Ag+ as a More Effective Elicitor for Production of Tanshinones than Phenolic Acids in Salvia miltiorrhiza Hairy Roots

Phenolic acids and tanshinones are two groups of bioactive ingredients in Salvia miltiorrhiza Bunge. As a heavy metal elicitor, it has been reported that Ag+ can induce accumulations of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. In this study, the effects of Ag+ treatment on accumulations of six phenolic acids and four tanshinones in S. miltiorrhiza hairy roots were investigated. To further elucidate the molecular mechanism, expressions of key genes involved in the biosynthesis of these ingredients were also detected. The results showed that although the total phenolic acids content was almost not affected by Ag+, accumulations of rosmarinic acid (RA), caffeic acid and ferulic acid were significantly increased, while accumulations of salvianolic acid B (LAB), danshensu (DSU) and cinnamic acid were decreased. We speculate that LAB probably derived from the branch pathway of DSU biosynthesis. Contents of four tanshinones were enhanced by Ag+ and their accumulations were more sensitive to Ag+ than phenolic acids. Genes in the upstream biosynthetic pathways of these ingredients responded to Ag+ earlier than those in the downstream biosynthetic pathways. Ag+ probably induced the whole pathways, upregulated gene expressions from the upstream pathways to the downstream pathways, and finally resulted in the enhancement of ingredient production. Compared with phenolic acids, tanshinone production was more sensitive to Ag+ treatments. This study will help us understand how secondary metabolism in S. miltiorrhiza responds to elicitors and provide a reference for the improvement of the production of targeted compounds in the near future.

As a group of efficient abiotic elicitors, the use of metal ions including Ca 2+ , Co 2+ , Ag + , Cd 2+ , Cu 2+ , Ce 3+ , La + , Mn 2+ and Zn 2+ to induce bioactive ingredient production in plants has been widely researched [21,25,26]. Among them, Ag + was the mostly studied. In Vitis vinifera cell suspension cultures, Ag + at a low concentration (5.0 μM) stimulated 3-O-glucosylresveratrol production, but it did not affect cell growth [26]. High concentrations of Ag + (30 μM) can enhance paclitaxel yield in suspension culture of Taxus spp. Cells [27]. Ag + can also improve echinacoside and acteoside production in cell suspension cultures of Cistanchede serticola [28]. In S. miltiorrhiza hairy roots, treatment with 15 μM Ag + significantly increased RA accumulation, as well as total phenolic acid content [12]. However, Xiao reported that LAB is more sensitive to Ag + than RA, and the content of RA was not affected [29]. In terms of tanshinone accumulation, the previous work showed that tanshinone production was significantly enhanced by Ag + probably via the non-MVA pathway [30]. There is currently a lack of information regarding the comparison of how the two groups of bioactive compounds in S. miltiorrhiza respond to Ag + .
In this study, to reveal the effects of Ag + treatments on both phenolic acid and tanshinone production, and elucidate the regulatory mechanisms of the biosynthesis of these ingredients, accumulations of six phenolic compounds and four tanshinone compounds were examined. Simultaneously, expressions of 15 genes involved in biosynthesis of these ingredients were investigated.

Effects of Ag + Elicitor on Cell Growth of S. miltiorrhiza Hairy Roots
Treatments with Ag + , the biomass of S. miltiorrhiza hairy roots in shake-flask cultures showed an increasing trend in both the control and the treatment groups ( Figure 3A). Compared with the control, the hairy roots growth was almost not affected by Ag + in the first 6 days after treatment and was slightly depressed on day 9. However, the dry weight (DW)/fresh weight (FW) was significantly increased at 1, 2, 6 and 9 days after treatment. This means that Ag + elicitor decreased the growth of S. miltiorrhiza hairy roots and reduced the water content in hairy roots ( Figure 3B). The asterisks indicate statistically significant differences at p < 0.05 (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01.) between the content in the elicitor treated cultures and that in the corresponding controls.

