Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

Piotr Szymczyk, Ewa Skała, Renata Grąbkowska, Agnieszka Jeleń, Marta Żebrowska, Ewa Balcerczak


The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data.

The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity.

Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.


promoter; cloning; trans-factor; bioinformatics; microarray data; RT-PCR

Full Text:



Chen F, Tholl D, Bohlmann J, Pichersky E. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011;66:212–229.

Zhou L, Zuo Z, Chow MS. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol. 2005;45:1345–1359.

Gao W, Hillwig ML, Huang L, Cui G, Wang X, Kong J, et al. A functional genomics approach to tanshinone biosynthesis provides stereochemical insights. Org Lett. 2009;11:5170–5173.

Cheng Q, He Y, Li G, Liu Y, Gao W, Huang L. Effects of combined elicitors on tanshinone metabolic profiling and SmCPS expression in Salvia miltiorrhiza hairy root cultures. Molecules. 2013;18:7473–7485.

Zhou YJ, Gao W, Rong Q, Jin G, Chu H, Liu W, et al. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc. 2012;134:3234−3241.

Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, et al. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol. 2008;26:1301–1308.

Dey N, Sarkar S, Acharya S, Maiti IB. Synthetic promoters in planta. Planta. 2015;242:1077–1094.

Chang CW, Sun TP. Characterization of cis-regulatory regions responsible for developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana. Plant Mol Biol. 2002;49:579–589.

Pugliesi C, Fambrini M, Salvini M. Molecular cloning and expression profile analysis of three sunflower (Helianthus annuus) diterpene synthase genes. Biochem Genet. 2011;49:46–62.

Khan S, Qureshi MI, Kamaluddin, Alam T, Abdin MZ. Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr J Biotechnol. 2007;6:175–178.

Chang WC, Lee TY, Huang HD, Huang HY, Pan RL. PlantPAN: plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene group. BMC Genomics. 2008;9:561–575.

Chow CN, Zheng HQ, Wu NY, Chien CH, Huang HD, Lee TY, et al. PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants. Nucleic Acids Res. 2016;44(D1):D1154–D1160.

Wang K, editor. Agrobacterium protocols. Totowa, NJ: Humana Press Inc.; 2006. (Methods in Molecular Biology; vol 343).

Yan Y, Wang Z. Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method. Plant Cell Tissue Organ Cult. 2007;88:175–184.

Kibbe WA. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 2007;35:W43–W46.

Pedros R, Moya I, Goulas Y, Jacquemoud S. Chlorophyll fluorescence emission spectrum inside a leaf. Photochem Photobiol Sci. 2008;7:498–502.

Ckurshumova W, Caragea AE, Goldstein RS, Berleth T. Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants. Mol Plant. 2011;4:794–804.

Yang Y, Hou S, Cui G, Chen S, Wei J, Huang L. Characterization of reference genes for quantitative real time PCR analysis in various tissues of Salvia miltiorrhiza. Mol Biol Rep. 2010;37:507–513.

Rajinikanth M, Harding SA, Tsai C. The glycine decarboxylase complex multienzyme family in Populus. J Exp Bot. 2007;58:1761–1770.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–408.

Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods. 200;46:69–81.

Toufighi K, Brady SM, Austin R, Ly E, Provart NJ. The botany array resource: e-Northerns, expression angling, and promoter analyses. Plant J. 2005;43:153–163.

Usadel B, Obayashi T, Mutwil M, Giorgi FM, Bassel GW, Tanimoto N, et al. Co-expression tools for plant biology: opportunities for hypothesis generation and caveats. Plant Cell Environ. 2009;32:1633–1651.

Shahmuradov IA, Gammerman AJ, Hancock JM, Bramley PM, Solovyev VV. PlantProm: a database of plant promoter sequences. Nucleic Acids Res. 2003;31:114–117.

Lam E, Chua NH. ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters. Plant Cell. 1989;1:1147–1156.

Sessa G, Morelli G, Ruberti I. The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities. EMBO J. 1993;12:3507–3517.

Aoyama T, Dong CH, Wu Y, Carabelli M, Sessa G, Ruberti I, et al. Ectopic expression of the Arabidopsis transcriptional activator Athb-1 alters leaf cell fate in tobacco. Plant Cell. 1995;7:1773–1785.

Kim SY, Chung HJ, Thomas TL. Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system. Plant J. 1997;11:1237–1251.

Solano R, Stepanova A, Chao Q, Ecker JR. Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev. 1998;12:3703–3714.

Brown RL, Kazan K, McGrath KC, Maclean DJ, Manners JM. A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiol. 2003;132:1020–1032.

Terzaghi WB, Cashmore AR. Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol. 1995;46:445–474.

Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, et al. Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiol. 2004;135:2150–2161.

Villain P, Mache R, Zhou DX. The mechanism of GT element-mediated cell type-specific transcriptional control. J Biol Chem. 1996;271:32593–32598.

Piechulla B, Merforth N, Rudolph B. Identification of tomato Lhc promoter regions necessary for circadian expression. Plant Mol Biol. 1998;38:655–662.

Barrett JW, Beech RN, Dancik BP, Strobeck C. A genomic clone of a type I cab gene encoding a light harvesting chlorophyll a/b binding protein of photosystem II identified from lodgepole pine. Genome. 1994;37:166–172.

Hudson ME, Quail PH. Identification of promoter motifs involved in the network of phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data. Plant Physiol. 2003;133:1605–1616.

Tepperman JM, Zhu T, Chang HS, Wang X, Quail PH. Multiple transcription-factor genes are early targets of phytochrome A signaling. Proc Natl Acad Sci USA. 2001;98:9437–9442.

