- Email: [email protected]
SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots
Phytochemistry 169 (2020) 112183
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots
T
Chenlu Zhanga,1,∗, Bingcong Xingb,c,1,∗∗, Dongfeng Yangd, Min Rene, Hui Guoe, Shushen Yanga, Zongsuo Liangb,d,∗∗∗ a
College of Biological Sciences & Engineering, Shaanxi University of Technology, Hanzhong, 723001, China Institute of Soil and Water Conservation, CAS & MWR, Yangling, 712100, China c University of Chinese Academy of Sciences, Beijing, 100049, China d College of Life Sciences, Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, 310018, China e Xinxiang University, Xinxiang, 453003, China b
ARTICLE INFO Keywords: Salvia miltiorrhiza Bunge (Lamiaceae) bHLH transcription factor Phenolic acids Tanshinones Transgenic Regulation
Phenolic acids and tanshinones are the two groups of pharmaceutically active metabolites in Salvia miltiorrhiza Bunge. Their contents are the key quality indicator to evaluate S. miltiorrhiza. bHLH transcription factors have important roles in regulation of plant specialised metabolism. In this study, an endogenous bHLH transcription factor, SmbHLH3, was identified and functionally analyzed. SmbHLH3 was presented in all the six tissues and mostly expressed in fibrous roots and flowers. It was localized to the nucleus. Overexpression of SmbHLH3 decreased both phenolic acids and tanshinones contents. Contents of caffeic acid and rosmarinic acid were both decreased to 50% of the control. And accumulation of salvianolic acid B was decreased as much as 62%. Content of cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA in SmbHLH3-overexpression lines were reduced 97%, 62%, 86% and 91%, respectively. In the transgenic lines, expression of C4H1, TAT and HPPR in phenolic acids pathways were reduced to about 43%, 66% and 77% of the control, respectively. For tanshinone biosynthetic pathways, transcripts of DXS3, DXR, HMGR1, KSL1, CPS1 and CYP76AH1 were reduced to 46%, 65%, 78%, 57%, 27% and 62% of the control, respectively. There was an E/G-box specific binding site in SmbHLH3, which may bind the E/G-box present in promoter region of these biosynthetic pathway genes. Y1H results indicated that SmbHLH3 could bind the promoter of TAT, HPPR, KSL1 and CYP76AH1. These findings indicated that SmbHLH3 downregulate both phenolic acids and tanshinone accumulation through directly suppressing the transcription of key enzyme genes.
1. Introduction 'Dan Shen', the roots of Salvia miltiorrhiza Bunge (Lamiaceae) is famous for the prevention and treatment of cardiovascular and
cerebrovascular diseases. Chemical and pharmacological research has found that it has two major groups of bioactive ingredients, the hydrophilic phenolic acids and the lipophilic tanshinones (Cheng et al., 2012). Phenolic acids mainly include caffeic acid (CA), rosmarinic acid
Abbreviations: 4CL, hydroxycinnamate coenzyme A ligase; ABA, abscisic acid; C4H, cinnamic acid 4-hydroxylase; CA, caffeic acid; DFR, dihydroflavonol reductase; CT, cryptotanshinone; CTAB, cetyltrimethylammonium bromide; DT-I, dihydrotanshinone I; DP, diterpenoid phytoalexins; DPF, diterpenoid phytoalexin factor; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; HMGR, 3-hydroxy3-methylglutaryl CoA reductase; HPPR, 4-hydroxyphenylpyruvate reductase; IPP, isopentyl diphosphate; CPS, copalyl diphosphate synthase; KSL, kaurene synthaselike; MeJA, methyl jasmonate; MEP, methylerythritol phosphate; MVA, mevalonate; ORF, open reading frame; PAL, phenylalanine ammonia-lyase; PDA, photodiode array detector; PEG, polyethylene glycol; PIF, phytochrome-interacting factor; RA, rosmarinic acid; RAS, rosmarinic acid synthase; Ri, root-inducing; RT-qPCR, quantitative real-time PCR; SA, salicylic acid; SAB, salvianolic acid B; T-I, tanshinone I; T-IIA, tanshinone IIA; T-DNA, transfer DNA; TAT, tyrosine aminotransferase; TF, transcription factors ∗ Corresponding author. ∗∗ Corresponding author. Institute of Soil and Water Conservation, CAS & MWR, Yangling 712100, China. ∗∗∗ Corresponding author. College of Life Sciences, Zhejiang Sci-Tech University, 928 Second Avenue, Xiasha Higher Education Zone, Hangzhou, 310018 , China E-mail addresses: [email protected] (C. Zhang), [email protected] (B. Xing), [email protected] (Z. Liang). 1 Bingcong Xing and Chenlu Zhang contributed equally to this work. https://doi.org/10.1016/j.phytochem.2019.112183 Received 15 May 2019; Received in revised form 15 October 2019; Accepted 19 October 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 1. The chemical structures of three water-soluble phenolic acids (CA, caffeic acid; RA, rosemarinic acid; SAB, salvianolic acid B) (A) and four lipid-soluble tanshinone (T-I, tanshinone I; T-IIA, tanshinone IIA; CT, cryptotanshinone; DT-I, dihydrotanshinone I) (B) studied in this article.
cis-elements of target genes' promoter regions and active/repress its transcription. TFs have functions in regulating plant development, specialised metabolism, abiotic and biotic stress. Besides, TFs could regulate expression of multiple genes simultaneously. It could be an effective strategy that using TFs enhances the production of pharmaceutically active metabolites in plant, such as flavonoids and terpenoid indole alkaloids (Gantet and Memelink, 2002). Some TFs have been investigated and found to regulate phenolic acids and tanshinone biosynthesis in S. miltiorrhiza. For instance, study of Zhang et al. (2013) indicated that SmMYB39 negatively regulated the biosynthesis of phenolic acids. Besides, overexpression of SmMYB9b and SmMYB36 inhibited the phenolic acids accumulation but induced tanshinone concentration in S. miltiorrhiza hairy roots (Ding et al., 2017; Zhang et al., 2017). Cao et al. cloned the SmWRKY1, which positive regulated tanshinone biosynthesis (Cao et al., 2018). The bHLH superfamily TFs are the proteins that has the basic helixloop-helix structural motif and it plays an important role in regulation of pharmaceutical terpenoids biosynthesis. In Arabidopsis thaliana, AtMYC2 activated expression of sesquiterpene synthase genes TPS21 and TPS11 and then increased sesquiterpene biosynthesis (Hong et al., 2012). Phytochrome-interacting factor 5 (PIF5), a bHLH TF of A. thaliana, enhanced isopentyl diphosphate (IPP) metabolism by positively regulating the MEP pathway (Mannen et al., 2014). Diterpenoid phytoalexin factor (DPF, a bHLH TF) could positively regulate diterpenoid phytoalexins (DP) accumulation via transcriptional regulation of DP biosynthetic genes in rice (Yamamura et al., 2015). Shen et al. found that overexpression of AaMYC2 significantly increased artemisinin content in Artemisia annua (Shen et al., 2016). The bHLHs also participated in the regulation of anthocyanin which share parts of the phenylpropanoid pathway with phenolic acids (Yang et al., 2012a). The
(RA), and salvianolic acid B (SAB) (Liu et al., 2006) (Fig. 1A). Tanshinones include tanshinone I (T-I), tanshinone IIA (T-IIA), cryptotanshinone (CT), and dihydrotanshinone I (DT-I) (Shi et al., 2005) (Fig. 1B). Phenolic acids and tanshinones share a range of pharmacological effects, such as anticancer, antioxidant, antibacterial, and antiinflammatory activities (Wang et al., 2015; Zhao et al., 2011; Zhou et al., 2011). Because of its pharmacological action and commercial value, the demand for high-quality S. miltiorrhiza is increasing. Numerous studies, from genetic engineering to metabolic engineering, have focused on improving the production of phenolic acids and tanshinones in S. miltiorrhiza. Phenolic acids are biosynthesized from the phenylpropanoid pathway and the tyrosine pathway (Fig. 2) (Di et al., 2013), while tanshinone components are derived via the mevalonate (MVA) pathway in cytosol and the methylerythritol phosphate (MEP) pathway in plastids (Fig. 3) (Gao et al., 2014). The genes involved in the biosynthesis pathways were key regulators of phenolic acids and tanshinone accumulation in S. miltiorrhiza. For instance, RNAi of the SmPAL1 caused a reduction of RA biosynthesis in S. miltiorrhiza (Song and Wang, 2011). In addition, overexpression of C4H, TAT or HPPR enhanced the production of RA and SAB (Xiao et al., 2011). Moreover, the accumulation of RA and SAB obviously decreased in RAS or CYP98A14 antisense transgenic S. miltiorrhiza hairy root lines (Di et al., 2013; Zhou et al., 2018). For the tanshinone biosynthesis, overexpression of GGPPS and/ or HMGR as well as DXS can significantly enhance the tanshinone accumulation (Kai et al., 2011). Overexpression of CPS1 improved tanshinone production, whereas, silencing CPS1, CYP76AH1 and KSL1 significantly decreased the content of tanshinones (Bai et al., 2018; Cheng et al., 2014; Cui et al., 2015; Ma et al., 2016). Transcription factors (TFs) are the proteins which could bind the 2
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 2. Relative expression levels of phenolic acids biosynthetic pathway genes in transgenic hairy roots lines and the control. The results were analyzed using the comparative Ct method. The S. miltiorrhiza Actin gene was used as an internal control to normalize expression levels. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
Anthocyanin l (An-1) of Petunia positively regulated anthocyanin biosynthesis in the flowers by activating transcription of structural anthocyanin genes (Spelt et al., 2000). The GL3, EGL3, and TT8 proteins of A. thaliana forming a transcriptional regulation complex with MYB proteins and WD40 repeat containing protein TTG1, also activated anthocyanin biosynthetic genes (Dubos et al., 2010; Gonzalez et al., 2008). Another bHLH of A. thaliana, AtMYC3, interacted with JAZs to upregulate the expression of dihydroflavonol reductase (DFR) and improve anthocyanin accumulation in seedlings (Cheng et al., 2011). There are more than 127 bHLH TFs in S. miltiorrhiza, some of which were supposed to regulate the biosynthesis of either phenolic acids or tanshinones (Zhang et al., 2015). Overexpression of SmMYC2 increased the production of phenolic acids, whereas knockdown of SmMYC2a and SmMYC2b extremely decreased yield of T-I, DT-I, T-IIA and CT (Yang et al., 2017; Zhou et al., 2016). We also found two bHLHs that could positively regulate phenolic acids or tanshinone biosynthesis (Xing et al., 2018a, 2018b). However, the detailed functions of the bHLH TFs large family remain unknown. In the present study, SmbHLH3 from S. miltiorrhiza, which responds to JA signaling, was cloned and analyzed. Overexpression of SmbHLH3 inhibited the accumulation of both tanshinone and phenolic acids in S.
miltiorrhiza hairy roots. Here, we present the analysis of the mechanism of SmbHLH3-mediated regulation of tanshinones and phenolic acids biosynthetic. 2. Results 2.1. Bioinformatics analysis of SmbHLH3 SmbHLH3 contained a 1478 bp ORF, encoding a protein of 491 amino acids with a predicted molecular mass of 54.367 kDa. Its GenBank accession number is MH717249. BLAST analysis revealed that SmbHLH3 has a conserved bHLH-MYC and R2R3-MYB TFs N-terminal (48–228 aa). The SMART analysis and multiple alignments of the amino acids of SmbHLH3 revealed that it had a Helix-Loop-Helix domain (347–396 aa) (Fig. 4A). To visually examine the evolutionary origins, the SmbHLH3, along with SibHLH3 (XP_011080018), EgbHLH3-like (XP_012836308.1), OebHLH3 (XP_022854140), StbHLH (XP_006352746), NsbHLH3 (XP_009803066), CbbHLH3 (PHT46573), AcbHLH3 (PSS14567), CabHLH3 (PHT80432), LnbHLH3-like (XP_019165044), OebHLH3-like (XP_022872401), CcbHLH3 (PHU16394), PabHLH3 (XP_021814813) and bHLH3 (AT4G16430), 3
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 3. Relative expression levels of tanshinone biosynthesis pathway genes in transgenic hairy roots lines and the control. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
bHLH13 (AAM10932), bHLH17 (AT2G46510) of A. thaliana and all of the SmbHLHs previously reported were used to construct a phylogenetic tree. The result showed that SmbHLH3 is most closely related to EgbHLH3 from Erythranthe guttata and that these are classified in the subgroup R (Fig. 4B). The identity between SmbHLH3 with the AtbHLH3, AtbHLH13, and AtbHLH17 is 49%, 38% and 39%, respectively.
nucleus. To detect whether SmbHLH3 is nuclear-localized, the GFP transient expression in the onion epidermis was used. Results showed that GFP fluorescence of the control existed in the nucleus and cytoplasm. The GFP fluorescence of SmbHLH3 was dispersed in the nucleus (Fig. 5).
