Skip to main content
SpringerLink
Log in

Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Tobacco is a commonly used heterologous system for studying combinatorial regulation of the flavonoid biosynthetic pathway by the bHLH–MYB transcription factor (TF) complex in plants. However, little is known about the endogenous tobacco bHLH and MYB TFs involved in the pathway. Ectopic expression in tobacco of heterologous bHLH TF genes, such as maize Lc, leads to increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with the Lc-like bHLH TFs. We isolated a gene (NtAn2) encoding a R2R3 MYB TF from developing tobacco flowers. NtAn2 shares high sequence homology with other known flavonoid-related MYB TFs and is mostly expressed in developing flowers. Constitutive ectopic expression of NtAn2 induces whole-plant anthocyanin production in tobacco and Arabidopsis. In transgenic tobacco and Arabidopsis expressing NtAn2, both subsets of early and late flavonoid pathway genes are up-regulated. Suppression of NtAn2 by RNAi in tobacco resulted in a white-flowered phenotype and the inhibition of the late pathway genes. Yeast two-hybrid assays demonstrated that NtAn2 can interact with five heterologous bHLH TFs known to induce anthocyanin synthesis in other species including maize, perilla, snapdragon and Arabidopsis. Bimolecular fluorescent complementation using split YFP demonstrated that NtAn2 interacts with Lc in tobacco cells and that the complex is localized to nuclei. Transient co-expression of NtAn2 and Lc or Arabidopsis TT8 in tobacco protoplasts activated the promoters of two key flavonoid pathway genes, chalcone synthase and dihydroflavonol reductase. These results suggest that NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

Abbreviations

ANS:

Anthocyanidin synthase

CHI:

Chalcone isomerase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol 4-reductase

F3H:

Flavanone 3-hydroxylase

4CL:

4-Coumarate-CoA ligase

PAL:

Phenylalanine ammonia-lyase

qPCR:

Quantitative real-time PCR

RNAi:

RNA interference

TF:

Transcription factor

YFP:

Yellow fluorescent protein

References

  • Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394

    Article  CAS  PubMed  Google Scholar 

  • Borovsky Y, Oren-Shamir M, Ovadia R, De Jong W, Paran I (2004) The A locus that controls anthocyanin accumulation in pepper encodes a MYB transcription factor homologous to anthocyanin2 of petunia. Theor Appl Genet 109:23–29

    Article  CAS  PubMed  Google Scholar 

  • Carey CC, Strahle JT, Selinger DA, Chandler VL (2004) Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana. Plant Cell 16:450–464

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Cone KC, Burr FA, Burr B (1986) Molecular analysis of the maize anthocyanin regulatory locus C1. Proc Natl Acad Sci USA 83:9631–9635

    Article  CAS  PubMed  Google Scholar 

  • Cultrone A, Cotroneo PS, Recupero GR (2009) Cloning and molecular characterization of R2R3-MYB and bHLH-MYC transcription factors from Citrus sinensis. Tree Genet Genomes 6:101–112

    Article  Google Scholar 

  • de Vetten N, Quattrocchio F, Mol J, Koes R (1997) The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals. Genes Dev 11:1422–1434

    Article  PubMed  Google Scholar 

  • Deluc L, Barrieu F, Marchive C, Lauvergeat V, Decendit A, Richard T, Carde JP, Merillon JM, Hamdi S (2006) Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol 140:499–511

    Article  CAS  PubMed  Google Scholar 

  • Deluc L, Bogs J, Walker AR, Ferrier T, Decendit A, Merillon JM, Robinson SP, Barrieu F (2008) The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol 147:2041–2053

    Article  CAS  PubMed  Google Scholar 

  • Dey N, Maiti IB (1999) Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants. Plant Mol Biol 40:771–782

    Article  CAS  PubMed  Google Scholar 

  • Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427

    Article  CAS  PubMed  Google Scholar 

  • Felsenstein J (1985) Confidence-limits on phylogenies—an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Galis I, Simek P, Narisawa T, Sasaki M, Horiguchi T, Fukuda H, Matsuoka K (2006) A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco. Plant J 46:573–592

    Article  CAS  PubMed  Google Scholar 

  • Gong ZZ, Yamagishi E, Yamazaki M, Saito K (1999a) A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization, heterologous expression in transgenic plants and transactivation in yeast cells. Plant Mol Biol 41:33–44

    Article  CAS  PubMed  Google Scholar 

  • Gong ZZ, Yamazaki M, Saito K (1999b) A light-inducible Myb-like gene that is specifically expressed in red Perilla frutescens and presumably acts as a determining factor of the anthocyanin formation. Mol Gen Genet 262:65–72

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Grotewold E (2005) Plant metabolic diversity: a regulatory perspective. Trends Plant Sci 10:57–62

    Article  CAS  PubMed  Google Scholar 

  • Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780

    Article  CAS  PubMed  Google Scholar 

  • Grotewold E, Drummond BJ, Bowen B, Peterson T (1994) The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76:543–553

