Abstract
Key message
SmbHLH148 activated the whole biosynthetic pathways of phenolic acids and tanshinones, thus upregulated the production of both the two groups of pharmaceutical ingredients in Salvia miltiorrhiza.
Abstract
Phenolic acids and tanshinones are the two important groups of pharmaceutical ingredients presented in Salvia miltiorrhiza Bunge. The bHLH transcription factors could regulate secondary metabolism efficiently in plants. However, there are only some MYCs have been studied on regulation of either phenolic acids or tanshinones biosynthesis. In this study, a bHLH TF named SmbHLH148, which is homologous to AtbHLH148, AtbHLH147 and CubHLH1, was isolated and functionally characterized from S. miltiorrhiza. Transcription of SmbHLH148 could be intensely induced by ABA and also be moderately induced by MeJA and GA. SmbHLH148 is present in all the six tissues and mostly expressed in fibrous root and flowers. Subcellular localization analysis found that SmbHLH148 was localized in the nucleus. Overexpression of SmbHLH148 significantly increased not only three phenolic acids components accumulation but also three tanshinones content. Content of caffeic acid, rosmarinic acid and salvianolic acid B were reached to 2.87-, 4.00- and 5.99-fold of the control in the ObHLH148-3, respectively. Content of dihydrotanshinone I, cryptotanshinone, and tanshinone I were also present highest in ObHLH148-3, reached 2.5-, 5.04- and 3.97-fold of the control, respectively. Expression analysis of pathway genes of phenolic acids and tanshinones in transgenic lines showed that most of them were obviously upregulated. Moreover, transcription of AREB and JAZs were also induced in SmbHLH148 overexpression lines. These results suggested that SmbHLH148 might be taken part in ABA and MeJA signaling and activated almost the whole biosynthetic pathways of phenolic acids and tanshinones, thus the production of phenolic acids and tanshinones were upregulated.
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References
Antje F, Katja M, Erich LBE G (2011) Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J 66:94–116
Antonio G, Mingzhe Z, LJ M, M. LA (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/MYB transcriptional complex in Arabidopsis seedlings. Plant J 53:814–827
Bai Z, Xia P, Wang R, Jiao J, Ru M, Liu J, Liang Z (2017) Molecular cloning and characterization of five SmGRAS genes associated with tanshinone biosynthesis in Salvia miltiorrhiza hairy roots. PLoS One 12:e0185322
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
Cheng QQ, Su P, Hu YT, He YF, Gao W, Huang LQ (2014) RNA interference-mediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS. Biotechnol Lett 36:363–369
Cui G, Duan L, Jin B, Qian J, Xue Z, Shen G, Snyder JH, Song J, Chen S, Huang L, Peters RJ, 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
Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15:573–581
Endo T, Fujii H, Sugiyama A, Nakano M, Nakajima N, Ikoma Y, Omura M, Shimada T (2016) Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1), which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes, modulates carotenoid metabolism in transgenic tomato. Plant Sci 243:35–48
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 ML, Jackson A, Wang X, Shen Y, Zhao N, Zhang L, Wang X-J, Peters RJ, Huang L (2014) Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza. BMC Genom 15:73
Hong GJ, Xue XY, Mao YB, Wang LJ, Chen XY (2012) Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell 24:2635–2648
Jia Y, Bai Z, Pei T, Ding K, Liang Z, Gong Y (2017) The protein kinase SmSnRK2.6 positively regulates phenolic acid biosynthesis in Salvia miltiorrhiza by interacting with SmAREB1. Front Plant Sci 8:1384
Kai G, Xu H, Zhou C, Liao P, Xiao J, Luo X, You L, Zhang L (2011) Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab Eng 13:319–327
Kai G, Liao P, Xu H, Wang J, Zhou C, Zhou W, Qi Y, Xiao J, Wang Y, Zhang L (2012) Molecular mechanism of elicitor-induced tanshinone accumulation in Salvia miltiorrhiza hairy root cultures. Acta Physiol Plant 34:1421–1433
Koes CSFQJMR (2000) Anthocyanin1 of Petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12:1619–1632
Liang Z, Ma Y, Xu T, Cui B, Liu Y, Guo Z, Yang D (2013) Effects of abscisic acid, gibberellin, ethylene and their interactions on production of phenolic acids in Salvia miltiorrhiza Bunge hairy roots. PLoS One 8:e72806
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 Anal 41:48–56
Liu X, Rodermel SR, 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 XH, Meng FY, Zhan ZL, Guo J, Huang LQ (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
Min S, Luo X, Ju G, Li L, Huang S, Tong Z, Wang H, Kai G (2016) Enhanced diterpene tanshinone accumulation and bioactivity of transgenic Salvia miltiorrhiza hairy roots by pathway engineering. J Agric Food Chem 64:2523–2530
Park KI (2012) A bHLH protein partially controls proanthocyanidin and phytomelanin pigmentation in the seed coats of morning glory Ipomoea tricolor. Hortic, Environ Biotechnol 53:304–309
Pei T, Ma P, Ding K, Liu S, Jia Y, Ru M, Dong J, Liang Z (2018) SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J Exp Bot 69:1663–1678
Seo JS, Joo J, Kim MJ, Kim YK, Nahm BH, Song SI, Cheong JJ, Lee JS, Kim JK, Choi YD (2011) OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. Plant J 65:907–921
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 ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis. J Plant Res 124:183–192
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
Xiao Y, Zhang L, Gao S, Saechao S, Di P, Chen J, Chen W (2010) 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, Liu L, Han R, Sun Y, Liang Z (2018) Phenolic acid production is more effectively enhanced than tanshinone production by methyl jasmonate in Salvia miltiorrhiza hairy roots. Plant Cell, Tissue Organ Cult (PCTOC) 134:119–129
Xue Y, Shao Y, Wei J, Wu F, Zhang H, Yang D, Liang Z, Jin W (2016) DsTRD: danshen transcriptional resource database. PLoS One 11:e0149747
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 CQ, Fang X, Wu XM, Mao YB, Wang LJ, Chen XY (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 H, Hedhili S, Montiel G, Zhang Y, Chatel G, Pre M, Gantet P, Memelink J (2011) The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus. Plant J 67:61–71
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 5:11244
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
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Nos. 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.
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Communicated by Salim Al-Babili.
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Xing, B., Liang, L., Liu, L. et al. Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots. Plant Cell Rep 37, 1681–1692 (2018). https://doi.org/10.1007/s00299-018-2339-9
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DOI: https://doi.org/10.1007/s00299-018-2339-9