2.2. SmbHLH3 is a nuclear-localized protein
Expression analysis of SmbHLH3 in different parts (stem, leaf, flower, root epidermis, xylem and fibrous root) of two years old flowering S. miltiorrhiza was performed. The result showed that SmbHLH3 expressed in all the six tissues of S. miltiorrhiza, with the highest
2.3. Tissue-specific expression and induction pattern of SmbHLH3
Subcellular localization of protein could help reveal its potential function. TFs mainly function by binding to the gene promoter in the 4
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 4. Sequence analysis of SmbHLH3. (A) Deduced amino acid sequence of SmbHLH3. Predicted HLH domain was present in shaded area. (B) Phylogenetic analysis of SmbHLH3. A phylogenetic tree was constructed based on the amino acid sequences of SmbHLH3 and SibHLH3 (XP_011080018), EgbHLH3-like (XP_012836308.1), OebHLH3 (XP_022854140), StbHLH (XP_006352746), NsbHLH3 (XP_009803066), CbbHLH3 (PHT46573), AcbHLH3 (PSS14567), CabHLH3 (PHT80432), LnbHLH3like (XP_019165044), OebHLH3-like (XP_022872401), CcbHLH3 (PHU16394), PabHLH3 (XP_021814813), AtbHLH3 (AT4G16430), AtbHLH13 (AAM10932), AtbHLH17 (AT2G46510) and all of the SmbHLHs that previously reported. These phylogenetic trees were constructed via MEGA6, using the neighbor-joining method with 500 bootstrap replicates.
5
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
analyzed with RT-qPCR. Among them, two overexpression lines (OEbHLH3-1, OE-bHLH3-2) presented higher expression level (2-fold greater than the WT) of SmbHLH3. Transcription level of SmbHLH3 in the two overexpressing lines (OE-bHLH3-1, OE-bHLH3-2) was approximately 9.09- and 7.43-fold with respect to the WT, respectively (Fig. 7C). 2.5. Both phenolic acids and tanshinone biosynthesis were inhibited in SmbHLH3 overexpression hairy roots Compared with the WT, accumulation of phenolic acids such as CA, RA and SAB decreased in SmbHLH3-overexpression hairy roots lines. CA content in OE-bHLH3-1 decreased to 0.078 mg/g DW, only 50% of the control. The RA content was also decreased to half of the control in OEbHLH3-1(2.56 mg/g DW) and OE-bHLH3-2 (2.75 mg/g DW). And content of SAB in OE-bHLH3-1 and OE-bHLH3-2 was decreased by 62% (4.17 mg/g DW) and 50%, respectively (Fig. 8A). To examine the effect of SmbHLH3 on tanshinone biosynthesis in S. miltiorrhiza, contents of DT-I, CT, T-I and T-II A were determined. Results showed that accumulation of all the four tanshinons ingredients was suppressed in SmbHLH3-overexpression lines. The CT was reduced as much as 97% in OE-bHLH3-2. And the decrease of T-II A was reached 91%. Content of DT- I and T- I were decreased to 38% and 14% of the control, respectively (Fig. 8B). In OE-bHLH3-1 transgenic line, accumulation of CT, DT- I, T- I, T-II A was reduced to 10%, 37%, 34%, 11% of the control, respectively (Fig. 8B). These results indicated that SmbHLH3 could inhibited both phenolic acids and tanshinone biosynthesis in S. miltiorrhiza hairy roots.
Fig. 5. Subcellular localization of SmbHLH3 protein in onion epidermal cells. Fluorescence was observed using a confocal laser scanning microscope at 24 h after incubation. The pictures showed bright field (TD), green fluorescent field (GFP), DAPI and overlay of three fields (Merge). The numerical reading of red ruler is 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.6. SmbHLH3 downregulated expression of genes involved in phenolic acids and tanshinone biosynthetic pathways To uncover the function of SmbHLH3 on phenolic acids and tanshinone accumulation, we analyzed the expression of genes for the main enzymes in their biosynthetic pathways by RT-qPCR. Overexpression of SmbHLH3 repressed genes expression of the phenylpropanoid pathway (C4H1), tyrosine pathway (TAT, HPPR) (Fig. 2). Expression of C4H1 was reduced about 45% and 57% in OE-bHLH3-1 and OE-bHLH3-2, respectively. Transcription of TAT and HPPR was suppressed to 66% and 77% of the control in OE-bHLH3-2. However, transcription levels of PAL1, 4CL2, RAS and CYP98A14 were present no changes in the transgenic lines when compared to the control. For tanshinone biosynthetic pathways, DXS3 and DXR of MEP pathway, HMGR1 of MVA pathway, and CPS1, KSL1 and CYP76AH1 of downstream pathway were all downregulated in SmbHLH3 overexpression lines compared with those in the control. And transcription levels of DXS2, HMGR2 and GGPPS were present no changes by overexpression of SmbHLH3 (Fig. 3). Expressions of DXS3, DXR, HMGR1 and KSL1 were also the lowest in line OE-bHLH3-1, 46%, 65%, 78% and 57% of the control, respectively. While CPS1 and CYP76AH1 expression was the lowest in line OE-bHLH3-2, reduced 27% and 62%, respectively. The variations of these pathway genes transcription were generally consistent with the active ingredients' contents.
Fig. 6. Expression pattern of SmbHLH3 in different tissues of S. miltiorrhiza. Bars are means ± SD from three independent biological replicates. One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
2.7. SmbHLH3 binds the predicted G-box motifs of TAT, HPPR, KSL1 and CYP76AH1
expression in fibrous root and the lowest expression in stem. (Fig. 6). 2.4. Generation of SmbHLH3-overexpression hairy roots
Y1H Gold reporter of strains which has G-box, mG-box, TAT, HPPR, KSL1 and CYP76AH1 promoters could be inhibited by 400, 400, 300, 900, 200 and 150 ng/ml concentration of aureobasidin A (AbA), respectively. And when transformed with SmbHLH3 prey plasmid, except for mG-box, these strains can grow on synthetic medium lacking leucine (SD/-Leu) under corresponding concentration of AbA (Fig. 9). These results showed that SmbHLH3 could successfully bind the G-box of TAT, HPPR, KSL1and CYP76AH1.
In the present study, 5 transgenic hairy roots lines were obtained through hygromycin resistance screening and PCR selection (Fig. 7A). The phenotypes of hairy roots presented by SmbHLH3 overexpression hairy roots showed a lighter color than the WT (the line infected by Agrobacterium rhizogenes, ATCC15834 only) (Fig. 7B). Relative expression level of SmbHLH3 in all the transgenic hairy root lines was 6
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 7. (A) Identification of positive transgenic hairy roots lines by PCR (A, 35S + SmbHLH3; B, hpt II; C, rol b; D, rol c). Numbers above represent individual transgenic lines and M represent DL2000 DNA marker. (B) The phenotypes of hairy roots. Hairy roots were cultured in 6,7-V liquid medium for 30 days before being photographed. (C) Relative quantitative analysis of SmbHLH3 expression in transgenic lines and control of S. miltiorrhiza hairy roots. Bars are means ± SD from three independent biological replicates. One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
3. Discussion
bioactive ingredients in S. miltiorrhiza, SmbHLH3-overexpression hairy roots were obtained by A. rhizogenes mediated method. The WT hairy roots were much redder than all the transgenic lines (Fig. 7B). It has been reported that more tanshinones would be obtained in the redder roots of S. miltiorrhiza (Wang et al., 2014). This result indicated that there is a lower level of tanshinones in the transgenic hairy roots lines than in the WT. HPLC results showed that the content of four tanshinones and three phenolic acids were all diminished in SmbHLH3 overexpression hairy roots (Fig. 8). Compared with the control, contents of CA, RA and SAB decreased by 50%, 50% and 62% in the OEbHLH3 lines, respectively (Fig. 8A). For all four tanshinones ingredients accumulation was suppressed in SmbHLH3-overexpression lines as well. The CT was reduced as much as 97% in OE-bHLH3-2. And the decrease of T-II A reached 91%. Contents of DT- I and T- I were decreased to 38% and 14.48% of the control, respectively (Fig. 8B). To uncover the downregulation mechanism mediated by SmbHLH3, the expression of phenolic acids and tanshinone biosynthetic genes was analyzed. The results indicated that transcription of C4H1, TAT and HPPR were repressed in SmbHLH3-overexpression lines (Fig. 2). Expression of C4H1 was reduced about 45% and 57% in OE-bHLH3-1 and OE-bHLH3-2, respectively. It was reported that C4H may represent regulatory bottlenecks in phenolic acid biosynthesis (Xiao et al., 2011). And overexpression of C4H significantly enhanced target phenolic acids accumulation. Transcription of TAT and HPPR were suppressed to 66% and 77% of the control in OE-bHLH3-2. TAT or HPPR overexpression and TAT-HPPR co-expression also improved the production of RA and SAB, and co-transformed lines produced more phenolic acids (Xiao
It was reported that 127 bHLH TF genes were identified in the genome of S. miltiorrhiza, and these SmbHLHs were classified into 25 subfamilies (Zhang et al., 2015). In the present study, an endogenous bHLH TF SmbHLH3 from S. miltiorrhiza was functionally identified. Phylogenetic analysis showed that SmbHLH3 is most closely related to SmbHLH13, SmbHLH37 and SmbHLH53 and is classified with these genes in the subgroup R (Fig. 4B). Most of the bHLHs in subfamily R were specific to S. miltiorrhiza, and MeJA responses (Zhang et al., 2015). MeJA could regulate the biosynthesis of both tanshinones and phenolic acids (Xing et al., 2018c). And the SmbHLH37 expression pattern was perfectly matched with the accumulation pattern of tanshinones. It was reported that MYC2 belongs to the subfamily R together with SmbHLH37, positively regulate the production of both phenolic acids and tanshinones in S. miltiorrhiza (Yang et al., 2017; Zhou et al., 2016). Therefore, SmbHLH3 could be a candidate TF for the regulation of the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza. SmbHLH3 expressed in all the six parts (stem, leaf, flower, root epidermis, xylem and fibrous root) of S. miltiorrhiza, with the highest expression in fibrous root and the lowest expression in stem (Fig. 6). Its expression pattern has correlations with phenolic acids and tanshinone accumulation. Phenolic acids and tanshinones were mainly present in the root epidermis. It was also being detected in leaves, flowers and stem with much lower content. To investigate whether SmbHLH3 could regulate biosynthesis of 7
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 8. Contents of CA, RA, SAB (A) and T-I, T-IIA, CT, DT-I (B) in transgenic and the control hairy roots lines of S. miltiorrhiza. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.05).