    Article  CAS  PubMed  Google Scholar 

  • Grotewold E, Sainz MB, Tagliani L, Hernandez JM, Bowen B, Chandler VL (2000) Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R. Proc Natl Acad Sci USA 97:13579–13584

    Article  CAS  PubMed  Google Scholar 

  • Harrison CJ, Langdale JA (2006) A step by step guide to phylogeny reconstruction. Plant J 45:561–572

    Article  CAS  PubMed  Google Scholar 

  • Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242

    Article  CAS  PubMed  Google Scholar 

  • Lloyd AM, Walbot V, Davis RW (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258:1773–1775

    Article  CAS  PubMed  Google Scholar 

  • Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86:7092–7096

    Article  CAS  PubMed  Google Scholar 

  • Martin C, Prescott A, Mackay S, Bartlett J, Vrijlandt E (1991) Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J 1:37–49

    Article  CAS  PubMed  Google Scholar 

  • Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, Matheis N, Schuster DK, Menasco DJ, Wagoner W, Lightner J, Wagner DR (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15:1689–1703

    Article  CAS  PubMed  Google Scholar 

  • Mehrtens F, Kranz H, Bednarek P, Weisshaar B (2005) The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis. Plant Physiol 138:1083–1096

    Article  CAS  PubMed  Google Scholar 

  • Mol J, Grotewold E, Koes R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217

    Article  Google Scholar 

  • Mooney M, Desnos T, Harrison K, Jones J, Carpenter R, Coen E (1995) Altered regulation of tomato and tobacco pigmentation genes caused by the Delila gene of Antirrhinum. Plant J 7:333–339

    Article  CAS  Google Scholar 

  • Nakatsuka T, Haruta KS, Pitaksutheepong C, Abe Y, Kakizaki Y, Yamamoto K, Shimada N, Yamamura S, Nishihara M (2008) Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. Plant Cell Physiol 49:1818–1829

    Article  CAS  PubMed  Google Scholar 

  • Nesi N, Debeaujon I, Jond C, Pelletier G, Caboche M, Lepiniec L (2000) The TT8 gene encodes a basic helix–loop–helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell 12:1863–1878

    Article  CAS  PubMed  Google Scholar 

  • Niu SS, Xu CJ, Zhang WS, Zhang B, Li X, Lin-Wang K, Ferguson IB, Allan AC, Chen KS (2009) Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor. Planta 6:101–112

    Google Scholar 

  • Ohad N, Shichrur K, Yalovsky S (2007) The analysis of protein–protein interactions in plants by bimolecular fluorescence complementation. Plant Physiol 145:1090–1099

    Article  CAS  PubMed  Google Scholar 

  • Pattanaik S, Xie CH, Kong Q, Shen KA, Yuan L (2006) Directed evolution of plant basic helix–loop–helix transcription factors for the improvement of transactivational properties. Biochim Biophys Acta-Gene Struct Express 1759:308–318

    CAS  Google Scholar 

  • Pattanaik S, Xie CH, Yuan L (2008) The interaction domains of the plant Myc-like bHLH transcription factors can regulate the transactivation strength. Planta 227:707–715

    Article  CAS  PubMed  Google Scholar 

  • Payne CT, Zhang F, Lloyd AM (2000) GL3 encodes a bHLH protein that regulates trichome development in arabidopsis through interaction with GL1 and TTG1. Genetics 156:1349–1362

    CAS  PubMed  Google Scholar 

  • Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H (1987) The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J 6:3553–3558

    CAS  PubMed  Google Scholar 

  • Quattrocchio F, Wing JF, Leppen H, Mol J, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5:1497–1512

    Article  CAS  PubMed  Google Scholar 

  • Quattrocchio F, Wing JF, van der Woude K, Mol JN, Koes R (1998) Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant J 13:475–488

    Article  CAS  PubMed  Google Scholar 

  • Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R (1999) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11:1433–1444

    Article  CAS  PubMed  Google Scholar 

  • Rabino I, Mancinelli AL (1986) Light, temperature, and anthocyanin production. Plant Physiol 81:922–924

    Article  CAS  PubMed  Google Scholar 

  • Radicella JP, Turks D, Chandler VL (1991) Cloning and nucleotide sequence of a cDNA encoding B-Peru, a regulatory protein of the anthocyanin pathway in maize. Plant Mol Biol 17:127–130

    Article  CAS  PubMed  Google Scholar 

  • Ramsay NA, Glover BJ (2005) MYB–bHLH–WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci 10:63–70

    Article  CAS  PubMed  Google Scholar 

  • Schardl CL, Byrd AD, Benzion G, Altschuler MA, Hildebrand DF, Hunt AG (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11

