Lipopolysaccharide Enhances Tanshinone Biosynthesis via a Ca2+-Dependent Manner in Salvia miltiorrhiza Hairy Roots

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca2+ signaling is essential for the LPS-triggered immune response, Ca2+ channel blocker LaCl3 and CaM antagonist W-7 were used to investigate the role of Ca2+ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl3 and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca2+ signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca2+ signaling. Ca2+ signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza.


LPS Enhances Tanshinone Accumulation in the Wild Type Hairy Roots of S. miltiorrhiza
Since the biosynthesis of secondary metabolites might be induced by microorganisms, the bacterial component LPS was applied as a novel elicitor to treat the wild type (WT) hairy roots of S. miltiorrhiza. After being treated by 50 µg/mL LPS for 10 days, the hairy roots and the culture medium showed a deep red color, which is the characteristic color of tanshinones ( Figure 1A). Compared to the control, LPS did not obviously affect the growth of hairy roots ( Figure 1A). Further, the content of the tanshinones, including dihydrotanshinone I (DTI), cryptotanshinone (CT), tanshinones I (TI) and tanshinones IIA (TIIA), was analyzed by HPLC. When the hairy roots were treated by LPS, the content of DTI significantly increased from 0.38 mg/g to 0.86 mg/g in contrast to the control ( Figure 1B), and the content of CT increased from 0.62 mg/g to 0.9 mg/g ( Figure 1C). However, the content of TI and TIIA showed no significant change ( Figure 1D,E). These results indicate that LPS can induce the accumulation of DTI and CT without markedly inhibiting the growth of S. miltiorrhiza WT hairy roots.

LPS Enhances Tanshinone Accumulation in the Wild Type Hairy Roots of S. miltiorrhiza
Since the biosynthesis of secondary metabolites might be induced by microorganisms, the bacterial component LPS was applied as a novel elicitor to treat the wild type (WT) hairy roots of S. miltiorrhiza. After being treated by 50 μg/mL LPS for 10 days, the hairy roots and the culture medium showed a deep red color, which is the characteristic color of tanshinones ( Figure 1A). Compared to the control, LPS did not obviously affect the growth of hairy roots ( Figure 1A). Further, the content of the tanshinones, including dihydrotanshinone I (DTI), cryptotanshinone (CT), tanshinones I (TI) and tanshinones IIA (TIIA), was analyzed by HPLC. When the hairy roots were treated by LPS, the content of DTI significantly increased from 0.38 mg/g to 0.86 mg/g in contrast to the control ( Figure  1B), and the content of CT increased from 0.62 mg/g to 0.9 mg/g ( Figure 1C). However, the content of TI and TIIA showed no significant change ( Figure 1D,E). These results indicate that LPS can induce the accumulation of DTI and CT without markedly inhibiting the growth of S. miltiorrhiza WT hairy roots. The hairy roots were treated by 50 μg/mL LPS for 10 days. H2O was used as a control. (B-E) The content of tanshinones in LPStreated hairy roots. The content of the tanshinones was analyzed by HPLC and presented by the means ± SD. The significant differences between different groups were calculated by the Student's ttest. (**) indicates a very significant difference (p ≤ 0.01). TI, tanshinone I; TIIA, tanshinone IIA; CT, cryptotanshinone; DT, dihydrotanshinone.

LPS Upregulates Key Gene's Expression in Tanshinone Biosynthesis Pathways
To elucidate the regulation mechanism of LPS, the key biosynthesis genes of tanshinones were analyzed by qRT-PCR. In the biosynthesis pathways of tanshinones, SmAACT and SmHMGS are from the MVA pathway, and SmDXS and SmHDR are from the MEP pathway ( Figure 2A). After being induced by LPS, the transcripts levels of SmAACT and SmHMGS were obviously upregulated at 6 h ( Figure 2B,C). Similarly, SmDXS and SmHDR showed the same response to LPS treatment ( Figure  2D,E). In the confirmed biosynthesis pathway of tanshinones, SmCPS, SmKSL and SmCYP76AH1 are located downstream the MVA and MEP pathways ( Figure 2A). Notably, the expression of these three genes also increased along with LPS treatment ( Figure 2F-H). SmCPS and SmCYP76AH1 were especially upregulated by LPS in a time-dependent manner ( Figure 2F,H). The content of tanshinones in LPS-treated hairy roots. The content of the tanshinones was analyzed by HPLC and presented by the means ± SD. The significant differences between different groups were calculated by the Student's t-test. (**) indicates a very significant difference (p ≤ 0.01). TI, tanshinone I; TIIA, tanshinone IIA; CT, cryptotanshinone; DT, dihydrotanshinone.