et al., 2011). It could indicate that TAT and HPPR also play a key role in phenolic acids biosynthesis. Most of the genes involved in tanshinone biosynthesis pathway, including DXS3 and DXR of MEP pathway, HMGR1 of MVA pathway, the CPS1, KSL1 and CYP76AH1 of downstream pathway, were downregulated in SmbHLH3 overexpression lines compared with those in the control (Fig. 3). In line OE-bHLH3-1, expression of DXS3, DXR, HMGR1 was decreased by 46%, 65% and 78% of the control, respectively. It has been reported that tanshinone production could be significantly improved by overexpression of HMGR as well as DXS (Kai et al., 2011). Also, these authors found SmDXS showed much more powerful effects than SmHMGR on tanshinone production. DXS and DXR always show closely similar effects on tanshinone accumulation. When treated with elicitors, such as MeJA, Ag+, polyethylene glycol (PEG), and ABA, variation of DXS and DXR were more associated with tanshinone level changes than HMGR (Xing et al., 2014, 2018c; Yang et al., 2012b), indicating that the MEP pathway plays a
more important role in tanshinone biosynthesis than the MVA pathway. Cheng et al. (2014) reported that when the transcription level of SmCPS was reduced to 26% in SmCPS–RNAi hairy roots, the DT- I and CT content were decreased by 53 and 38% of the control, and T- IIA was not detected. In the present study, CPS1 expression was reduced 27% in line OE-bHLH3-2. Silencing of CYP76AH1 also decreased the content of tanshinones (Ma et al., 2016). CYP76AH1 expression was reduced 62% in SmbHLH3 overexpression lines. These findings indicated that SmbHLH3 could be decrease the biosynthesis of phenolic acids and tanshinones by repressing the transcription of pathway genes. The TFs active/repress the gene expression by binding to gene promoters. We found a G-box binding site in the amino acid of SmbHLH3 (Fig. 4A). A G-box was present in the promoter regions of PAL1, C4H1, TAT, HPPR, RAS, CYP98A14 of the phenolic acids biosynthetic pathway, and DXS, DXR, HMGR, CPS1, KSL1, CYP76AH1 of the tanshinone biosynthetic pathway. Y1H results showed that 8
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 9. SmbHLH3 binding with the G-box motifs of the TAT, HPPR, KSL1and CYP76AH1.
the MEGA 6 software program by employing the neighbor joining method, with 500 bootstrap replicates. All amino acid sequences of the other species were downloaded from the NCBI database.
SmbHLH3 could binding to the G-box motifs of the TAT, HPPR, KSL1and CYP76AH1 promoters in yeast cells. bHLH TFs often interact with MYB family proteins to form a complex, and then regulate the transcription of target genes (Feller et al., 2011). We found a bHLHMYC_N-domain in SmbHLH3, which was also called MYB interactive region, in the SmbHLH3 (Fig. 4A). It was essential for binding with MYB proteins (Pattanaik et al., 2008). This indicated that there might be some MYBs involved in the progress of SmbHLH3 regulate phenolic acids and tanshinone biosynthesis. However, the function mode of SmbHLH3 is by complex with the MYBs or independently needs further study.
5.2. Total RNA and DNA extraction Total complete RNA was isolated from frozen S. miltiorrhiza hairy roots or plants by using the RNAprep Pure Plant Kit (TIANGEN, China). The RNA was then reversely transcribed to generate the first strand cDNA, according to the manufacturer's instructions of the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Japan). An improved cetyltrimethylammonium bromide (CTAB) method was used to isolate the genomic DNA. The quality and concentration of the genomic DNA and RNA were examined by agarose gel electrophoresis and spectrophotometer analysis (Thermo Scientific NanoDrop, 2000).
4. Conclusion An endogenous bHLH TF named SmbHLH3 was identified and functionally analyzed in S. miltiorrhiza. It belongs to the subfamily R of SmbHLHs. Overexpression of SmbHLH3 decreased the biosynthesis of both the two groups of bioactive ingredients. Transcriptions of almost all the key genes involved in phenolic acids and tanshinone biosynthesis were repressed by SmbHLH3. These findings indicated that SmbHLH3 decreased phenolic acids and tanshinone biosynthesis by repressing the transcription of pathway genes. Moreover, SmbHLH3 binding site (Gbox) was present in the promoter of these genes, and Y1H results showed that SmbHLH3 bind to the G-box motifs of TAT, HPPR, KSL1and CYP76AH1 promoters in yeast cells. These findings indicated that SmbHLH3 directly bind to the pathway genes promoter, and repress their expression and then downregulate phenolic acids and tanshinone biosynthesis.
5.3. Subcellular localization analysis The ORF of SmbHLH3 was amplified with primers SmGFP1Sal I (5′-ACGCGTCGACATGGGGAGTAAGTTTTGGTTGAATG-3′) and SmGFP2-BamH I (5′-CGGGATCCCGCACCACCACCACCACCATTTAAGA GAGCAGCAGCCAACTTA-3′) using PrimeSTAR® HS DNA Polymerase (Takara, Japan). The amplification sequence was ligated with Sal I and BamH I -digested pTF486 vector to generate a SmbHLH3-GFP fusion construct under the control of cauliflower mosaic virus 35S (CaMV 35S) promoter (Liu et al., 2010). The construct was confirmed by sequencing and used for transient transformation of onion epidermis via a gene gun (Bio-Rad, Hercules, CA, USA). After 24 h of incubation, GFP fluorescence in transformed onion cells was observed under a confocal microscope (Nikon A1, Tokyo, Japan).
5. Materials and methods
5.4. Construction of plant expression vectors and acquisition of positive transgenic hairy roots
5.1. Bioinformatics analysis of SmbHLH3 SmbHLH3 was isolated from the roots of Salvia miltiorrhiza Bunge (Lamiaceae) transcriptome database (Shao et al., 2016) and the open reading frame (ORF) was found with the OFP-finder (https://www. ncbi.nlm.nih.gov/orffinder/). BLAST was used to determine the differences between SmbHLH3 sequences and the NCBI database (https:// www.ncbi.nlm.nih.gov/). Its conserved domain was identified by BLASTP of NCBI database and searched with SMART server (http:// smart.embl-heidelberg.de/). Phylogenetic trees were generated with
The coding region of SmbHLH3 was amplified and cloned into the restriction sites Xba I and Sac I of the pCAMBIA1300 binary vector under the control of the CaMV35S promoter and the NOS terminator. The recombinant plasmids SmbHLH3-1300 was transformed into A. rhizogenes (ATCC15834). The hairy roots lines were acquired from the transformation of leaf explants of the S. miltiorrhiza sterile plantlets. The positive transgenic lines were identified by PCR using rolB, rolC, HPT 9
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
and 35S-SmbHLH3 specific primer. All the primers used for the overexpression vector construction and the PCR identification of transgenic lines are listed in Table S1. Samples of the hairy roots weighing 0.2 g were inoculated into 100 mL beaker flasks containing 50 mL of 6,7-V liquid medium (with 30 g L−1 sucrose). The beaker flasks containing the hairy roots were then placed on an orbital shaker at 110 rpm min−1, 25 °C in the dark. These latter lines were used for RNA and HPLC analysis and they were regularly sub-cultured (every 30 days).
triplicate, and the results were represented by their means ± SD. 5.8. Yeast One-Hybrid (Y1H) assays The Y1H was used to detect whether SmbHLH3 directly interacted with the promoters of key genes in the phenolic acids and tanshinone biosynthetic pathways according the MATCHMAKER Gold Yeast OneHybrid system manual (Clontech). The promoter elements of C4H1, TAT, HPPR, DXS3, DXR, CPS1 and CYP76AH1 (which expressions were suppressed by overexpression of SmbHLH3) were analyzed with the Plant CARE online sever (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/). And fragments from the promoters of C4H1, TAT, HPPR, DXS3, DXR, KSL1, CPS1 and CYP76AH1 were amplified, which contain the G-box-like elements. The fragments obtained were inserted into pAbAi vector with In-Fusion Cloning Kit (Clontech). All bait constructs were linearized and integrated into the genome of the Y1H Gold yeast stain, selected on synthetic defined SD/-Ura agar medium plate and cultured at 30 °C for 2 days. The prey construct was the ORF of SmbHLH3 fused in-frame with the yeast GAL4 transcription activation domain (GAL4 AD) in pGADT7 with In-Fusion assay. The prey construct was then introduced into yeast cells previously transformed with the bait constructs, with blank pGADT7 plasmid as control. The positively transformed clones were cultured at 30 °C on SD/-Leu medium with different concentrations of AbA. The primers used for amplification of the G-box-like fragments and construct prey were designed with online in-fusion tools (http://www.clontech.com/US/Products/Cloning_and_ Competent_Cells/Cloning_Resources/Online_In-Fusion_Tools) are listed in Table S3. The G-box (CACGTG) and mG-box (ACCGTA) were performed as positive and negative control, respectively as Shen et al. (2016) described.
5.5. HPLC analysis The dried hairy roots were powdered using a homogenizer (Bioprep24, Hangzhou, China). The sample powder (20 mg) was extracted with 70% methanol (2 mL) under ultrasonic treatment for 45 min and the resulting mixture was centrifuged at 8000×g for 5 min. The supernatant was filtered through a 0.45 μm organic membrane filter and analyzed by HPLC. The metabolite contents were determined on a Waters HPLC e2695 system (Waters, Milford, MA, USA) equipped with an automatic sample injector and a Waters 2996 photodiode array detector. Chromatographic separation was performed using a SunFire C18 column (4.6 mm × 250 mm, 5 μm particle size) at 30 °C. Empower 3 software was used for data acquisition and processing. The sample injection volume was 10 μL and the PDA wavelengths used for the detection of the lipid-soluble diterpenoids and phenolic acids were 270 and 288 nm, respectively. Separation was achieved by linear gradient elution with solvents A (acetonitrile) and B (0.026% phosphoric acid solution). The gradient was as follows (all concentrations were v/v): 0–10 min, 5–20% A; 10–15 min, 20–25% A; 15–20 min, 25% A; 20–25 min, 25–20% A; 25–28 min, 20–30% A; 28–40 min, 30% A; 40–45 min, 30–45% A; 45–58 min, 45–58% A; 58–67 min, 58–50% A; 67–70 min, 50–60% A; 70–80 min, 60–65% A; 80–85 min, 65–95% A; and 85–95 min, 95% A.
Author contributions ZL, SY conceived and designed the research. BX, DY and CZ conducted experiments and analyzed the data. BX wrote the manuscript. MR and HG contributed advice and revised.