Effects of Ag + Elicitor on Accumulation of Phenolic Acids in S. miltiorrhiza Hairy Roots
In this study, six phenolic acids compounds were examined (RA, LAB, DSU, caffeic acid, ferulic acid and cinnamic acid). The asterisks indicate statistically significant differences at p < 0.05 (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01.) between the content in the elicitor treated cultures and that in the corresponding controls.
With Ag + treatment, RA content was significantly increased to 15.27 mg/g DW on day 6 after treatment (1.3-fold of the control). Accumulations of caffeic acid and ferulic acid were also stimulated by Ag + , and the maximum contents were 0.37 mg/g DW and 0.11 mg/g DW, respectively. However, contents of DSU, cinnamic acid and LAB were significantly inhibited by Ag + , and were just 22%, 65% and 70% of the control levels, respectively. The results indicated that Ag + induced accumulations of RA, caffeic acid and ferulic acid, but decreased contents of LAB, DSU and cinnamic acid. However, accumulation of the total phenolic acids was almost not affected by Ag + treatment ( Figure 4).  The asterisks indicate statistically significant differences at p < 0.05 (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001.) between the content in the elicitor treated cultures and that in the corresponding controls.

Effects of Ag + Elicitor on Genes Expressions Involved in the Two Parallel Pathways of LAB Biosynthesis
Expressions of seven key genes in RA biosynthesis pathways were determined in this study, including PAL, C4H, 4CL, TAT, HPPR, RAS and CYP98A14 ( Figure 6). The results showed that expression levels of genes in the upstream pathway (PAL, C4H, 4CL, TAT and HPPR) were greatly promoted by Ag + and reached their maximum on day 1 post-treatment. Their expression levels were 29.18-fold, 5.43-fold, 12.76-fold, 13.15-fold and 22.83-fold of the control levels, respectively. Then, expressions of those genes decreased gradually ( Figure 6). This indicated PAL was more sensitive to Ag + than C4H, 4CL, TAT and HPPR. However, responses of two genes in the downstream pathway (RAS and CYP98A14) were later and reached to their maximum expression levels on day 6 after treatment, 16.14-fold (RAS) and 1.44-fold (CYP98A14) of the control levels, respectively ( Figure 6). The results indicated that the upstream genes responded to Ag + earlier than the downstream genes. The asterisks indicate statistically significant differences at p < 0.05 (*, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001.) between the content in the elicitor treated cultures and that in the corresponding controls.

Effects of Ag + Elicitor on Genes Expression Involved in Pathways of Tanshinones Biosynthesis
HMGR, DXS1, DXS2, DXR, GGPPS were key genes in the upstream pathways of tanshinones biosynthesis. CPS, KSL and CYP76AH1 were key genes in the downstream pathways. In this study, expression levels of these genes were detected by RT-qPCR. The results showed that expressions of HMGR, DXS1, DXS2 and DXR were dramatically induced by Ag + on day 1 (Figure 7). Then their expressions decreased to control levels on day 2 and 6, but sharply increased on day 3 and 9. HMGR gene expression level increased by 9.32-fold, while DXS1, DXS2, DXR and GGPPS increased by 5.12-fold, 39.13-fold, 88.38-fold and 37.10-fold on day 1 post-treatment, respectively. However, CPS, KSL and CYP76AH1 reached their maximum expression levels until day 9 after treatment, and increased by 78.67-fold, 220.50-fold and 4.02-fold of the control level, respectively. The results indicated that genes in the upstream pathway responded to Ag + earlier than the downstream genes.