LPS Upregulates Key Gene's Expression in Tanshinone Biosynthesis Pathways
To elucidate the regulation mechanism of LPS, the key biosynthesis genes of tanshinones were analyzed by qRT-PCR. In the biosynthesis pathways of tanshinones, SmAACT and SmHMGS are from the MVA pathway, and SmDXS and SmHDR are from the MEP pathway ( Figure 2A). After being induced by LPS, the transcripts levels of SmAACT and SmHMGS were obviously upregulated at 6 h ( Figure 2B,C). Similarly, SmDXS and SmHDR showed the same response to LPS treatment ( Figure 2D,E). In the confirmed biosynthesis pathway of tanshinones, SmCPS, SmKSL and SmCYP76AH1 are located downstream the MVA and MEP pathways ( Figure 2A). Notably, the expression of these three genes also increased along with LPS treatment ( Figure 2F-H). SmCPS and SmCYP76AH1 were especially upregulated by LPS in a time-dependent manner ( Figure 2F,H). To further explore the stimulation mechanism of these biosynthesis genes by LPS, the expression of transcription factors SmWRKY1 and SmWRKY2 was analyzed by qRT-PCR. In S. miltiorrhiza, SmWRKY1 can bind with the promoter of SmDXR, and SmWRKY2 can bind with SmDXS and SmCPS, to positively regulate tanshinone biosynthesis [5,17,18]. Our further analysis indicated that LPS upregulated the transcript levels of SmWRKY1 and SmWRKY2 in the same time-dependent manner. The expression of these transcription factors responded to LPS and reached a peak at 6 h ( Figure  3A,B). Hence, SmDXS, SmCPS and SmDXR can be highly transcribed due to the activation of SmWRKY1 and SmWRKY2.
Taken together, the secondary metabolite tanshinones can be induced by the immune regulator LPS. LPS enhances tanshinone accumulation through stimulating SmWRKY1-and SmWRKY2regulated gene expression in tanshinone biosynthesis pathways. To further explore the stimulation mechanism of these biosynthesis genes by LPS, the expression of transcription factors SmWRKY1 and SmWRKY2 was analyzed by qRT-PCR. In S. miltiorrhiza, SmWRKY1 can bind with the promoter of SmDXR, and SmWRKY2 can bind with SmDXS and SmCPS, to positively regulate tanshinone biosynthesis [5,17,18]. Our further analysis indicated that LPS upregulated the transcript levels of SmWRKY1 and SmWRKY2 in the same time-dependent manner. The expression of these transcription factors responded to LPS and reached a peak at 6 h ( Figure 3A,B). Hence, SmDXS, SmCPS and SmDXR can be highly transcribed due to the activation of SmWRKY1 and SmWRKY2.
Taken together, the secondary metabolite tanshinones can be induced by the immune regulator LPS. LPS enhances tanshinone accumulation through stimulating SmWRKY1and SmWRKY2-regulated gene expression in tanshinone biosynthesis pathways.

Ca 2+ Inhibitors Affect Tanshinone Accumulation
Ca 2+ signal transduction is essential for the LPS-triggered plant immune response [15]. Thus, three Ca 2+ signal inhibitors, including Ca 2+ channel blocker LaCl3, CaM antagonist W-7 and Ca 2+ chelator EGTA, were applied to analyze the role of Ca 2+ signaling in tanshinone biosynthesis [15,19]. Since tanshinones can generate a deep red color in the roots of S. miltiorrhiza, we preliminarily observed the color of the hairy roots treated by different Ca 2+ reagents. As shown in Figure 4A,B, the hairy roots treated by 1 mmol/L LaCl3 apparently showed a light color compared to the H2O control. Similarly, 100 μmol/L W-7 also led to light color in contrast to the DMSO control. Nevertheless, 1mmol/L EGTA did not obviously affect the color of the hairy roots compared to the H2O control. These results suggest that Ca 2+ signaling is closely associated with tanshinone biosynthesis. The accumulation of tanshinones might be inhibited by blocking Ca 2+ influx or repressing CaM-mediated signaling in the hairy roots of S. miltiorrhiza.