5.6. Standard curves A series of standard solutions of CA, RA, SAB, T-I, CT, DT-I and T-IIA were used to determine the linearity and the linearity range of the analytes in the developed method. The detector response was linearly correlated with content, in the ranges of 0.02–1 mg mL−1 for CA, RA, SAB, T-I, CT, DT-I and T-IIA. The linearity of each standard curve was confirmed by plotting the peak area (y) and the corresponding concentration (x, g·mL-1) of the analytes. The regression equations and correlation coefficients were [y = 44,272,869.30912 x + 657,954.18145] (R2 = 0.99825) for CA, [y = 32,773,318.17664 x - 238,200.78917] (R2 = 0.98605) for RA, [y = 13,281,967.79415 x + 297,278.96047] (R2 = 0.99967) for SAB, [y = 1,806,477.7934 x + 20,298.5506] (R2 = 0.9999) for T-I, [y = 3,229,227.8047 x + 29,146.1676] (R2 = 0.9988) for CT, [y = 3,387,376.8337 x 39,835.6132] (R2 = 0.9997) for DT-I and [y = 5,190,705.9762 x + 9846.8041] (R2 = 0.9997) for T-IIA. In this study, authentic standards were obtained from the National Institute for the Control of Biological and Pharmaceutical Products (Beijing, China).
Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81703646, 81773835 and 81673536), Natural Science Foundation of Zhejiang Provincial (No. LZ16H280001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112183.
5.7. Real-time quantitative PCR analysis
References
The first strand cDNA for RT-qPCR were synthesized using the PrimeScript™ RT Master Mix (Perfect Real Time) (Takara, Tokyo, Japan). Primers used for RT-qPCR were list at supplement Table S2. The SmActin gene was used as reference. RT-qPCR was performed according to the manufacturer's instruction of TB Green™ Premix Ex Taq™ II (TliRNaseH Plus, Takara) using the following protocol: 95 °C, 30 s, 1 cycle; 95 °C, 5 s, 58 °C, 30 s, 40 cycles. The program was performed on the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Massachusetts, USA). Quantification of the gene's expression was done with comparative CT method (2−ΔΔCT). Experiments were performed in
Bai, Z., Li, W., Jia, Y., Yue, Z., Jiao, J., Huang, W., Xia, P., Liang, Z., 2018. The ethylene response factor SmERF6 co-regulates the transcription of SmCPS1 and SmKSL1 and is involved in tanshinone biosynthesis in Salvia miltiorrhiza hairy roots. Planta 248, 243–255. Cao, W., Wang, Y., Shi, M., Hao, X., Zhao, W., Wang, Y., Ren, J., Kai, G., 2018. Transcription factor SmWRKY1 positively promotes the biosynthesis of tanshinones in Salvia miltiorrhiza. Front. Plant Sci. 9, 554. Cheng, H.T., Li, X.L., Li, X.R., Li, Y.H., Wang, L.J., Xue, M., 2012. Simultaneous quantification of selected compounds from Salvia herbs by HPLC method and their application. Food Chem. 130, 1031–1035. Cheng, Q.Q., Su, P., Hu, Y.T., He, Y.F., Gao, W., Huang, L.Q., 2014. RNA interferencemediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy
10
Phytochemistry 169 (2020) 112183
C. Zhang, et al. roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS. Biotechnol. Lett. 36, 363–369. Cheng, Z., Sun, L., Qi, T., Zhang, B., Peng, W., Liu, Y., Xie, D., 2011. The bHLH transcription factor MYC3 interacts with the Jasmonate ZIM-domain proteins to mediate jasmonate response in Arabidopsis. Mol. Plant 4, 279–288. Cui, G., Duan, L., Jin, B., Qian, J., Xue, Z., Shen, G., Snyder, J.H., Song, J., Chen, S., Huang, L., Peters, R.J., Qi, X., 2015. Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza. Plant Physiol. 169, 1607–1618. Di, P., Zhang, L., Chen, J., Tan, H., Xiao, Y., Dong, X., Zhou, X., Chen, W., 2013. 13C tracer reveals phenolic acids biosynthesis in hairy root cultures of Salvia miltiorrhiza. ACS Chem. Biol. 8, 1537–1548. Ding, K., Pei, T., Bai, Z., Jia, Y., Ma, P., Liang, Z., 2017. SmMYB36, a novel R2R3-MYB transcription factor, enhances tanshinone accumulation and decreases phenolic acid content in Salvia miltiorrhiza hairy roots. Sci. Rep. 7, 5104. Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., Lepiniec, L., 2010. MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573–581. Feller, A., Machemer, K., Braun, E.L., Grotewold, E., 2011. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J. 66, 94–116. Gantet, P., Memelink, J., 2002. Transcription factors: tools to engineer the production of pharmacologically active plant metabolites. Trends Pharmacol. Sci. 23, 563–569. Gao, W., Sun, H.X., Xiao, H., Cui, G., Hillwig, M.L., Jackson, A., Wang, X., Shen, Y., Zhao, N., Zhang, L., Wang, X.J., Peters, R.J., Huang, L., 2014. Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza. BMC Genomics 15, 73. Gonzalez, A., Zhao, M., Leavitt, J.M., Lloyd, A.M., 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 53, 814–827. Hong, G.J., Xue, X.Y., Mao, Y.B., Wang, L.J., Chen, X.Y., 2012. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell 24, 2635–2648. Kai, G.Y., Xu, H., Zhou, C.C., Liao, P., Xiao, J.B., Luo, X.Q., You, L.J., Zhang, L., 2011. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab. Eng. 13, 319–327. Liu, A.H., Li, L., Xu, M., Lin, Y.H., Guo, H.Z., Guo, D.A., 2006. Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC–DAD method. J. Pharm. Biomed. 41, 48–56. Liu, X., Rodermel, S.R., Yu, F., 2010. A var2 leaf variegation suppressor locus, SUPPRESSOR OF VARIEGATION3, encodes a putative chloroplast translation elongation factor that is important for chloroplast development in the cold. BMC Plant Biol. 10 287-287. Ma, Y., Ma, X.H., Meng, F.Y., Zhan, Z.L., Guo, J., Huang, L.Q., 2016. RNA interference targeting CYP76AH1 in hairy roots of Salvia miltiorrhiza reveals its key role in the biosynthetic pathway of tanshinones. Biochem. Biophys. Res. Commun. 477, 155–160. Mannen, K., Matsumoto, T., Takahashi, S., Yamaguchi, Y., Tsukagoshi, M., Sano, R., Suzuki, H., Sakurai, N., Shibata, D., Koyama, T., Nakayama, T., 2014. Coordinated transcriptional regulation of isopentenyl diphosphate biosynthetic pathway enzymes in plastids by phytochrome-interacting factor 5. Biochem. Bioph. Res. Co. 443, 768–774. Pattanaik, S., Xie, C.H., Yuan, L., 2008. The interaction domains of the plant Myc-like bHLH transcription factors can regulate the transactivation strength. Planta 227, 707–715. Shao, Y., Wei, J., Wu, F., Zhang, H., Yang, D., Liang, Z., Jin, W., 2016. DsTRD: danshen transcriptional resource database. PLoS One 11. Shen, Q., Lu, X., Yan, T., Fu, X., Lv, Z., Zhang, F., Pan, Q., Wang, G., Sun, X., Tang, K., 2016. The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol. 210, 1269–1281. Shi, Z., He, J., Yao, T., Chang, W., Zhao, M., 2005. Simultaneous determination of cryptotanshinone, tanshinone I and tanshinone IIA in traditional Chinese medicinal preparations containing Radix Salvia miltiorrhiza by HPLC. J. Pharm. Biomed. Anal. 37, 481–486.
Song, J., Wang, Z., 2011. RNAi-mediated suppression of the phenylalanine ammonialyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis. J. Plant Res. 124, 183–192. Spelt, C., Quattrocchio, F., Mol, J.N.M., Koes, R., 2000. anthocyanin1 of Petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12, 1619–1631. Wang, D., Zhang, W., Wang, T., Li, N., Mu, H., Zhang, J., Duan, J., 2015. Unveiling the mode of action of two antibacterial tanshinone derivatives. Int. J. Mol. Sci. 16, 17668–17681. Wang, Hai, Yan, Zhuyun, Shen, Yuxiang, He, Dongmei, Lan, Ying, Deguang, W., 2014. Correlation of color feature with effective constituents of radix salviae miltiorrhizae. Tradit. Chin. Drug Res. Clin. Pharmacol. 3, 333–338. Xiao, Y., Zhang, L., Gao, S., Saechao, S., Di, P., Chen, J., Chen, W., 2011. The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. PLoS One 6, e29713. Xing, B., Yang, D., Yu, H., Zhang, B., Yan, K., Zhang, X., Han, R., Liang, Z., 2018a. Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots. Plant Sci. 229–238. Xing, B., Liang, L., Liu, L., Hou, Z., Yang, D., Yan, K., Zhang, X., Liang, Z., 2018b. Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots. Plant Cell Rep. 37, 1681–1692. Xing, B., Yang, D., Guo, W., Liang, Z., Yan, X., Zhu, Y., Liu, Y., 2014. Ag+ as a more effective elicitor for production of tanshinones than phenolic acids in Salvia miltiorrhiza hairy roots. Molecules 20, 309–324. Xing, B., Yang, D., Liu, L., Han, R., Sun, Y., Liang, Z., 2018c. Phenolic acid production is more effectively enhanced than tanshinone production by methyl jasmonate in Salvia miltiorrhiza hairy roots. Plant Cell Tissue Organ Cult. 134, 119–129. Yamamura, C., Mizutani, E., Okada, K., Nakagawa, H., Fukushima, S., Tanaka, A., Maeda, S., Kamakura, T., Yamane, H., Takatsuji, H., Mori, M., 2015. Diterpenoid phytoalexin factor, a bHLH transcription factor, plays a central role in the biosynthesis of diterpenoid phytoalexins in rice. Plant J. 84, 1100–1113. Yang, C.Q., Fang, X., Wu, X.M., Mao, Y.B., Wang, L.J., Chen, X.Y., 2012a. Transcriptional regulation of plant secondary metabolism. J. Integr. Plant Biol. 54, 703–712. Yang, D., Ma, P., Liang, X., Wei, Z., Liang, Z., Liu, Y., Liu, F., 2012b. PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots. Physiol. Plant. 146, 173–183. Yang, N., Zhou, W., Su, J., Wang, X., Li, L., Wang, L., Cao, X., Wang, Z., 2017. Overexpression of SmMYC2 increases the production of phenolic acids in Salvia miltiorrhiza. Front. Plant Sci. 8, 1804. Zhang, J., Zhou, L., Zheng, X., Zhang, J., Yang, L., Tan, R., Zhao, S., 2017. Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots. Plant Cell Rep. 36, 1297–1309. Zhang, S., Ma, P., Yang, D., Li, W., Liang, Z., Liu, Y., Liu, F., 2013. Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza. PLoS One 8, e73259. Zhang, X., Luo, H., Xu, Z., Zhu, Y., Ji, A., Song, J., Chen, S., 2015. Genome-wide characterisation and analysis of bHLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza. Sci. Rep. 5. Zhao, J., Lou, J., Mou, Y., Li, P., Wu, J., Zhou, L., 2011. Diterpenoid tanshinones and phenolic acids from cultured hairy roots of Salvia miltiorrhiza Bunge and their antimicrobial activities. Molecules 16, 2259–2267. Zhou, Y., Li, W., Xu, L., Chen, L., 2011. In Salvia miltiorrhiza, phenolic acids possess protective properties against amyloid beta-induced cytotoxicity, and tanshinones act as acetylcholinesterase inhibitors. Environ. Toxicol. Pharmacol. 31, 443–452. Zhou, Y., Sun, W., Chen, J., Tan, H., Xiao, Y., Li, Q., Ji, Q., Gao, S., Chen, L., Chen, S., Zhang, L., Chen, W., 2016. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza. Sci. Rep. 6, 22852. Zhou, Z., Tan, H., Li, Q., Chen, J., Gao, S., Wang, Y., Chen, W., Zhang, L., 2018. CRISPR/ Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza. Phytochemistry 148, 63–70.