Ballas N, Wong LM, Malcolm K, Theologis A. Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5. Proc Natl Acad Sci USA. 1995;86:3483–3487.

Ballas N, Wong LM, Theologis A. Identification of the auxin-responsive element, AuxRE, in the primary indoleacetic acid-inducible gene, PS-IAA4/5, of pea (Pisum sativum). J Mol Biol. 1993;233:580–596.

Mohanty B, Krishnan SP, Swarup S, Bajic VB. Detection and preliminary analysis of motifs in promoters of anaerobically induced genes of different plant species. Ann Bot. 2005;96:66–81.

Yu D, Chen C, Chen Z. Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell. 2001;13:1527–1540.

Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell. 1993;5:1529–1539.

Martínez IM, Chrispeels MJ. Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. Plant Cell. 2003;15:561–576.

Punwani JA, Rabiger DS, Drews GN. MYB98 positively regulates a battery of synergid-expressed genes encoding filiform apparatus-localized proteins. Plant Cell. 2007;19:2557–2568.

Cui D, Zhao J, Jing Y, Fan M, Liu J, Wang Z, et al. The Arabidopsis IDD14, IDD15, and IDD16 cooperatively regulate lateral organ morphogenesis and gravitropism by promoting auxin biosynthesis and transport. PLoS Genet. 2013;9(9):e1003759.

Furuta KM, Yadav SR, Lehesranta S, Belevich I, Miyashima S, Heo J, et al. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation. Science. 2014;345:933–937.

Shirzadi R, Andersen ED, Bjerkan KN, Gloeckle BM, Heese M, Ungru A, et al. Genome-wide transcript profiling of endosperm without paternal contribution identifies parent-of-origin-dependent regulation of AGAMOUS-LIKE36. PLoS Genet. 2011;7(2):e1001303.

Pařenicová L, de Folter S, Kieffer M, Horner DS, Favalli C, Busscher J, et al. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell. 2003;15:1538–1551.

Rabiger DS, Drews GN. MYB64 and MYB119 are required for cellularization and differentiation during female gametogenesis in Arabidopsis thaliana. PLoS Genet. 2013;9(9):e1003783.

Yoo SK, Lee JS, Ahn JH. Overexpression of AGAMOUS-LIKE 28 (AGL28) promotes flowering by upregulating expression of floral promoters within the autonomous pathway. Biochem Biophys Res Commun. 2006;348:929–936.

Chardin C, Girin T, Roudier F, Meyer C, Krapp A. The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development. J Exp Bot. 2014;65:5577–5587.

Jang IC, Yang SW, Yang JY, Chua NH. Independent and interdependent functions of LAF1 and HFR1 in phytochrome A signaling. Genes Dev. 2007;21:2100–2111.

Nakamichi N, Kiba T, Kamioka M, Suzuki T, Yamashino T, Higashiyama T, et al. Transcriptional repressor PRR5 directly regulates clock-output pathways. Proc Natl Acad Sci USA. 2012;109:17123–17128.

Shirasu K. The HSP90-SGT1 chaperone complex for NLR immune sensors. Annu Rev Plant Biol. 2009;60:139–164.

Lippold F, Sanchez DH, Musialak M, Schlereth A, Scheible WR, Hincha DK, et al. AtMyb41 regulates transcriptional and metabolic responses to osmotic stress in Arabidopsis. Plant Physiol. 2009;149:1761–1772.

Andersen P, Kragelund BB, Olsen AN, Larsen FH, Chua NH, Poulsen FM, et al. Structure and biochemical function of a prototypical Arabidopsis U-box domain. J Biol Chem. 2004;279:40053–40061.

Craig KL, Tyers M. The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction. Prog Biophys Mol Biol. 1999;72:299–328.

Gonzalez A, Zhao M, Leavitt JM, Lloyd AM. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 2008;53:814–827.

Para A, Lia Y, Marshall-Colón A, Varala K, Francoeur NJ, Moran TM, et al. Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis. Proc Natl Acad Sci USA. 2014;111:10371–10376.

Thomas SG, Phillips AL, Hedden P. Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc Natl Acad Sci USA. 1999;96:4698–4703.

Abel S, Nguyen MD, Chow W, Theologis A. ASC4, a primary indoleacetic acid responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis thaliana. J Biol Chem. 1995;270:19093–19099.

Vinces MD, Legendre M, Caldara M, Hagihara M, Verstrepen KJ. Unstable tandem repeats in promoters confer transcriptional evolvability. Science. 2009;324:1213–1216.

Reinders J, Paszkowski J. Unlocking the Arabidopsis epigenome. Epigenetics. 2009;4(8):557–563.

Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qia Z. Rice microRNA effector complexes and targets. Plant Cell. 2009;21:3421–3435.

Saulierè J, Sureau A, Expert-Bezancon A, Marie J. The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the β-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit. Mol Cell Biol. 2006;26:8755–8769.

Fleet CM, Yamaguchi S, Hanada A, Kawaide H, David CJ, Kamiya Y, et al. Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins. Plant Physiol. 2003;132:830–839.

Qiu J, Sun S, Luo S, Zhang J, Xiao X, Zhang L, et al. Arabidopsis AtPAP1 transcription factor induces anthocyanin production in transgenic Taraxacum brevicorniculatum. Plant Cell Rep. 2014;33:669–680.

Pyvovarenko T, Lopato S. Isolation of plant transcription factors using a yeast one-hybrid system. Methods Mol Biol. 2011;754:45–60.