Ca 2+ Channel Blocker Inhibits LPS-Induced Tanshinone Accumulation
LaCl3 is capable of suppressing cytoplasmic Ca 2+ elevation via blocking Ca 2+ influx [15,19]. Thus, LaCl3 was synergistically utilized with LPS to analyze the role of Ca 2+ signaling in tanshinone

Ca 2+ Inhibitors Affect Tanshinone Accumulation
Ca 2+ signal transduction is essential for the LPS-triggered plant immune response [15]. Thus, three Ca 2+ signal inhibitors, including Ca 2+ channel blocker LaCl 3 , CaM antagonist W-7 and Ca 2+ chelator EGTA, were applied to analyze the role of Ca 2+ signaling in tanshinone biosynthesis [15,19]. Since tanshinones can generate a deep red color in the roots of S. miltiorrhiza, we preliminarily observed the color of the hairy roots treated by different Ca 2+ reagents. As shown in Figure 4A,B, the hairy roots treated by 1 mmol/L LaCl 3 apparently showed a light color compared to the H 2 O control. Similarly, 100 µmol/L W-7 also led to light color in contrast to the DMSO control. Nevertheless, 1mmol/L EGTA did not obviously affect the color of the hairy roots compared to the H 2 O control. These results suggest that Ca 2+ signaling is closely associated with tanshinone biosynthesis. The accumulation of tanshinones might be inhibited by blocking Ca 2+ influx or repressing CaM-mediated signaling in the hairy roots of S. miltiorrhiza.

Ca 2+ Inhibitors Affect Tanshinone Accumulation
Ca 2+ signal transduction is essential for the LPS-triggered plant immune response [15]. Thus, three Ca 2+ signal inhibitors, including Ca 2+ channel blocker LaCl3, CaM antagonist W-7 and Ca 2+ chelator EGTA, were applied to analyze the role of Ca 2+ signaling in tanshinone biosynthesis [15,19]. Since tanshinones can generate a deep red color in the roots of S. miltiorrhiza, we preliminarily observed the color of the hairy roots treated by different Ca 2+ reagents. As shown in Figure 4A,B, the hairy roots treated by 1 mmol/L LaCl3 apparently showed a light color compared to the H2O control. Similarly, 100 μmol/L W-7 also led to light color in contrast to the DMSO control. Nevertheless, 1mmol/L EGTA did not obviously affect the color of the hairy roots compared to the H2O control. These results suggest that Ca 2+ signaling is closely associated with tanshinone biosynthesis. The accumulation of tanshinones might be inhibited by blocking Ca 2+ influx or repressing CaM-mediated signaling in the hairy roots of S. miltiorrhiza.

Ca 2+ Channel Blocker Inhibits LPS-Induced Tanshinone Accumulation
LaCl3 is capable of suppressing cytoplasmic Ca 2+ elevation via blocking Ca 2+ influx [15,19]. Thus, LaCl3 was synergistically utilized with LPS to analyze the role of Ca 2+ signaling in tanshinone