11
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots
T
Chenlu Zhanga,1,∗, Bingcong Xingb,c,1,∗∗, Dongfeng Yangd, Min Rene, Hui Guoe, Shushen Yanga, Zongsuo Liangb,d,∗∗∗ a
College of Biological Sciences & Engineering, Shaanxi University of Technology, Hanzhong, 723001, China Institute of Soil and Water Conservation, CAS & MWR, Yangling, 712100, China c University of Chinese Academy of Sciences, Beijing, 100049, China d College of Life Sciences, Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Zhejiang Sci-Tech University, Hangzhou, 310018, China e Xinxiang University, Xinxiang, 453003, China b
ARTICLE INFO Keywords: Salvia miltiorrhiza Bunge (Lamiaceae) bHLH transcription factor Phenolic acids Tanshinones Transgenic Regulation
Phenolic acids and tanshinones are the two groups of pharmaceutically active metabolites in Salvia miltiorrhiza Bunge. Their contents are the key quality indicator to evaluate S. miltiorrhiza. bHLH transcription factors have important roles in regulation of plant specialised metabolism. In this study, an endogenous bHLH transcription factor, SmbHLH3, was identified and functionally analyzed. SmbHLH3 was presented in all the six tissues and mostly expressed in fibrous roots and flowers. It was localized to the nucleus. Overexpression of SmbHLH3 decreased both phenolic acids and tanshinones contents. Contents of caffeic acid and rosmarinic acid were both decreased to 50% of the control. And accumulation of salvianolic acid B was decreased as much as 62%. Content of cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA in SmbHLH3-overexpression lines were reduced 97%, 62%, 86% and 91%, respectively. In the transgenic lines, expression of C4H1, TAT and HPPR in phenolic acids pathways were reduced to about 43%, 66% and 77% of the control, respectively. For tanshinone biosynthetic pathways, transcripts of DXS3, DXR, HMGR1, KSL1, CPS1 and CYP76AH1 were reduced to 46%, 65%, 78%, 57%, 27% and 62% of the control, respectively. There was an E/G-box specific binding site in SmbHLH3, which may bind the E/G-box present in promoter region of these biosynthetic pathway genes. Y1H results indicated that SmbHLH3 could bind the promoter of TAT, HPPR, KSL1 and CYP76AH1. These findings indicated that SmbHLH3 downregulate both phenolic acids and tanshinone accumulation through directly suppressing the transcription of key enzyme genes.
1. Introduction 'Dan Shen', the roots of Salvia miltiorrhiza Bunge (Lamiaceae) is famous for the prevention and treatment of cardiovascular and
cerebrovascular diseases. Chemical and pharmacological research has found that it has two major groups of bioactive ingredients, the hydrophilic phenolic acids and the lipophilic tanshinones (Cheng et al., 2012). Phenolic acids mainly include caffeic acid (CA), rosmarinic acid
Abbreviations: 4CL, hydroxycinnamate coenzyme A ligase; ABA, abscisic acid; C4H, cinnamic acid 4-hydroxylase; CA, caffeic acid; DFR, dihydroflavonol reductase; CT, cryptotanshinone; CTAB, cetyltrimethylammonium bromide; DT-I, dihydrotanshinone I; DP, diterpenoid phytoalexins; DPF, diterpenoid phytoalexin factor; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; HMGR, 3-hydroxy3-methylglutaryl CoA reductase; HPPR, 4-hydroxyphenylpyruvate reductase; IPP, isopentyl diphosphate; CPS, copalyl diphosphate synthase; KSL, kaurene synthaselike; MeJA, methyl jasmonate; MEP, methylerythritol phosphate; MVA, mevalonate; ORF, open reading frame; PAL, phenylalanine ammonia-lyase; PDA, photodiode array detector; PEG, polyethylene glycol; PIF, phytochrome-interacting factor; RA, rosmarinic acid; RAS, rosmarinic acid synthase; Ri, root-inducing; RT-qPCR, quantitative real-time PCR; SA, salicylic acid; SAB, salvianolic acid B; T-I, tanshinone I; T-IIA, tanshinone IIA; T-DNA, transfer DNA; TAT, tyrosine aminotransferase; TF, transcription factors ∗ Corresponding author. ∗∗ Corresponding author. Institute of Soil and Water Conservation, CAS & MWR, Yangling 712100, China. ∗∗∗ Corresponding author. College of Life Sciences, Zhejiang Sci-Tech University, 928 Second Avenue, Xiasha Higher Education Zone, Hangzhou, 310018 , China E-mail addresses: [email protected] (C. Zhang), [email protected] (B. Xing), [email protected] (Z. Liang). 1 Bingcong Xing and Chenlu Zhang contributed equally to this work. https://doi.org/10.1016/j.phytochem.2019.112183 Received 15 May 2019; Received in revised form 15 October 2019; Accepted 19 October 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 1. The chemical structures of three water-soluble phenolic acids (CA, caffeic acid; RA, rosemarinic acid; SAB, salvianolic acid B) (A) and four lipid-soluble tanshinone (T-I, tanshinone I; T-IIA, tanshinone IIA; CT, cryptotanshinone; DT-I, dihydrotanshinone I) (B) studied in this article.
cis-elements of target genes' promoter regions and active/repress its transcription. TFs have functions in regulating plant development, specialised metabolism, abiotic and biotic stress. Besides, TFs could regulate expression of multiple genes simultaneously. It could be an effective strategy that using TFs enhances the production of pharmaceutically active metabolites in plant, such as flavonoids and terpenoid indole alkaloids (Gantet and Memelink, 2002). Some TFs have been investigated and found to regulate phenolic acids and tanshinone biosynthesis in S. miltiorrhiza. For instance, study of Zhang et al. (2013) indicated that SmMYB39 negatively regulated the biosynthesis of phenolic acids. Besides, overexpression of SmMYB9b and SmMYB36 inhibited the phenolic acids accumulation but induced tanshinone concentration in S. miltiorrhiza hairy roots (Ding et al., 2017; Zhang et al., 2017). Cao et al. cloned the SmWRKY1, which positive regulated tanshinone biosynthesis (Cao et al., 2018). The bHLH superfamily TFs are the proteins that has the basic helixloop-helix structural motif and it plays an important role in regulation of pharmaceutical terpenoids biosynthesis. In Arabidopsis thaliana, AtMYC2 activated expression of sesquiterpene synthase genes TPS21 and TPS11 and then increased sesquiterpene biosynthesis (Hong et al., 2012). Phytochrome-interacting factor 5 (PIF5), a bHLH TF of A. thaliana, enhanced isopentyl diphosphate (IPP) metabolism by positively regulating the MEP pathway (Mannen et al., 2014). Diterpenoid phytoalexin factor (DPF, a bHLH TF) could positively regulate diterpenoid phytoalexins (DP) accumulation via transcriptional regulation of DP biosynthetic genes in rice (Yamamura et al., 2015). Shen et al. found that overexpression of AaMYC2 significantly increased artemisinin content in Artemisia annua (Shen et al., 2016). The bHLHs also participated in the regulation of anthocyanin which share parts of the phenylpropanoid pathway with phenolic acids (Yang et al., 2012a). The
(RA), and salvianolic acid B (SAB) (Liu et al., 2006) (Fig. 1A). Tanshinones include tanshinone I (T-I), tanshinone IIA (T-IIA), cryptotanshinone (CT), and dihydrotanshinone I (DT-I) (Shi et al., 2005) (Fig. 1B). Phenolic acids and tanshinones share a range of pharmacological effects, such as anticancer, antioxidant, antibacterial, and antiinflammatory activities (Wang et al., 2015; Zhao et al., 2011; Zhou et al., 2011). Because of its pharmacological action and commercial value, the demand for high-quality S. miltiorrhiza is increasing. Numerous studies, from genetic engineering to metabolic engineering, have focused on improving the production of phenolic acids and tanshinones in S. miltiorrhiza. Phenolic acids are biosynthesized from the phenylpropanoid pathway and the tyrosine pathway (Fig. 2) (Di et al., 2013), while tanshinone components are derived via the mevalonate (MVA) pathway in cytosol and the methylerythritol phosphate (MEP) pathway in plastids (Fig. 3) (Gao et al., 2014). The genes involved in the biosynthesis pathways were key regulators of phenolic acids and tanshinone accumulation in S. miltiorrhiza. For instance, RNAi of the SmPAL1 caused a reduction of RA biosynthesis in S. miltiorrhiza (Song and Wang, 2011). In addition, overexpression of C4H, TAT or HPPR enhanced the production of RA and SAB (Xiao et al., 2011). Moreover, the accumulation of RA and SAB obviously decreased in RAS or CYP98A14 antisense transgenic S. miltiorrhiza hairy root lines (Di et al., 2013; Zhou et al., 2018). For the tanshinone biosynthesis, overexpression of GGPPS and/ or HMGR as well as DXS can significantly enhance the tanshinone accumulation (Kai et al., 2011). Overexpression of CPS1 improved tanshinone production, whereas, silencing CPS1, CYP76AH1 and KSL1 significantly decreased the content of tanshinones (Bai et al., 2018; Cheng et al., 2014; Cui et al., 2015; Ma et al., 2016). Transcription factors (TFs) are the proteins which could bind the 2
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 2. Relative expression levels of phenolic acids biosynthetic pathway genes in transgenic hairy roots lines and the control. The results were analyzed using the comparative Ct method. The S. miltiorrhiza Actin gene was used as an internal control to normalize expression levels. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
Anthocyanin l (An-1) of Petunia positively regulated anthocyanin biosynthesis in the flowers by activating transcription of structural anthocyanin genes (Spelt et al., 2000). The GL3, EGL3, and TT8 proteins of A. thaliana forming a transcriptional regulation complex with MYB proteins and WD40 repeat containing protein TTG1, also activated anthocyanin biosynthetic genes (Dubos et al., 2010; Gonzalez et al., 2008). Another bHLH of A. thaliana, AtMYC3, interacted with JAZs to upregulate the expression of dihydroflavonol reductase (DFR) and improve anthocyanin accumulation in seedlings (Cheng et al., 2011). There are more than 127 bHLH TFs in S. miltiorrhiza, some of which were supposed to regulate the biosynthesis of either phenolic acids or tanshinones (Zhang et al., 2015). Overexpression of SmMYC2 increased the production of phenolic acids, whereas knockdown of SmMYC2a and SmMYC2b extremely decreased yield of T-I, DT-I, T-IIA and CT (Yang et al., 2017; Zhou et al., 2016). We also found two bHLHs that could positively regulate phenolic acids or tanshinone biosynthesis (Xing et al., 2018a, 2018b). However, the detailed functions of the bHLH TFs large family remain unknown. In the present study, SmbHLH3 from S. miltiorrhiza, which responds to JA signaling, was cloned and analyzed. Overexpression of SmbHLH3 inhibited the accumulation of both tanshinone and phenolic acids in S.
miltiorrhiza hairy roots. Here, we present the analysis of the mechanism of SmbHLH3-mediated regulation of tanshinones and phenolic acids biosynthetic. 2. Results 2.1. Bioinformatics analysis of SmbHLH3 SmbHLH3 contained a 1478 bp ORF, encoding a protein of 491 amino acids with a predicted molecular mass of 54.367 kDa. Its GenBank accession number is MH717249. BLAST analysis revealed that SmbHLH3 has a conserved bHLH-MYC and R2R3-MYB TFs N-terminal (48–228 aa). The SMART analysis and multiple alignments of the amino acids of SmbHLH3 revealed that it had a Helix-Loop-Helix domain (347–396 aa) (Fig. 4A). To visually examine the evolutionary origins, the SmbHLH3, along with SibHLH3 (XP_011080018), EgbHLH3-like (XP_012836308.1), OebHLH3 (XP_022854140), StbHLH (XP_006352746), NsbHLH3 (XP_009803066), CbbHLH3 (PHT46573), AcbHLH3 (PSS14567), CabHLH3 (PHT80432), LnbHLH3-like (XP_019165044), OebHLH3-like (XP_022872401), CcbHLH3 (PHU16394), PabHLH3 (XP_021814813) and bHLH3 (AT4G16430), 3
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 3. Relative expression levels of tanshinone biosynthesis pathway genes in transgenic hairy roots lines and the control. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
bHLH13 (AAM10932), bHLH17 (AT2G46510) of A. thaliana and all of the SmbHLHs previously reported were used to construct a phylogenetic tree. The result showed that SmbHLH3 is most closely related to EgbHLH3 from Erythranthe guttata and that these are classified in the subgroup R (Fig. 4B). The identity between SmbHLH3 with the AtbHLH3, AtbHLH13, and AtbHLH17 is 49%, 38% and 39%, respectively.
nucleus. To detect whether SmbHLH3 is nuclear-localized, the GFP transient expression in the onion epidermis was used. Results showed that GFP fluorescence of the control existed in the nucleus and cytoplasm. The GFP fluorescence of SmbHLH3 was dispersed in the nucleus (Fig. 5).