Discussion
Phenolics and tanshinones are two important groups of bioactive compounds in S. miltiorrhiza. The biosynthetic mechanisms of these compounds has been widely studied because of their medicinal value. Phenolic acids were mainly produced through the phenylpropanoid and tyrosine pathways, and tanshinones are biosynthesized via the MVA and MEP pathways. Various elicitors have been used to improve the accumulation of these bioactive ingredients in S. miltiorrhiza [31]. However, the regulation mechanisms of phenolic acid and tanshinone biosynthesis by elicitors is still unclear.
Among abiotic elicitors, heavy metals have a significant impact on plant growth and secondary metabolism. It was reported that Ca 2+ , Ag + , and Cd 2+ could improve the production of tropane alkaloids in hairy roots cultures of Brugmansia candida [32]. Many kinds of heavy metal were also used as elicitors to induce accumulations of bioactive compounds in S. miltiorrhiza, such as Co 2+ , Ag + , Cd 2+ , Cu 2+ , Ce 3+ , La, Mn 2+ and Zn 2+ etc., [12,21,24,33]. Among them, Ag + was considered as an effective elicitor for phenolic compound and tanshinone production in S. miltiorrhiza hairy roots, and could improve RA, LAB and tanshinones production. But it was also reported that RA accumulation was almost not affected by Ag + [29], so whether Ag + have significant effect on phenolic acids remains unclear. No report compares how the two groups of bioactive compounds respond to Ag + .
LAB was considered as a derivative of RA, and its accumulation was affected by RA content [9]. In this study, we found that accumulations of all the six phenolic compounds were influenced by Ag + (Figure 4). RA content reached 1.57-fold of the control (on day 9), but LAB content was reduced to 70% of the control. This result was the same as Yan's report [12]. DSU was derived from 4-hydroxphenyllactic acid, and is a main degradation product of LAB [34]. In this work, we found that DSU accumulation was decreased by Ag + as well as LAB. In another experiment of our lab, overexpression of RAS resulted in a decrease of LAB content, but increased RA content (unpublished data). Therefore, we speculated that DSU was probably a precursor of LAB biosynthesis and LAB was probably not biosynthesized from RA pathway but from DSU branch pathway ( Figure 2). However, Xiao found that LAB was dramatically induced by Ag + while RA content was not affected [29]. The reason of inconsistency may be the difference between bacterial strains used to obtain the hairy roots and the different harvest time we selected. Caffeic acid, ferulic acid and cinnamic acid are three upstream metabolites in the phenyl-propanoid pathway. Cinnamic acid is a precursor of caffeic acid and ferulic acid biosynthesis. Cinnamic acid accumulation was decreased, while accumulations of the two derivatives (caffeic acid and ferulic acid) were increased by Ag + . However, content of total phenolic acids was almost unaffected by Ag + (Figure 5). It was probably because that metabolic flux among various phenolic acids was influenced by Ag + .
Changes of the six phenolic acids' accumulations may be caused by gene expression in the phenylpropanoid and tyrosine pathways. PAL is the first key enzyme in the phenylpropanoid pathway, and its expression is closely related to accumulations of RA and other phenolics. Down-regulation of PAL markedly reduced accumulations of RA and LAB [35]. C4H, TAT and HPPR expressions were also related to accumulations of RA and LAB [36]. Our gene expression results indicated that both the phenylpropanoid and tyrosine pathways were affected by Ag + . Gene expressions in the upstream pathway responded to Ag + earlier than those in the downstream pathway (Figure 7). This demonstrated Ag + probably induced the whole phenolic acid biosynthesis pathway, and up-regulated gene expression from the upstream pathway to the downstream pathway, and this finally resulted in an enhancement of phenolic acid production.
As another group of bioactive compounds in S. miltiorrhiza, tanshinone production was also promoted by various elicitors (YE, fungi, PEG and ABA etc.) [10,33,37]. Ag + has been widely reported to enhance tanshinone production in S. miltiorrhiza hairy roots [22,24,38]. Ge used 30 µM Ag + to treat S. miltiorrhiza hairy roots, and found that the maximum content of the total tanshinones was 1.2-fold of the control [30]. In another report, 25 µM Ag + could improve CT, T-I and T-IIA contents by 30-fold, 0.87-fold, 3.9-fold, respectively [33]. In this study, accumulations of four tanshinones components were also induced by Ag + (Figure 5), and the maximum contents of T-I, T-IIA, CT and DT-I was 1.45-fold, 1.63-fold, 4.4-fold and 2.55-fold of the control, respectively. The maximum content of the total tanshinones was increased by 2-fold, which was much higher than the present reports. This indicated that Ag + was an effective elicitor of tanshinone production, and CT was more sensitive to Ag + than other tanshinone compounds.
The increase of tanshinone accumulation probably resulted from up-regulation of gene expression in the MEP and MVA pathways. With 30 µM Ag + treatment, expression of many genes such as HMGR, DXS2, GGPPS and CPS in S. miltiorrhiza hairy roots was induced [22,39]. Our results showed that key genes in the two pathways were all induced by Ag + (Figure 7). Expression of HMGR was increased by 9.31-fold (day 1) and expressions of DXS1, DXS2, DXR increased by 5.12-fold, 39.13-fold, 88.38-fold and 37.10-fold, respectively (day 1). It was indicated that the MEP pathway was more sensitive to Ag + than the MVA pathway. This result was similar to previous reports [18]. However, expression of genes in the downstream pathway responded later than the upstream genes. The results were the same as those of the genes involved in phenolic acid biosynthesis.