Ca 2+ Channel Blocker Inhibits LPS-Induced Tanshinone Accumulation
LaCl 3 is capable of suppressing cytoplasmic Ca 2+ elevation via blocking Ca 2+ influx [15,19]. Thus, LaCl 3 was synergistically utilized with LPS to analyze the role of Ca 2+ signaling in tanshinone biosynthesis. As shown in Figure 5A, the LPS-treated hairy roots showed the deepest color and LaCl 3 treatment resulted in the lightest color. LPS-induced deep red was apparently decreased by LaCl 3 synergetic treatment ( Figure 5A). Further, the content of tanshinones was examined by HPLC. Compared to the LPS treatment, the content of DTI in the LaCl 3 +LPS-treated sample significantly reduced from 0.71 mg/g to 0.28 mg/g, and CT reduced from 0.88 mg/g to 0.63 mg/g ( Figure 5B,C). The LPS+LaCl 3 treatment also led to a significant reduction in TI and TIIA in a similar way ( Figure 5D,E). These results confirmed that with the inhibition of Ca 2+ influx by LaCl 3 , LPS-induced tanshinone accumulation was accordingly diminished. Therefore, the Ca 2+ influx signal is involved in regulating tanshinone accumulation. biosynthesis. As shown in Figure 5A, the LPS-treated hairy roots showed the deepest color and LaCl3 treatment resulted in the lightest color. LPS-induced deep red was apparently decreased by LaCl3 synergetic treatment ( Figure 5A). Further, the content of tanshinones was examined by HPLC. Compared to the LPS treatment, the content of DTI in the LaCl3+LPS-treated sample significantly reduced from 0.71 mg/g to 0.28 mg/g, and CT reduced from 0.88 mg/g to 0.63 mg/g ( Figure 5B,C). The LPS+LaCl3 treatment also led to a significant reduction in TI and TIIA in a similar way ( Figure 5D,E). These results confirmed that with the inhibition of Ca 2+ influx by LaCl3, LPS-induced tanshinone accumulation was accordingly diminished. Therefore, the Ca 2+ influx signal is involved in regulating tanshinone accumulation.

CaM Antagonist Inhibits LPS-Induced Tanshinone Accumulation
CaM serves as a crucial sensor in Ca 2+ signal transduction. Through binding with Ca 2+ , the Ca 2+ -CaM complex interacts with target proteins such as CNGC, CDPK, and MAPK to regulate numerous metabolism reactions [8,20]. Hence, the CaM antagonist W-7 was utilized to corporately treat hairy roots with LPS. As shown in Figure 6A, W-7 treatment partly decreased the LPS-induced deep red of the hairy roots and generated the lightest color, while showing no obvious growth inhibition. Compared to LPS treatment, the content of DTI, CT and TI significantly declined from 0.52 mg/g to 0.23 mg/g, 1.15 mg/g to 0.52 mg/g, and 0.29 mg/g to 0.20 mg/g in LPS+W-7 treated hairy roots ( Figure 6B-D). Notably, the separate W-7 treatment resulted in extreme inhibition of these four tanshinones, especially CT and TI ( Figure 6B-E). Taken together, CaM-mediated signaling is essential for LPS-induced tanshinone accumulation in S. miltiorrhiza hairy roots.

CaM Antagonist Inhibits LPS-Induced Tanshinone Accumulation
CaM serves as a crucial sensor in Ca 2+ signal transduction. Through binding with Ca 2+ , the Ca 2+ -CaM complex interacts with target proteins such as CNGC, CDPK, and MAPK to regulate numerous metabolism reactions [8,20]. Hence, the CaM antagonist W-7 was utilized to corporately treat hairy roots with LPS. As shown in Figure 6A, W-7 treatment partly decreased the LPS-induced deep red of the hairy roots and generated the lightest color, while showing no obvious growth inhibition. Compared to LPS treatment, the content of DTI, CT and TI significantly declined from 0.52 mg/g to 0.23 mg/g, 1.15 mg/g to 0.52 mg/g, and 0.29 mg/g to 0.20 mg/g in LPS+W-7 treated hairy roots ( Figure  6B-D). Notably, the separate W-7 treatment resulted in extreme inhibition of these four tanshinones, especially CT and TI ( Figure 6B-E). Taken together, CaM-mediated signaling is essential for LPSinduced tanshinone accumulation in S. miltiorrhiza hairy roots.

LPS Induces the Expression of Key Tanshinone Biosynthesis Genes in a Ca 2+ -Dependent Manner
To further investigate the role of Ca 2+ signaling in tanshinone biosynthesis, the expression levels of key genes in LPS and LaCl 3 treated WT hairy roots were analyzed by qRT-PCR. When synergistically treated by LaCl 3 and LPS for 6 h, the expression levels of SmCPS and SmCYP76AH1 reduced approximately 40-fold and 37-fold, respectively, compared to the LPS separate treatment ( Figure 7A,B). However, SmHDR and SmDXS did not show apparent reduction by LaCl 3 +LPS treatment ( Figure 7C,D). Comparatively, LaCl 3 preferentially inhibits the downstream genes (SmCPS and SmCYP76AH1) in tanshinone biosynthesis pathways. This suggests that the downstream genes of the tanshinone biosynthesis pathway are more likely to be regulated by Ca 2+ signaling than the MEP pathway genes.