2.2. SmbHLH3 is a nuclear-localized protein
Expression analysis of SmbHLH3 in different parts (stem, leaf, flower, root epidermis, xylem and fibrous root) of two years old flowering S. miltiorrhiza was performed. The result showed that SmbHLH3 expressed in all the six tissues of S. miltiorrhiza, with the highest
2.3. Tissue-specific expression and induction pattern of SmbHLH3
Subcellular localization of protein could help reveal its potential function. TFs mainly function by binding to the gene promoter in the 4
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 4. Sequence analysis of SmbHLH3. (A) Deduced amino acid sequence of SmbHLH3. Predicted HLH domain was present in shaded area. (B) Phylogenetic analysis of SmbHLH3. A phylogenetic tree was constructed based on the amino acid sequences of SmbHLH3 and SibHLH3 (XP_011080018), EgbHLH3-like (XP_012836308.1), OebHLH3 (XP_022854140), StbHLH (XP_006352746), NsbHLH3 (XP_009803066), CbbHLH3 (PHT46573), AcbHLH3 (PSS14567), CabHLH3 (PHT80432), LnbHLH3like (XP_019165044), OebHLH3-like (XP_022872401), CcbHLH3 (PHU16394), PabHLH3 (XP_021814813), AtbHLH3 (AT4G16430), AtbHLH13 (AAM10932), AtbHLH17 (AT2G46510) and all of the SmbHLHs that previously reported. These phylogenetic trees were constructed via MEGA6, using the neighbor-joining method with 500 bootstrap replicates.
5
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
analyzed with RT-qPCR. Among them, two overexpression lines (OEbHLH3-1, OE-bHLH3-2) presented higher expression level (2-fold greater than the WT) of SmbHLH3. Transcription level of SmbHLH3 in the two overexpressing lines (OE-bHLH3-1, OE-bHLH3-2) was approximately 9.09- and 7.43-fold with respect to the WT, respectively (Fig. 7C). 2.5. Both phenolic acids and tanshinone biosynthesis were inhibited in SmbHLH3 overexpression hairy roots Compared with the WT, accumulation of phenolic acids such as CA, RA and SAB decreased in SmbHLH3-overexpression hairy roots lines. CA content in OE-bHLH3-1 decreased to 0.078 mg/g DW, only 50% of the control. The RA content was also decreased to half of the control in OEbHLH3-1(2.56 mg/g DW) and OE-bHLH3-2 (2.75 mg/g DW). And content of SAB in OE-bHLH3-1 and OE-bHLH3-2 was decreased by 62% (4.17 mg/g DW) and 50%, respectively (Fig. 8A). To examine the effect of SmbHLH3 on tanshinone biosynthesis in S. miltiorrhiza, contents of DT-I, CT, T-I and T-II A were determined. Results showed that accumulation of all the four tanshinons ingredients was suppressed in SmbHLH3-overexpression lines. The CT was reduced as much as 97% in OE-bHLH3-2. And the decrease of T-II A was reached 91%. Content of DT- I and T- I were decreased to 38% and 14% of the control, respectively (Fig. 8B). In OE-bHLH3-1 transgenic line, accumulation of CT, DT- I, T- I, T-II A was reduced to 10%, 37%, 34%, 11% of the control, respectively (Fig. 8B). These results indicated that SmbHLH3 could inhibited both phenolic acids and tanshinone biosynthesis in S. miltiorrhiza hairy roots.
Fig. 5. Subcellular localization of SmbHLH3 protein in onion epidermal cells. Fluorescence was observed using a confocal laser scanning microscope at 24 h after incubation. The pictures showed bright field (TD), green fluorescent field (GFP), DAPI and overlay of three fields (Merge). The numerical reading of red ruler is 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.6. SmbHLH3 downregulated expression of genes involved in phenolic acids and tanshinone biosynthetic pathways To uncover the function of SmbHLH3 on phenolic acids and tanshinone accumulation, we analyzed the expression of genes for the main enzymes in their biosynthetic pathways by RT-qPCR. Overexpression of SmbHLH3 repressed genes expression of the phenylpropanoid pathway (C4H1), tyrosine pathway (TAT, HPPR) (Fig. 2). Expression of C4H1 was reduced about 45% and 57% in OE-bHLH3-1 and OE-bHLH3-2, respectively. Transcription of TAT and HPPR was suppressed to 66% and 77% of the control in OE-bHLH3-2. However, transcription levels of PAL1, 4CL2, RAS and CYP98A14 were present no changes in the transgenic lines when compared to the control. For tanshinone biosynthetic pathways, DXS3 and DXR of MEP pathway, HMGR1 of MVA pathway, and CPS1, KSL1 and CYP76AH1 of downstream pathway were all downregulated in SmbHLH3 overexpression lines compared with those in the control. And transcription levels of DXS2, HMGR2 and GGPPS were present no changes by overexpression of SmbHLH3 (Fig. 3). Expressions of DXS3, DXR, HMGR1 and KSL1 were also the lowest in line OE-bHLH3-1, 46%, 65%, 78% and 57% of the control, respectively. While CPS1 and CYP76AH1 expression was the lowest in line OE-bHLH3-2, reduced 27% and 62%, respectively. The variations of these pathway genes transcription were generally consistent with the active ingredients' contents.
Fig. 6. Expression pattern of SmbHLH3 in different tissues of S. miltiorrhiza. Bars are means ± SD from three independent biological replicates. One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
2.7. SmbHLH3 binds the predicted G-box motifs of TAT, HPPR, KSL1 and CYP76AH1
expression in fibrous root and the lowest expression in stem. (Fig. 6). 2.4. Generation of SmbHLH3-overexpression hairy roots
Y1H Gold reporter of strains which has G-box, mG-box, TAT, HPPR, KSL1 and CYP76AH1 promoters could be inhibited by 400, 400, 300, 900, 200 and 150 ng/ml concentration of aureobasidin A (AbA), respectively. And when transformed with SmbHLH3 prey plasmid, except for mG-box, these strains can grow on synthetic medium lacking leucine (SD/-Leu) under corresponding concentration of AbA (Fig. 9). These results showed that SmbHLH3 could successfully bind the G-box of TAT, HPPR, KSL1and CYP76AH1.
In the present study, 5 transgenic hairy roots lines were obtained through hygromycin resistance screening and PCR selection (Fig. 7A). The phenotypes of hairy roots presented by SmbHLH3 overexpression hairy roots showed a lighter color than the WT (the line infected by Agrobacterium rhizogenes, ATCC15834 only) (Fig. 7B). Relative expression level of SmbHLH3 in all the transgenic hairy root lines was 6
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 7. (A) Identification of positive transgenic hairy roots lines by PCR (A, 35S + SmbHLH3; B, hpt II; C, rol b; D, rol c). Numbers above represent individual transgenic lines and M represent DL2000 DNA marker. (B) The phenotypes of hairy roots. Hairy roots were cultured in 6,7-V liquid medium for 30 days before being photographed. (C) Relative quantitative analysis of SmbHLH3 expression in transgenic lines and control of S. miltiorrhiza hairy roots. Bars are means ± SD from three independent biological replicates. One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.01).
3. Discussion
bioactive ingredients in S. miltiorrhiza, SmbHLH3-overexpression hairy roots were obtained by A. rhizogenes mediated method. The WT hairy roots were much redder than all the transgenic lines (Fig. 7B). It has been reported that more tanshinones would be obtained in the redder roots of S. miltiorrhiza (Wang et al., 2014). This result indicated that there is a lower level of tanshinones in the transgenic hairy roots lines than in the WT. HPLC results showed that the content of four tanshinones and three phenolic acids were all diminished in SmbHLH3 overexpression hairy roots (Fig. 8). Compared with the control, contents of CA, RA and SAB decreased by 50%, 50% and 62% in the OEbHLH3 lines, respectively (Fig. 8A). For all four tanshinones ingredients accumulation was suppressed in SmbHLH3-overexpression lines as well. The CT was reduced as much as 97% in OE-bHLH3-2. And the decrease of T-II A reached 91%. Contents of DT- I and T- I were decreased to 38% and 14.48% of the control, respectively (Fig. 8B). To uncover the downregulation mechanism mediated by SmbHLH3, the expression of phenolic acids and tanshinone biosynthetic genes was analyzed. The results indicated that transcription of C4H1, TAT and HPPR were repressed in SmbHLH3-overexpression lines (Fig. 2). Expression of C4H1 was reduced about 45% and 57% in OE-bHLH3-1 and OE-bHLH3-2, respectively. It was reported that C4H may represent regulatory bottlenecks in phenolic acid biosynthesis (Xiao et al., 2011). And overexpression of C4H significantly enhanced target phenolic acids accumulation. Transcription of TAT and HPPR were suppressed to 66% and 77% of the control in OE-bHLH3-2. TAT or HPPR overexpression and TAT-HPPR co-expression also improved the production of RA and SAB, and co-transformed lines produced more phenolic acids (Xiao
It was reported that 127 bHLH TF genes were identified in the genome of S. miltiorrhiza, and these SmbHLHs were classified into 25 subfamilies (Zhang et al., 2015). In the present study, an endogenous bHLH TF SmbHLH3 from S. miltiorrhiza was functionally identified. Phylogenetic analysis showed that SmbHLH3 is most closely related to SmbHLH13, SmbHLH37 and SmbHLH53 and is classified with these genes in the subgroup R (Fig. 4B). Most of the bHLHs in subfamily R were specific to S. miltiorrhiza, and MeJA responses (Zhang et al., 2015). MeJA could regulate the biosynthesis of both tanshinones and phenolic acids (Xing et al., 2018c). And the SmbHLH37 expression pattern was perfectly matched with the accumulation pattern of tanshinones. It was reported that MYC2 belongs to the subfamily R together with SmbHLH37, positively regulate the production of both phenolic acids and tanshinones in S. miltiorrhiza (Yang et al., 2017; Zhou et al., 2016). Therefore, SmbHLH3 could be a candidate TF for the regulation of the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza. SmbHLH3 expressed in all the six parts (stem, leaf, flower, root epidermis, xylem and fibrous root) of S. miltiorrhiza, with the highest expression in fibrous root and the lowest expression in stem (Fig. 6). Its expression pattern has correlations with phenolic acids and tanshinone accumulation. Phenolic acids and tanshinones were mainly present in the root epidermis. It was also being detected in leaves, flowers and stem with much lower content. To investigate whether SmbHLH3 could regulate biosynthesis of 7
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 8. Contents of CA, RA, SAB (A) and T-I, T-IIA, CT, DT-I (B) in transgenic and the control hairy roots lines of S. miltiorrhiza. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P < 0.05).