    Article  CAS  PubMed  Google Scholar 

  • Shinya T, Galis I, Narisawa T, Sasaki M, Fukuda H, Matsuoka H, Saito M, Matsuoka K (2007) Comprehensive analysis of glucan elicitor-regulated gene expression in tobacco BY-2 cells reveals a novel MYB transcription factor involved in the regulation of phenylpropanoid metabolism. Plant Cell Physiol 48:1404–1413

    Article  CAS  PubMed  Google Scholar 

  • Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456

    Article  CAS  PubMed  Google Scholar 

  • Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677

    Article  CAS  PubMed  Google Scholar 

  • Sugimoto K, Takeda S, Hirochika H (2000) MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes. Plant Cell 12:2511–2528

    Article  CAS  PubMed  Google Scholar 

  • Suttipanta N, Pattanaik S, Gunjan S, Xie CH, Littleton J, Yuan L (2007) Promoter analysis of the Catharanthus roseus geraniol 10-hydroxylase gene involved in terpenoid indole alkaloid biosynthesis. Biochim Biophys Acta 1769:139–148

    CAS  PubMed  Google Scholar 

  • Tamura K, Dudley J, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599

    Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  CAS  PubMed  Google Scholar 

  • Tohge T, Matsui K, Ohme-Takagi M, Yamazaki M, Saito K (2005) Enhanced radical scavenging activity of genetically modified Arabidopsis seeds. Biotechnol Lett 27:297–303

    Article  CAS  PubMed  Google Scholar 

  • Zhou LL, Zeng HN, Shi MZ, Xie DY (2008) Development of tobacco callus cultures over expressing Arabidopsis PAP1/MYB75 transcription factor and characterization of anthocyanin biosynthesis. Planta 229:37–51

    Article  CAS  PubMed  Google Scholar 

  • Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40:22–34

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. G. Collins of the University of Kentucky for the RNAi vector and Dr. E. Grotewold of the Ohio State University of the split YFP vector. We also express our appreciation to Dr. K. Saito for providing the Myc-RP cDNA, Dr. S. Wessler for the Lc cDNA and the John Innes Research Center for the Delila cDNA. This work is supported by a grant from the Kentucky Tobacco Research and Development Center to L.Y.

Author information

Authors and Affiliations

  1. Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546, USA

    Sitakanta Pattanaik, Que Kong, Joshua R. Werkman, Claire H. Xie & Ling Yuan

  2. Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, 40546, USA

    Sitakanta Pattanaik, David Zaitlin, Claire H. Xie, Barunava Patra & Ling Yuan

Authors
  1. Sitakanta Pattanaik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Que Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Zaitlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joshua R. Werkman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Claire H. Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Barunava Patra
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ling Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ling Yuan.

Additional information

S. Pattanaik and Q. Kong contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

425_2010_1108_MOESM1_ESM.tif

SI Fig. 1a Schematic diagram of the expression vector containing the full-length NtAn2 cDNA expressed under the control of a modified Mirabilis mosaic virus (MMV) full-length transcript promoter and ribulose bisphosphate carboxylase (rbcS) terminator. b Schematic diagram of the RNAi vector containing a part of the NtAn2 cDNA both in sense and anti sense orientation with a Glycine max FAD3 intron expressed under the control of CaMV 35S promoter and rbcS terminator. 35S-T,CaMV35S terminator; NPT II, neomycin phosphotransferase; LB, left T-DNA border; RB, right T-DNA border (TIFF 1069 kb)

425_2010_1108_MOESM2_ESM.tif

SI Fig. 2 Relative expression levels of NtAn2 transcripts in different floral parts. CX, calyx; CL corolla limb; CT corolla tube; AN, anther; OV, ovary. The gene expression levels were measured by quantitative PCR (qPCR). The results were analyzed using the comparative Ct method and presented as fold-changes compared with the wild-type control (TIFF 7741 kb)

425_2010_1108_MOESM3_ESM.tif

SI Fig. 3 Tobacco flowers of empty-vector control plant (a) and homozygous transgenic tobacco plants overexpressing the bHLH transcription factors Delila (b), Myc-RP (c) and Lc (d) (Pattanaik et al. 2008; Pattanaik et al. unpublished data) (TIFF 463 kb)

425_2010_1108_MOESM4_ESM.tif

SI Fig. 4 Protein-protein interactions between NtAn2 and Arabidopsis bHLH TFs. a-b. In a yeast two hybrid assay, the NtAn2/GAL4-activation domain fusion (pAD-NtAn2) was co-transformed with fusion constructs of GAL4-DNA-binding domain with MYB-interaction domain (ID) of GL3 or TT8. The transformants were grown in media with double (A) or quadruple (B) selections. 1, pAD-NtAn2+pBD-GL3aa 1-209; 2, pAD-NtAn2+pBD-TT8aa 1-204; 3, pAD+pBD-TT8aa1-204; 4, pAD-NtAn2+pBD (TIFF 239 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pattanaik, S., Kong, Q., Zaitlin, D. et al. Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231, 1061–1076 (2010). https://doi.org/10.1007/s00425-010-1108-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-010-1108-y

Keywords

Search

Navigation