LPS Induces the Expression of Key Tanshinone Biosynthesis Genes in a Ca 2+ -Dependent Manner
To further investigate the role of Ca 2+ signaling in tanshinone biosynthesis, the expression levels of key genes in LPS and LaCl3 treated WT hairy roots were analyzed by qRT-PCR. When synergistically treated by LaCl3 and LPS for 6 h, the expression levels of SmCPS and SmCYP76AH1 reduced approximately 40-fold and 37-fold, respectively, compared to the LPS separate treatment ( Figure 7A,B). However, SmHDR and SmDXS did not show apparent reduction by LaCl3+LPS treatment (Figure 7C,D). Comparatively, LaCl3 preferentially inhibits the downstream genes (SmCPS and SmCYP76AH1) in tanshinone biosynthesis pathways. This suggests that the downstream genes of the tanshinone biosynthesis pathway are more likely to be regulated by Ca 2+ signaling than the MEP pathway genes. Therefore, we present the mechanism of LPS-induced tanshinone biosynthesis in Figure 8. Firstly, LPS induces the generation of Ca 2+ signaling in the cytoplasm, which is accordingly decoded by the Ca 2+ -dependent regulators. Then, SmWRKY1 and SmWRKY2 are upregulated and activated by some undefined Ca 2+ -dependent regulators. Eventually, the key biosynthesis genes of tanshinones such as SmCPS, SmDXS, SmDXR and SmCYP76AH1 are transcribed in a high level that in turn synthesizes the tanshinones in the hairy roots of S. miltiorrhiza. Therefore, we present the mechanism of LPS-induced tanshinone biosynthesis in Figure 8. Firstly, LPS induces the generation of Ca 2+ signaling in the cytoplasm, which is accordingly decoded by the Ca 2+ -dependent regulators. Then, SmWRKY1 and SmWRKY2 are upregulated and activated by some undefined Ca 2+ -dependent regulators. Eventually, the key biosynthesis genes of tanshinones such as SmCPS, SmDXS, SmDXR and SmCYP76AH1 are transcribed in a high level that in turn synthesizes the tanshinones in the hairy roots of S. miltiorrhiza.

Discussion
The dry roots of S. miltiorrhiza (Danshen) have been used in Traditional Chinese Medicine (TCM) since 200-300AD [2]. Because of slow growth and a low content of bioactive components, the wild resources of S. miltiorrhiza cannot meet the growing requirements from pharmaceutical markets. Therefore, improving the content of pharmacological ingredients is the main purpose of metabolic research. Up to now, many approaches have been applied to enhance the content of phenolic acids and tanshinones in S. miltiorrhiza [21]. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. According to the biosynthesis pathway of tanshinones, cryptotanshinone (CT) is the first tanshinone to be generated, and then tanshinone IIA (TIIA), tanshinone IIB (TIIB), tanshinone I (TI), and dihydrotanshinone I (DTI) [3,22]. We have found that LPS significantly enhances the accumulation of tanshinones CT and DTI. Furthermore, the gene expression analysis has shown that key genes from the MVA pathway (SmAACT, SmHMGS), the MEP pathway (SmDXS, smHDR) and the downstream biosynthesis pathway (SmCPS, SmKSL, SmCYP76AH1) respond to LPS treatment in a time-dependent manner. These results demonstrate that LPS is capable of activating key genes' expression in the tanshinone biosynthesis process. It is worth noting that LPS does not obviously inhibit the growth of hairy roots. Thus, LPS can be applied as a positive elicitor to enhance the content of tanshinones without affecting the growth of the S. miltiorrhiza hairy roots. This is valuable for increasing the content of metabolites.