et al., 2011). It could indicate that TAT and HPPR also play a key role in phenolic acids biosynthesis. Most of the genes involved in tanshinone biosynthesis pathway, including DXS3 and DXR of MEP pathway, HMGR1 of MVA pathway, the CPS1, KSL1 and CYP76AH1 of downstream pathway, were downregulated in SmbHLH3 overexpression lines compared with those in the control (Fig. 3). In line OE-bHLH3-1, expression of DXS3, DXR, HMGR1 was decreased by 46%, 65% and 78% of the control, respectively. It has been reported that tanshinone production could be significantly improved by overexpression of HMGR as well as DXS (Kai et al., 2011). Also, these authors found SmDXS showed much more powerful effects than SmHMGR on tanshinone production. DXS and DXR always show closely similar effects on tanshinone accumulation. When treated with elicitors, such as MeJA, Ag+, polyethylene glycol (PEG), and ABA, variation of DXS and DXR were more associated with tanshinone level changes than HMGR (Xing et al., 2014, 2018c; Yang et al., 2012b), indicating that the MEP pathway plays a
more important role in tanshinone biosynthesis than the MVA pathway. Cheng et al. (2014) reported that when the transcription level of SmCPS was reduced to 26% in SmCPS–RNAi hairy roots, the DT- I and CT content were decreased by 53 and 38% of the control, and T- IIA was not detected. In the present study, CPS1 expression was reduced 27% in line OE-bHLH3-2. Silencing of CYP76AH1 also decreased the content of tanshinones (Ma et al., 2016). CYP76AH1 expression was reduced 62% in SmbHLH3 overexpression lines. These findings indicated that SmbHLH3 could be decrease the biosynthesis of phenolic acids and tanshinones by repressing the transcription of pathway genes. The TFs active/repress the gene expression by binding to gene promoters. We found a G-box binding site in the amino acid of SmbHLH3 (Fig. 4A). A G-box was present in the promoter regions of PAL1, C4H1, TAT, HPPR, RAS, CYP98A14 of the phenolic acids biosynthetic pathway, and DXS, DXR, HMGR, CPS1, KSL1, CYP76AH1 of the tanshinone biosynthetic pathway. Y1H results showed that 8
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
Fig. 9. SmbHLH3 binding with the G-box motifs of the TAT, HPPR, KSL1and CYP76AH1.
the MEGA 6 software program by employing the neighbor joining method, with 500 bootstrap replicates. All amino acid sequences of the other species were downloaded from the NCBI database.
SmbHLH3 could binding to the G-box motifs of the TAT, HPPR, KSL1and CYP76AH1 promoters in yeast cells. bHLH TFs often interact with MYB family proteins to form a complex, and then regulate the transcription of target genes (Feller et al., 2011). We found a bHLHMYC_N-domain in SmbHLH3, which was also called MYB interactive region, in the SmbHLH3 (Fig. 4A). It was essential for binding with MYB proteins (Pattanaik et al., 2008). This indicated that there might be some MYBs involved in the progress of SmbHLH3 regulate phenolic acids and tanshinone biosynthesis. However, the function mode of SmbHLH3 is by complex with the MYBs or independently needs further study.
5.2. Total RNA and DNA extraction Total complete RNA was isolated from frozen S. miltiorrhiza hairy roots or plants by using the RNAprep Pure Plant Kit (TIANGEN, China). The RNA was then reversely transcribed to generate the first strand cDNA, according to the manufacturer's instructions of the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Japan). An improved cetyltrimethylammonium bromide (CTAB) method was used to isolate the genomic DNA. The quality and concentration of the genomic DNA and RNA were examined by agarose gel electrophoresis and spectrophotometer analysis (Thermo Scientific NanoDrop, 2000).
4. Conclusion An endogenous bHLH TF named SmbHLH3 was identified and functionally analyzed in S. miltiorrhiza. It belongs to the subfamily R of SmbHLHs. Overexpression of SmbHLH3 decreased the biosynthesis of both the two groups of bioactive ingredients. Transcriptions of almost all the key genes involved in phenolic acids and tanshinone biosynthesis were repressed by SmbHLH3. These findings indicated that SmbHLH3 decreased phenolic acids and tanshinone biosynthesis by repressing the transcription of pathway genes. Moreover, SmbHLH3 binding site (Gbox) was present in the promoter of these genes, and Y1H results showed that SmbHLH3 bind to the G-box motifs of TAT, HPPR, KSL1and CYP76AH1 promoters in yeast cells. These findings indicated that SmbHLH3 directly bind to the pathway genes promoter, and repress their expression and then downregulate phenolic acids and tanshinone biosynthesis.
5.3. Subcellular localization analysis The ORF of SmbHLH3 was amplified with primers SmGFP1Sal I (5′-ACGCGTCGACATGGGGAGTAAGTTTTGGTTGAATG-3′) and SmGFP2-BamH I (5′-CGGGATCCCGCACCACCACCACCACCATTTAAGA GAGCAGCAGCCAACTTA-3′) using PrimeSTAR® HS DNA Polymerase (Takara, Japan). The amplification sequence was ligated with Sal I and BamH I -digested pTF486 vector to generate a SmbHLH3-GFP fusion construct under the control of cauliflower mosaic virus 35S (CaMV 35S) promoter (Liu et al., 2010). The construct was confirmed by sequencing and used for transient transformation of onion epidermis via a gene gun (Bio-Rad, Hercules, CA, USA). After 24 h of incubation, GFP fluorescence in transformed onion cells was observed under a confocal microscope (Nikon A1, Tokyo, Japan).
5. Materials and methods
5.4. Construction of plant expression vectors and acquisition of positive transgenic hairy roots
5.1. Bioinformatics analysis of SmbHLH3 SmbHLH3 was isolated from the roots of Salvia miltiorrhiza Bunge (Lamiaceae) transcriptome database (Shao et al., 2016) and the open reading frame (ORF) was found with the OFP-finder (https://www. ncbi.nlm.nih.gov/orffinder/). BLAST was used to determine the differences between SmbHLH3 sequences and the NCBI database (https:// www.ncbi.nlm.nih.gov/). Its conserved domain was identified by BLASTP of NCBI database and searched with SMART server (http:// smart.embl-heidelberg.de/). Phylogenetic trees were generated with
The coding region of SmbHLH3 was amplified and cloned into the restriction sites Xba I and Sac I of the pCAMBIA1300 binary vector under the control of the CaMV35S promoter and the NOS terminator. The recombinant plasmids SmbHLH3-1300 was transformed into A. rhizogenes (ATCC15834). The hairy roots lines were acquired from the transformation of leaf explants of the S. miltiorrhiza sterile plantlets. The positive transgenic lines were identified by PCR using rolB, rolC, HPT 9
Phytochemistry 169 (2020) 112183
C. Zhang, et al.
and 35S-SmbHLH3 specific primer. All the primers used for the overexpression vector construction and the PCR identification of transgenic lines are listed in Table S1. Samples of the hairy roots weighing 0.2 g were inoculated into 100 mL beaker flasks containing 50 mL of 6,7-V liquid medium (with 30 g L−1 sucrose). The beaker flasks containing the hairy roots were then placed on an orbital shaker at 110 rpm min−1, 25 °C in the dark. These latter lines were used for RNA and HPLC analysis and they were regularly sub-cultured (every 30 days).
triplicate, and the results were represented by their means ± SD. 5.8. Yeast One-Hybrid (Y1H) assays The Y1H was used to detect whether SmbHLH3 directly interacted with the promoters of key genes in the phenolic acids and tanshinone biosynthetic pathways according the MATCHMAKER Gold Yeast OneHybrid system manual (Clontech). The promoter elements of C4H1, TAT, HPPR, DXS3, DXR, CPS1 and CYP76AH1 (which expressions were suppressed by overexpression of SmbHLH3) were analyzed with the Plant CARE online sever (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/). And fragments from the promoters of C4H1, TAT, HPPR, DXS3, DXR, KSL1, CPS1 and CYP76AH1 were amplified, which contain the G-box-like elements. The fragments obtained were inserted into pAbAi vector with In-Fusion Cloning Kit (Clontech). All bait constructs were linearized and integrated into the genome of the Y1H Gold yeast stain, selected on synthetic defined SD/-Ura agar medium plate and cultured at 30 °C for 2 days. The prey construct was the ORF of SmbHLH3 fused in-frame with the yeast GAL4 transcription activation domain (GAL4 AD) in pGADT7 with In-Fusion assay. The prey construct was then introduced into yeast cells previously transformed with the bait constructs, with blank pGADT7 plasmid as control. The positively transformed clones were cultured at 30 °C on SD/-Leu medium with different concentrations of AbA. The primers used for amplification of the G-box-like fragments and construct prey were designed with online in-fusion tools (http://www.clontech.com/US/Products/Cloning_and_ Competent_Cells/Cloning_Resources/Online_In-Fusion_Tools) are listed in Table S3. The G-box (CACGTG) and mG-box (ACCGTA) were performed as positive and negative control, respectively as Shen et al. (2016) described.
5.5. HPLC analysis The dried hairy roots were powdered using a homogenizer (Bioprep24, Hangzhou, China). The sample powder (20 mg) was extracted with 70% methanol (2 mL) under ultrasonic treatment for 45 min and the resulting mixture was centrifuged at 8000×g for 5 min. The supernatant was filtered through a 0.45 μm organic membrane filter and analyzed by HPLC. The metabolite contents were determined on a Waters HPLC e2695 system (Waters, Milford, MA, USA) equipped with an automatic sample injector and a Waters 2996 photodiode array detector. Chromatographic separation was performed using a SunFire C18 column (4.6 mm × 250 mm, 5 μm particle size) at 30 °C. Empower 3 software was used for data acquisition and processing. The sample injection volume was 10 μL and the PDA wavelengths used for the detection of the lipid-soluble diterpenoids and phenolic acids were 270 and 288 nm, respectively. Separation was achieved by linear gradient elution with solvents A (acetonitrile) and B (0.026% phosphoric acid solution). The gradient was as follows (all concentrations were v/v): 0–10 min, 5–20% A; 10–15 min, 20–25% A; 15–20 min, 25% A; 20–25 min, 25–20% A; 25–28 min, 20–30% A; 28–40 min, 30% A; 40–45 min, 30–45% A; 45–58 min, 45–58% A; 58–67 min, 58–50% A; 67–70 min, 50–60% A; 70–80 min, 60–65% A; 80–85 min, 65–95% A; and 85–95 min, 95% A.
Author contributions ZL, SY conceived and designed the research. BX, DY and CZ conducted experiments and analyzed the data. BX wrote the manuscript. MR and HG contributed advice and revised.