Hairy Roots Culture
Agrobacterium rhizogenes (ATCC15834) with Ri (root inducing) T-DNA (transfer DNA) was used to infect S. miltiorrhiza aseptic leaves, then the hairy roots of S. miltiorrhiza were obtained. 6,7-V liquid medium (with 30 g·L −1 sucrose) was used to culture the hairy roots. The hairy roots were maintained in a 100 mL beaker flask with 50 mL 6,7-V liquid medium. Each beaker flask was inoculated with 0.2 g fresh hairy roots and placed on an orbital shaker at 110 rpm·min −1 , 25 °C in the dark. Elicitation was performed on the 18th day after inoculation. The hairy roots were harvested from the culture medium on days 1, 2, 3, 6 and 9 post-treatment. The fresh weight of each sample was recorded. Take half of each sample to isolate RNA, while the others were dried at 50 °C in an oven until constant dry weight for metabolites content analysis. Untreated hairy roots were designated as the control. The experiment was performed in triplicate, and the results are means ± SD.

Preparation of Ag + Elicitor
Silver ions (Ag + ) were supplied to the culture at a final concentration of 15 μM, using a concentrated silver thiosulfate (Ag2S2O3) solution prepared by mixing AgNO3 and Na2S2O3 at a molar ratio of 1:4. Then it was sterilized by filtration (0.22 μm membrane).

Metabolite Extraction and HPLC Analysis
Compound extraction and analysis followed the methods described by Yang [10] with minor modifications. The dried hairy root was ground to a powder with a mortar and pestle. The sample powder (50 mg) was extracted with 70% methanol (5 mL) under sonication for 45 min, then centrifuged at 8000 rpm (6010× g) for 10 min. The supernatant was ready to use for HPLC analysis. The metabolite contents were determined on a Waters HPLC system (Waters, Milford, MA, USA) equipped with a 1525 binary pump, a manual sample injector and a Waters 2996 photodiode array detector (PDA). Chromatography separation was performed with a SunFire C18 column (4.6 mm × 250 mm, 5 mm particle size) at 30 °C. Empower 2 software was used for data acquisition and processing. The sample injection volume was 20 µL and the PDA detection wavelength for the lipid-soluble diterpenoids were 270 nm, and for the water-soluble phenolic acids was 280 nm. Separation was achieved by elution using a linear gradient with solvent-A (acetonitrile) and solvent-B (0.02% phosphoric acid solution). The gradient was as follows: t = 0 min, 5% A; t = 10 min, 20% A; t = 15 min, 25% A; t = 20 min, 25% A; t = 25 min, 20% A; t = 28 min, 30% A; t = 36 min, 30% A. The flow rate was 1.0 mL·min −1 . Standards of metabolite compounds were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) [8].

RNA Isolation and Real-Time Quantitative PCR Analysis
Total RNAs from S. miltiorrhiza hairy roots were extracted at selected time points using RNAiso TM Plus (Takara, Tokyo, Japan) according to the manufacturer's instructions. The quality and concentration of RNA were examined by ethidium bromide (EB)-stained agarose gel electrophoresis and spectrophotometer analysis (Thermo Scientific NanoDrop 2000). The first strand cDNA for RT-qPCR was synthesized from 1 µg total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, Takara, Tokyo, Japan). Primers used for RT-qPCR were list at Table 1. The constitutively expressed 18S rRNA with specific primers F18S (5'-ATGATAACTCGACGGATCGC-3') and R18S (5'-CTTGGATGTGGTAGCCGTTT-3') were used as control. Real-time PCR was performed according to the manufacturer's instruction of SYBR ® Premix Ex Taq™ II (TliRNaseH Plus, Takara) using the following protocol: 95 °C, 30 s, 1 cycle; 95 °C, 5 s, annealing temperature (Tm), 30 s, 40 cycles. The program was performed on the Mastercyclerep realplex4 system (Eppendorf, Hamburg, Germany). Quantification of the gene expression was done with comparative CT method (2 −ΔΔCT ). Experiments were performed in triplicate, and the results were represented by their means ± SD.

Conclusions
In conclusion, the production of both phenolic acids and tanshinones except DSU, LAB and cinnamic acid were enhanced by Ag + treatment, but Ag + was more effective at stimulating tanshinone production than that of phenolic acids. LAB was probably derived from the branch pathway of DSU biosynthesis, and the MEP pathway was more sensitive to Ag + than the MVA pathway. Genes in the upstream pathways responded to Ag + earlier than those in the downstream pathway. Ag + probably induced the whole pathways of those ingredients' biosyntheses, upregulated gene expressions from the upstream pathway to the downstream pathway, and this finally resulted in the enhancement of ingredient production. This study will help us understand how secondary metabolism in S. miltiorrhiza responds to elicitors and provide a reference for the improvement of targeted compound production in the near future.