Abstract
Key message
A Salvia miltiorrhiza R2R3-MYB gene, SmMYB9b , has been cloned and characterized. Overexpression of SmMYB9b resulted in a significant improvement of tanshinones, the lipophilic active ingredients in danshen hairy roots.
Abstract
Plant R2R3-MYB transcription factors play important roles in various physiological and biochemical processes. Danshen (Salvia miltiorrhiza bunge) is a valuable medicinal herb with tanshinones and salvianolic acids as the principal bioactive ingredients. A number of putative R2R3-MYB transcription factors have been identified in the plant, but their function remains to be studied. Here, we report the cloning of SmMYB9b, an S20 R2R3-MYB member and its regulatory properties. SmMYB9b contains an open reading frame of 792 bp in length and encodes a 264-amino acid protein. Its transcripts were most abundant in blooming flowers (except for calyces) and increased with flower development. Exogenous abscisic acid strongly activated its transcription. Gibberellins and methyl jasmonate also showed a time-dependent activation effect on its transcription, but to a weaker degree. Overexpression of SmMYB9b in danshen hairy roots enhanced tanshinone concentration to 2.16 ± 0.39 mg/g DW, a 2.2-fold improvement over the control. In addition to increased tanshinone concentration, the hairy root growth and lateral hairy root formation were also suppressed. KEGG pathway enrichment analysis with de novo RNAseq data indicated that stress-response-related metabolic pathways, such as the terpenoid and plant hormone signal transduction pathways, were significantly enriched, implying possible implication of SmMYB9b in such processes. Quantitative RT-PCR analysis showed that the transcription of terpenoid biosynthetic genes SmDXS2, SmDXR, SmGGPPS, and SmKSL1 was significantly up-regulated in danshen hairy roots over expressing SmMYB9b. These data suggest that overexpression of SmMYB9b results in enhanced tanshinone concentration through stimulation of the MEP pathway. The present findings shed new light on elucidating the roles of R2R3-MYB in the biosynthesis of diterpenoids in S. miltiorrhiza.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abe H, Urao T, Ito T et al (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78. doi:10.1105/tpc.006130
Aharoni A, De Vos CHR, Wein M et al (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J 28:319–332
Bedon F, Bomal C, Caron S et al (2010) Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid-and flavonoid-oriented responses. J Exp Bot 61:3847–3864
Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnol Adv 26:318–324. doi:10.1016/j.biotechadv.2008.03.001
Chen H, Chen F (2000) Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures. Plant Cell Rep 19:710–717. doi:10.1007/s002999900166
Chen J, Wang F, Lee FSC et al (2006) Separation and identification of water-soluble salvianolic acids from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography and ESI-MS analysis. Talanta 69:172–179
Cheng C, Jiao C, Singer SD et al (2015) Gibberellin-induced changes in the transcriptome of grapevine (Vitis labrusca × V. vinifera) cv. Kyoho flowers. BMC Genom 16:1–16. doi:10.1186/s12864-015-1324-8
Colquhoun TA, Kim JY, Wedde AE et al (2011) PhMYB4 fine-tunes the floral volatile signature of Petunia hybrida through PhC4H. J Exp Bot 62:1133–1143. doi:10.1093/jxb/erq342
Cui B, Liang Z, Liu Y et al (2012) Effects of ABA and its biosynthetic inhibitor fluridone on accumulation of phenolic acids and activity of PAL and TAT in hairy root of Salvia miltiorrhiza. China J Chin Mater Med 37:754–759
Dai Z, Liu Y, Huang L, Zhang X (2012) Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae. Biotechnol Bioeng 109:2845–2853. doi:10.1002/bit.24547
Danquah A, de Zelicourt A, Colcombet J, Hirt H (2014) The role of ABA and MAPK signaling pathways in plant abiotic stress responses. Biotechnol Adv 32:40–52. doi:10.1016/j.biotechadv.2013.09.006