5.6. Standard curves A series of standard solutions of CA, RA, SAB, T-I, CT, DT-I and T-IIA were used to determine the linearity and the linearity range of the analytes in the developed method. The detector response was linearly correlated with content, in the ranges of 0.02–1 mg mL−1 for CA, RA, SAB, T-I, CT, DT-I and T-IIA. The linearity of each standard curve was confirmed by plotting the peak area (y) and the corresponding concentration (x, g·mL-1) of the analytes. The regression equations and correlation coefficients were [y = 44,272,869.30912 x + 657,954.18145] (R2 = 0.99825) for CA, [y = 32,773,318.17664 x - 238,200.78917] (R2 = 0.98605) for RA, [y = 13,281,967.79415 x + 297,278.96047] (R2 = 0.99967) for SAB, [y = 1,806,477.7934 x + 20,298.5506] (R2 = 0.9999) for T-I, [y = 3,229,227.8047 x + 29,146.1676] (R2 = 0.9988) for CT, [y = 3,387,376.8337 x 39,835.6132] (R2 = 0.9997) for DT-I and [y = 5,190,705.9762 x + 9846.8041] (R2 = 0.9997) for T-IIA. In this study, authentic standards were obtained from the National Institute for the Control of Biological and Pharmaceutical Products (Beijing, China).
Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81703646, 81773835 and 81673536), Natural Science Foundation of Zhejiang Provincial (No. LZ16H280001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112183.
5.7. Real-time quantitative PCR analysis
References
The first strand cDNA for RT-qPCR were synthesized using the PrimeScript™ RT Master Mix (Perfect Real Time) (Takara, Tokyo, Japan). Primers used for RT-qPCR were list at supplement Table S2. The SmActin gene was used as reference. RT-qPCR was performed according to the manufacturer's instruction of TB Green™ Premix Ex Taq™ II (TliRNaseH Plus, Takara) using the following protocol: 95 °C, 30 s, 1 cycle; 95 °C, 5 s, 58 °C, 30 s, 40 cycles. The program was performed on the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Massachusetts, USA). Quantification of the gene's expression was done with comparative CT method (2−ΔΔCT). Experiments were performed in
Bai, Z., Li, W., Jia, Y., Yue, Z., Jiao, J., Huang, W., Xia, P., Liang, Z., 2018. The ethylene response factor SmERF6 co-regulates the transcription of SmCPS1 and SmKSL1 and is involved in tanshinone biosynthesis in Salvia miltiorrhiza hairy roots. Planta 248, 243–255. Cao, W., Wang, Y., Shi, M., Hao, X., Zhao, W., Wang, Y., Ren, J., Kai, G., 2018. Transcription factor SmWRKY1 positively promotes the biosynthesis of tanshinones in Salvia miltiorrhiza. Front. Plant Sci. 9, 554. Cheng, H.T., Li, X.L., Li, X.R., Li, Y.H., Wang, L.J., Xue, M., 2012. Simultaneous quantification of selected compounds from Salvia herbs by HPLC method and their application. Food Chem. 130, 1031–1035. Cheng, Q.Q., Su, P., Hu, Y.T., He, Y.F., Gao, W., Huang, L.Q., 2014. RNA interferencemediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy
10
Phytochemistry 169 (2020) 112183
C. Zhang, et al. roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS. Biotechnol. Lett. 36, 363–369. Cheng, Z., Sun, L., Qi, T., Zhang, B., Peng, W., Liu, Y., Xie, D., 2011. The bHLH transcription factor MYC3 interacts with the Jasmonate ZIM-domain proteins to mediate jasmonate response in Arabidopsis. Mol. Plant 4, 279–288. Cui, G., Duan, L., Jin, B., Qian, J., Xue, Z., Shen, G., Snyder, J.H., Song, J., Chen, S., Huang, L., Peters, R.J., Qi, X., 2015. Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza. Plant Physiol. 169, 1607–1618. Di, P., Zhang, L., Chen, J., Tan, H., Xiao, Y., Dong, X., Zhou, X., Chen, W., 2013. 13C tracer reveals phenolic acids biosynthesis in hairy root cultures of Salvia miltiorrhiza. ACS Chem. Biol. 8, 1537–1548. Ding, K., Pei, T., Bai, Z., Jia, Y., Ma, P., Liang, Z., 2017. SmMYB36, a novel R2R3-MYB transcription factor, enhances tanshinone accumulation and decreases phenolic acid content in Salvia miltiorrhiza hairy roots. Sci. Rep. 7, 5104. Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., Lepiniec, L., 2010. MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573–581. Feller, A., Machemer, K., Braun, E.L., Grotewold, E., 2011. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J. 66, 94–116. Gantet, P., Memelink, J., 2002. Transcription factors: tools to engineer the production of pharmacologically active plant metabolites. Trends Pharmacol. Sci. 23, 563–569. Gao, W., Sun, H.X., Xiao, H., Cui, G., Hillwig, M.L., Jackson, A., Wang, X., Shen, Y., Zhao, N., Zhang, L., Wang, X.J., Peters, R.J., Huang, L., 2014. Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza. BMC Genomics 15, 73. Gonzalez, A., Zhao, M., Leavitt, J.M., Lloyd, A.M., 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 53, 814–827. Hong, G.J., Xue, X.Y., Mao, Y.B., Wang, L.J., Chen, X.Y., 2012. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell 24, 2635–2648. Kai, G.Y., Xu, H., Zhou, C.C., Liao, P., Xiao, J.B., Luo, X.Q., You, L.J., Zhang, L., 2011. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab. Eng. 13, 319–327. Liu, A.H., Li, L., Xu, M., Lin, Y.H., Guo, H.Z., Guo, D.A., 2006. Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC–DAD method. J. Pharm. Biomed. 41, 48–56. Liu, X., Rodermel, S.R., Yu, F., 2010. A var2 leaf variegation suppressor locus, SUPPRESSOR OF VARIEGATION3, encodes a putative chloroplast translation elongation factor that is important for chloroplast development in the cold. BMC Plant Biol. 10 287-287. Ma, Y., Ma, X.H., Meng, F.Y., Zhan, Z.L., Guo, J., Huang, L.Q., 2016. RNA interference targeting CYP76AH1 in hairy roots of Salvia miltiorrhiza reveals its key role in the biosynthetic pathway of tanshinones. Biochem. Biophys. Res. Commun. 477, 155–160. Mannen, K., Matsumoto, T., Takahashi, S., Yamaguchi, Y., Tsukagoshi, M., Sano, R., Suzuki, H., Sakurai, N., Shibata, D., Koyama, T., Nakayama, T., 2014. Coordinated transcriptional regulation of isopentenyl diphosphate biosynthetic pathway enzymes in plastids by phytochrome-interacting factor 5. Biochem. Bioph. Res. Co. 443, 768–774. Pattanaik, S., Xie, C.H., Yuan, L., 2008. The interaction domains of the plant Myc-like bHLH transcription factors can regulate the transactivation strength. Planta 227, 707–715. Shao, Y., Wei, J., Wu, F., Zhang, H., Yang, D., Liang, Z., Jin, W., 2016. DsTRD: danshen transcriptional resource database. PLoS One 11. Shen, Q., Lu, X., Yan, T., Fu, X., Lv, Z., Zhang, F., Pan, Q., Wang, G., Sun, X., Tang, K., 2016. The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol. 210, 1269–1281. Shi, Z., He, J., Yao, T., Chang, W., Zhao, M., 2005. Simultaneous determination of cryptotanshinone, tanshinone I and tanshinone IIA in traditional Chinese medicinal preparations containing Radix Salvia miltiorrhiza by HPLC. J. Pharm. Biomed. Anal. 37, 481–486.
Song, J., Wang, Z., 2011. RNAi-mediated suppression of the phenylalanine ammonialyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis. J. Plant Res. 124, 183–192. Spelt, C., Quattrocchio, F., Mol, J.N.M., Koes, R., 2000. anthocyanin1 of Petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12, 1619–1631. Wang, D., Zhang, W., Wang, T., Li, N., Mu, H., Zhang, J., Duan, J., 2015. Unveiling the mode of action of two antibacterial tanshinone derivatives. Int. J. Mol. Sci. 16, 17668–17681. Wang, Hai, Yan, Zhuyun, Shen, Yuxiang, He, Dongmei, Lan, Ying, Deguang, W., 2014. Correlation of color feature with effective constituents of radix salviae miltiorrhizae. Tradit. Chin. Drug Res. Clin. Pharmacol. 3, 333–338. Xiao, Y., Zhang, L., Gao, S., Saechao, S., Di, P., Chen, J., Chen, W., 2011. The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. PLoS One 6, e29713. Xing, B., Yang, D., Yu, H., Zhang, B., Yan, K., Zhang, X., Han, R., Liang, Z., 2018a. Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots. Plant Sci. 229–238. Xing, B., Liang, L., Liu, L., Hou, Z., Yang, D., Yan, K., Zhang, X., Liang, Z., 2018b. Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots. Plant Cell Rep. 37, 1681–1692. Xing, B., Yang, D., Guo, W., Liang, Z., Yan, X., Zhu, Y., Liu, Y., 2014. Ag+ as a more effective elicitor for production of tanshinones than phenolic acids in Salvia miltiorrhiza hairy roots. Molecules 20, 309–324. Xing, B., Yang, D., Liu, L., Han, R., Sun, Y., Liang, Z., 2018c. Phenolic acid production is more effectively enhanced than tanshinone production by methyl jasmonate in Salvia miltiorrhiza hairy roots. Plant Cell Tissue Organ Cult. 134, 119–129. Yamamura, C., Mizutani, E., Okada, K., Nakagawa, H., Fukushima, S., Tanaka, A., Maeda, S., Kamakura, T., Yamane, H., Takatsuji, H., Mori, M., 2015. Diterpenoid phytoalexin factor, a bHLH transcription factor, plays a central role in the biosynthesis of diterpenoid phytoalexins in rice. Plant J. 84, 1100–1113. Yang, C.Q., Fang, X., Wu, X.M., Mao, Y.B., Wang, L.J., Chen, X.Y., 2012a. Transcriptional regulation of plant secondary metabolism. J. Integr. Plant Biol. 54, 703–712. Yang, D., Ma, P., Liang, X., Wei, Z., Liang, Z., Liu, Y., Liu, F., 2012b. PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots. Physiol. Plant. 146, 173–183. Yang, N., Zhou, W., Su, J., Wang, X., Li, L., Wang, L., Cao, X., Wang, Z., 2017. Overexpression of SmMYC2 increases the production of phenolic acids in Salvia miltiorrhiza. Front. Plant Sci. 8, 1804. Zhang, J., Zhou, L., Zheng, X., Zhang, J., Yang, L., Tan, R., Zhao, S., 2017. Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots. Plant Cell Rep. 36, 1297–1309. Zhang, S., Ma, P., Yang, D., Li, W., Liang, Z., Liu, Y., Liu, F., 2013. Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza. PLoS One 8, e73259. Zhang, X., Luo, H., Xu, Z., Zhu, Y., Ji, A., Song, J., Chen, S., 2015. Genome-wide characterisation and analysis of bHLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza. Sci. Rep. 5. Zhao, J., Lou, J., Mou, Y., Li, P., Wu, J., Zhou, L., 2011. Diterpenoid tanshinones and phenolic acids from cultured hairy roots of Salvia miltiorrhiza Bunge and their antimicrobial activities. Molecules 16, 2259–2267. Zhou, Y., Li, W., Xu, L., Chen, L., 2011. In Salvia miltiorrhiza, phenolic acids possess protective properties against amyloid beta-induced cytotoxicity, and tanshinones act as acetylcholinesterase inhibitors. Environ. Toxicol. Pharmacol. 31, 443–452. Zhou, Y., Sun, W., Chen, J., Tan, H., Xiao, Y., Li, Q., Ji, Q., Gao, S., Chen, L., Chen, S., Zhang, L., Chen, W., 2016. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza. Sci. Rep. 6, 22852. Zhou, Z., Tan, H., Li, Q., Chen, J., Gao, S., Wang, Y., Chen, W., Zhang, L., 2018. CRISPR/ Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza. Phytochemistry 148, 63–70.
11