshinone biosynthesis related genes (SmHMGR-M6, SmDXRM9, 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).


a

0.08

0.06

0.04

1 2

0.02

17.081

10.476

AU

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)

1427

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

Elution time (min)

b

0.10 0.08

AU

0.06 0.04 0.02

1 2

0.00 2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

20.00

22.00

Elution time (min)

c

0.15

1

AU

0.10

0.05

2

0.00 2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

Elution time (min)

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 gradually 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’ production, implying that the activation of these six genes coding for the respective six enzymes contribute to the improvement 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?.

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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

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’ production. Hence, co-activation of these six genes (SmHMGS, SmDXR, SmDXS2, SmCMK, SmIPPI, SmCPS) coding for the respective six enzymes dramatically improved the tanshinones’ production under the treatment of YE.

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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?

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 promote 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 stimulated (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


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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?

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’ production. 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 contribute 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?.

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 accumulation 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 pathway 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 accumulation enhanced after 3 days (Fig. 2c), and mRNA expressions of six genes in the tanshinone biosynthetic pathway including SmHMGS, SmDXR, SmDXS2, SmCMK,

Discussion In a word, both of our experimental results and other previous studies (Ge and Wu 2005a, b; Wang et al. 2007a;

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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 combined 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 tanshinone 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 accumulation 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 tanshinone 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 biosynthesis because of their low mRNA expression in uninduced 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 tanshinone 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-hydroxymethylglutaryl-CoA (HMG-CoA) to mevalonate, has been

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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 treatment of Ag? in S. miltiorrhiza hairy roots. Therefore, SmHMGR is most likely one of the key genes in the tanshinone biosynthetic pathway in S. miltiorrhiza hairy roots. DXS catalyze the first-limiting step in the DXP biosynthetic pathway (Este´vez et al. 2001). Over-expression of DXS in Arabidopsis thaliana and Lavandula latifolia dramatically enhanced terpenoid production respectively (Este´vez et al. 2001; Lois et al. 2000; Munõz-Bertomeu et al. 2006), confirming that DXS is one of the key regulatory 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 treatment 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 experiments could also come to the same conclusion (unpublished 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.


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 MEPderived 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 confirmed 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 parallel with increased tanshinone accumulation in S. miltiorrhiza hairy roots (Figs. 2c, 6). The above studies suggest that SmDXR may also be a target for the exploitation 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 tanshinone 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 previous work demonstrated that SmGGPPS encoded a functional 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,

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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). Transcriptional 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 treatment 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 rearrangement of normal CPP to form an abietane-type diterpene 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? respectively, 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 accumulation was dramatically stimulated by MJ (Fig. 2a), and almost all the tested genes encoding related enzymes

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involved in tanshinone biosynthesis were coordinately induced by MJ (Fig. 4), providing direct molecular evidence for improving tanshinone content by MJ. ORCAsimilar transcript factors may exist in S. miltiorrhiza for global control of several tanshinone biosynthetic genes, as illustrated for terpenoid indole alkaloids (TIA) in Catharanthus roseus (Van der Fits and Memelink 2000). This current work leads us in a direction where it will be possible to clone upstream regulatory regions of the MJinduced genes. Then we will be able to analyze, identify the cis-acting elements and trans-acting factors of promoters 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 tanshinone 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 pathway for better understanding the whole tanshinone biosynthetic 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 regulation 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 bottlenecks to be targeted for metabolic engineering that can

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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 experiments, analyzed the data and drafts the manuscript. HX and JW participated in RNA extracting and gene expression analysis. CCZ and WZ helped to culture hairy roots. YPQ drafted part of the manuscript and revised the manuscript. 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 Committee 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 Transgenic Organism New Variety Culture Key Project (2009ZX08012002B), 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.

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