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(7):1537–1548
De Smet I, Signora L, Beeckman T et al (2003) An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis. Plant J 33:543–555. doi:10.1046/j.1365-313X.2003.01652.x
Devaiah BN, Madhuvanthi R, Karthikeyan AS, Raghothama KG (2009) Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis. Mol Plant 2:43–58
Dubos C, Stracke R, Grotewold E et al (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15:573–581. doi:10.1016/j.tplants.2010.06.005
Gao W, Hillwig ML, Huang L et al (2009) A functional genomics approach to tanshinone biosynthesis provides stereochemical insights. Org Lett 11:5170–5173
Gao W, Sun H-X, Xiao H et al (2014) Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza. BMC Genom 15:73. doi:10.1186/1471-2164-15-73
Guo Y, Gan S (2011) AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis. Plant Physiol 156:1612–1619. doi:10.1104/pp.111.177022
Hantarachot THC, Uaboocha TEB, Ongya HGU, Hadchawan SUC (2012) Putative calmodulin-binding R2R3-MYB transcription factors in rice (Oryza sativa L.). Thai J Bot 4:101–112
Hao G, Jiang X, Feng L et al (2016) Cloning, molecular characterization and functional analysis of a putative R2R3-MYB transcription factor of the phenolic acid biosynthetic pathway in S. miltiorrhiza Bge. f. alba. Plant Cell Tissue Organ Cult 124:151–168. doi:10.1007/s11240-015-0883-3
He Y, Li W, Lv J et al (2012) Ectopic expression of a wheat MYB transcription factor gene, TaMYB73, improves salinity stress tolerance in Arabidopsis thaliana. J Exp Bot 63:1511–1522. doi:10.1093/jxb/err389
Hu Z-B, Alfermann AW (1993) Diterpenoid production in hairy root cultures of Salvia miltiorrhiza. Phytochemistry 32:699–703. doi:10.1016/S0031-9422(00)95156-2
Hu P, Luo G-A, Zhao Z-Z, Jiang Z-H (2005) Quantitative determination of four diterpenoids in Radix Salviae Miltiorrhizae using LC–MS–MS. Chem Pharm Bull (Tokyo) 53:705–709
Kai G, Xu H, Zhou C et al (2011) Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab Eng 13:319–327. doi:10.1016/j.ymben.2011.02.003
Kim JH, Hyun WY, Nguyen HN et al (2015) AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5. Plant Cell Environ 38:559–571. doi:10.1111/pce.12415
Li C, Lu S (2014) Genome-wide characterization and comparative analysis of R2R3-MYB transcription factors shows the complexity of MYB-associated regulatory networks in Salvia miltiorrhiza. BMC Genom 15:277. doi:10.1186/1471-2164-15-277
Li L, Luo H (2008) Phytochemistry of Danshen. People’s Medical Publishing House, Beijing
Liang Z, Ma Y, Xu T et al (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. doi:10.1371/journal.pone.0072806
Liu P, Hu P, Deng R et al (2011) Triterpenoids from the flowers of Salvia miltiorrhiza. Helv Chim Acta 94:136–141
Ma Y, Yuan L, Wu B et al (2012) Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza. J Exp Bot 63:2809–2823. doi:10.1093/jxb/err466
Ma P, Liu J, Zhang C, Liang Z (2013) Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza bunge. Appl Biochem Biotechnol 170:1253–1262. doi:10.1007/s12010-013-0265-4
Mandaokar A, Browse J (2009) MYB108 acts together with MYB24 to regulate jasmonate-mediated stamen maturation in Arabidopsis. Plant Physiol 149:851–862. doi:10.1104/pp.108.132597
Mohr PG, Cahill DM (2007) Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes, in Arabidopsis infected with Pseudomonas syringae pv. tomato. Funct Integr Genom 7:181–191. doi:10.1007/s10142-006-0041-4
Okada K (2011) The biosynthesis of isoprenoids and the mechanisms regulating it in plants. Biosci Biotechnol Biochem 75:1219–1225. doi:10.1271/bbb.110228
Petersen M (2013) Rosmarinic acid: new aspects. Phytochem Rev 12:207–227. doi:10.1007/s11101-013-9282-8
Schmiderer C, Grassi P, Novak J et al (2008) Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae). Plant Biol 10:433–440. doi:10.1111/j.1438-8677.2008.00053.x