Discussion
The dry roots of S. miltiorrhiza (Danshen) have been used in Traditional Chinese Medicine (TCM) since 200-300AD [2]. Because of slow growth and a low content of bioactive components, the wild resources of S. miltiorrhiza cannot meet the growing requirements from pharmaceutical markets. Therefore, improving the content of pharmacological ingredients is the main purpose of metabolic research. Up to now, many approaches have been applied to enhance the content of phenolic acids and tanshinones in S. miltiorrhiza [21]. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. According to the biosynthesis pathway of tanshinones, cryptotanshinone (CT) is the first tanshinone to be generated, and then tanshinone IIA (TIIA), tanshinone IIB (TIIB), tanshinone I (TI), and dihydrotanshinone I (DTI) [3,22]. We have found that LPS significantly enhances the accumulation of tanshinones CT and DTI. Furthermore, the gene expression analysis has shown that key genes from the MVA pathway (SmAACT, SmHMGS), the MEP pathway (SmDXS, smHDR) and the downstream biosynthesis pathway (SmCPS, SmKSL, SmCYP76AH1) respond to LPS treatment in a time-dependent manner. These results demonstrate that LPS is capable of activating key genes' expression in the tanshinone biosynthesis process. It is worth noting that LPS does not obviously inhibit the growth of hairy roots. Thus, LPS can be applied as a positive elicitor to enhance the content of tanshinones without affecting the growth of the S. miltiorrhiza hairy roots. This is valuable for increasing the content of metabolites.
The bacterial component LPS is an immune activator. It is capable of inducing cytoplasm Ca 2+ elevation, which is essential for the plant innate immune response [15,34]. To analyze the role of Ca 2+ signaling in tanshinone biosynthesis, S. miltiorrhiza hairy roots were collaboratively treated by LPS and Ca 2+ inhibitors. Both Ca 2+ channel blocker LaCl 3 and CaM antagonist W-7 can significantly inhibit the accumulation of tanshinones. Further analysis has shown that the downstream biosynthetic genes (SmCPS, SmCYP76AH1) are presumably regulated by Ca 2+ signaling in priority. Based on these data, we present the pathway of LPS-induced tanshinone biosynthesis as Ca 2+ signal-Ca 2+ -dependent regulators-SmWRKY1,2-downstream genes axis in S. miltiorrhiza. Our study provides a new insight into the essential role of Ca 2+ signaling in tanshinone biosynthesis. However, the exact mechanism of how SmWRKY1 and SmWRKY2 are modulated by Ca 2+ -dependent regulators remains unresolved. In the future, searching for the Ca 2+ -dependent master regulators, which are capable of activating SmWRKY1 and SmWRKY2, might be the key to uncovering the mechanism of Ca 2+ -mediated tanshinone biosynthesis in S. miltiorrhiza. For the purpose of promoting the content of valuable metabolites, the Ca 2+ transduction pathway might be the potential regulation target.
Based on our findings, the LPS-induced Ca 2+ signal is highly associated with ion influx sourced from apoplast. In plant tissues, the arabinogalactan proteins (AGPs), negatively charged and anchored to the extracellular side of the plasma membrane, can reversibly bind with Ca 2+ and hypothetically serve as the calcium capacitor [35]. Triggered by a low pH related to plasma membrane (PM) H + -ATPases, the AGPs-Ca 2+ complex can release free Ca 2+ into the cell-surface apoplast and in turn lead to [Ca 2+ ] cyt signal generation [35]. In recent in-depth research, knockouts of the key β-glucuronosyltransferases (GlcATs), which are responsible for adding glucuronic acid (GlcA) to AGPs, resulted in reduced AGPs glucuronidation, impaired Ca 2+ signaling and consequent deficient plant development [36,37]. This AGPs-Ca 2+ interaction model highlights the crucial role of the proton pump in modulating Ca 2+ signaling. The post-translational regulation, especially phosphorylation, is central to alternating PM H + -ATPases between the auto-inhibited state and active state [38]. For instance, fusicoccin, the secreta of fungi Fusicoccum amygdali, is able to activate plant PM H + -ATPases by increasing the phosphorylation level [38,39]. Notably, LPS-induced phosphorylation of key proteins such as AMPK [40], p53 [41] and mTOR [42], has been proved by massive studies in animals. In plants, LPS might similarly regulate phosphorylation of crucial proteins and might be a potential activator of PM H + -ATPases. Thus, we further hypothesize that LPS induces Ca 2+ influx via the regulation of PM H + -ATPases. The phosphorylation modification of PM H + -ATPases could be the key to uncover LPS-generated Ca 2+ influx in S. miltiorrhiza.
In addition, in comparison with the elaborated studies in animals, LPS-regulated pathways in plants are still elusive. Up to date, several proteins including AtLBR1,2 (LPS binding protein) and OsCERK1 (LysM-type receptor-like kinase) have been determined as the key players in LPS-induced immune responses [43,44]. However, the potential correlations between these LPS-related regulators and secondary metabolism have not been deeply elucidated. Consequently, the regulatory network of LPS in plants still needs to be illuminated by more comprehensive research in the future.

Hairy Roots Culture and Treatment
The S. miltiorrhiza wild type (WT) hairy roots were generated by Agrobacterium rhizogenes (ATCC15834). The generation and culture of hairy roots were based on previous research [45]. Before analysis, hairy roots weighing 0.3 g were cultured in 50 mL 6, 7-V liquid medium [46] containing an amount of 30 g/L sucrose for 21 days at 25 • C. LPS and other reagents were added into the culturing medium on the 10th day, and then the hairy roots were harvested on the 21st day. The hairy roots were dried at 45 • C for 4 days before HPLC analysis.

Reverse Transcription and Quantitative Real-Time PCR Analysis
To investigate the expression of key biosynthesis genes, the WT hairy roots were treated by 50 µg/mL LPS in a time gradient, and the 0 h treatment was used as a control. The total RNAs of the control and LPS-treated hairy roots were extracted by a SteadyPure Plant RNA Extraction Kit (AG21019). Total RNA (1 µg) was reversely transcribed by an Evo M-MLV RT Kit (with gDNA Clean) (AG11601). The gene expression was analyzed by a SYBR ® Green Premix Pro Taq HS qPCR Kit (AG11701). qRT-PCR was conducted on a real-time PCR system (Bio-RAD CFX96, Hercules, CA, USA). SmACT was used as a reference gene. The relative expression level of a gene was calculated by the 2 −∆∆Ct method. The gene's expression level of 0 h was set to 1. Gene-specific primers were shown in the Appendix A Table A1.
To investigate the regulation of Ca 2+ signaling in gene expression, the WT hairy roots were treated by 50 µg/mL LPS and 50 µg/mL LPS + 1 mmol/L LaCl 3 for 6 h, and H 2 O was used as a control. The total RNAs of the control and the different treated hairy roots were extracted, reversely transcribed and analyzed by qRT-PCR, as mentioned above. The gene's expression level of control at 0 h was set to 1.

HPLC Analysis
The dried hairy roots powder (0.02 g) was extracted by 70% methyl alcohol (4 mL) overnight and treated by ultrasonic for 45 min. Then, the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.45 µm membrane before HPLC analysis. The content of DTI, CT, TI and TIIA was determined by the Waters 1525/2489 HPLC system (Milford, MA, USA) equipped with an InertSustain ® C18 column (5 um, 250 mm × 4.6 mm, SHIMADZU-GL, Tokyo, Japan).
The HPLC operation software was Empower 2. The detection wavelength for tanshinones was 270 nm. Elution gradients are as follows

Statistical Analysis
All experiments were conducted over three times. The results were described as the mean ± standard deviation (SD). The significant differences between different groups were calculated by the Student's t-test. (*) indicates a significant difference (0.01 < p < 0.05). (**) indicates a very significant difference (p ≤ 0.01).

Conclusions
On the basis of the data in this study, we proposed the model of LPS-enhanced tanshinone biosynthesis in S. miltiorrhiza. Firstly, LPS induces a cytoplasmic Ca 2+ signal which consequently activates the expression of the transcription factors SmWRKY1 and SmWRKY2 via some undefined Ca 2+ sensors. Then, the key biosynthesis genes of tanshinones are upregulated by SmWRKY1 and SmWRKY2. To summarize, the Ca 2+ signal-Ca 2+ -dependent regulators-SmWRKY1,2-downstream genes axis might be central to regulate tanshinone biosynthesis in S. miltiorrhiza.   LPS  lipopolysaccharide  TI  tanshinone I  TIIA  tanshinone IIA  TIIB  tanshinone IIB  CT  cryptotanshinone  DTI dihydrotanshinone I Appendix A Table A1. Primers for qRT-PCR.