Seo PJ, Xiang F, Qiao M et al (2009) The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol 151:275–289. doi:10.1104/pp.109.144220
Song J, Wang Z (2009) Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza. Mol Biol Rep 36:939–952. doi:10.1007/s11033-008-9266-8
Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456. doi:10.1016/S1369-5266(00)00199-0
Sun Z, Kuczek T, Zhu Y (2014) Statistical calibration of QRT-PCR, microarray and RNA-Seq gene expression data with measurement error models. Ann Appl Stat 8:1022–1044. doi:10.1214/14-AOAS721
Tamura K, Stecher G, Peterson D et al (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi:10.1093/molbev/mst197
Urao T, Yamaguchi-shinozaki K, Urao CS et al (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5:1529–1539. doi:10.1105/tpc.5.11.1529
Wenping H, Yuan Z, Jie S et al (2011) De novo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients. Genomics 98:272–279. doi:10.1016/j.ygeno.2011.03.012
Wise A, Liu Z, Binns AN (2006) Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol 343:43–53. doi:10.1385/1-59745-130-4:43
Wu SJ, Shi M, Wu JY (2009) Cloning and characterization of the 1-deoxy-d-xylulose 5-phosphate reductoisomerase gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza (Chinese sage) hairy roots. Biotechnol Appl Biochem 52:89–95. doi:10.1042/BA20080004
Xiao Y, Gao S, Di P et al (2009) Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures. Physiol Plant 137:1–9. doi:10.1111/j.1399-3054.2009.01257.x
Xu R (1990) Danshen, biology and application. Science Press, Beijing
Xu H, Song J, Luo H et al (2016) Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza. Mol Plant 9:949–952. doi:10.1016/j.molp.2016.03.010
Yang D, Ma P, Liang X et al (2012) 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. doi:10.1111/j.1399-3054.2012.01603.x
Yang L, Ding G, Lin H et al (2013) Transcriptome analysis of medicinal plant Salvia miltiorrhiza and identification of genes related to tanshinone biosynthesis. PLoS One 8:1–13. doi:10.1371/journal.pone.0080464
Zhang S, Ma P, Yang D et al (2013) Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza. PLoS One 8:e73259. doi:10.1371/journal.pone.0073259
Zhao S, Zhang J, Tan R et al (2015) Enhancing diterpenoid concentration in Salvia miltiorrhiza hairy roots through pathway engineering with maize C1 transcription factor. J Exp Bot 66:7211–7226. doi:10.1093/jxb/erv418
Zhou L, Zuo Z, Chow MSS (2005) Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol 45:1345–1359. doi:10.1177/0091270005282630
Zhou YJ, Gao W, Rong Q et al (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 134:3234–3241. doi:10.1021/ja2114486
Zhang Y, Yan YP, Wu YC, Hua WP, Chen C, Ge Q, Wang ZZ, (2014) Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation. Metab Eng 21:71–80
Acknowledgements
This manuscript was supported by National Natural Science Foundation of China (No. 30973878, 81673540), Natural Science Foundation of Shanghai (No. 16ZR1434100), and Innovation Program of Shanghai Municipal Education Commission (No. 14ZZ117). The authors greatly appreciate the help of Dr. J. Andrew Jones, visiting assistant Professor at Hamilton College (Clinton, NY, USA) and Professor Mattheos A. G. Koffas, Rensselaer Polytechnic Institute (Troy, NY, USA) for their help with editing the manuscript, Professor Zhibi Hu, Professor Zhengtao Wang, and Mrs. Jiyan Zhou, Shanghai University of Traditional Chinese Medicine, for their valuable comments and suggestions.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Additional information
Communicated by Maike Petersen.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, J., Zhou, L., Zheng, X. et al. Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots. Plant Cell Rep 36, 1297–1309 (2017). https://doi.org/10.1007/s00299-017-2154-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-017-2154-8