Planta

, 234:363 | Cite as

Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active

  • Yanhong Bai
  • Sitakanta Pattanaik
  • Barunava Patra
  • Joshua R. Werkman
  • Claire H. Xie
  • Ling Yuan
Original Article

Abstract

The basic helix-loop-helix (bHLH) transcription factors (TFs) comprise one of the largest families of TFs involved in developmental and physiological processes in plants. Here, we describe the functional characterization of two bHLH TFs (NtAn1a and NtAn1b) isolated from tobacco (Nicotiana tabacum) flowers. NtAn1a and NtAn1b originate from two ancestors of tobacco, N. sylvestris and N. tomentosiformis, respectively. NtAn1a and NtAn1b share high sequence similarity with other known flavonoid-related bHLH TFs and are predominantly expressed in flowers. GUS expression driven by the NtAn1a promoter is consistent with NtAn1 transcript profile in tobacco flowers. Both NtAn1a and NtAn1b are transcriptional activators as demonstrated by transactivation assays using yeast cells and tobacco protoplasts. Ectopic expression of NtAn1a or NtAn1b enhances anthocyanin accumulation in tobacco flowers. In transgenic tobacco expressing NtAn1a or NtAn1b, both subsets of early and late flavonoid pathway genes were up-regulated. Yeast two-hybrid assays showed that NtAn1 proteins interact with the previously characterized R2R3-MYB TF, NtAn2. The NtAn1–NtAn2 complex activated the promoters of two key anthocyanin pathway genes, dihydroflavonol reductase and chalcone synthase. The promoter activation is severely repressed by dominant repressive forms of either NtAn1a or NtAn2, created by fusing the SRDX repressor domain to the TFs. Our results show that NtAn1 and NtAn2 act in concert to regulate the anthocyanin pathway in tobacco flowers and NtAn2 up-regulates NtAn1 gene expression.

Keywords

Anthocyanin bHLH transcription factor Flavonoids Transcriptional regulation MYB transcription factor 

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

TF

Transcription factor

Supplementary material

425_2011_1407_MOESM1_ESM.tif (260 kb)
Supplementary Figure (TIFF 259 kb)
425_2011_1407_MOESM2_ESM.doc (34 kb)
Supplementary Table (DOC 33 kb)

References

  1. Allan AC, Hellens RP, Laing WA (2008) MYB transcription factors that colour our fruit. Trends Plant Sci 13:99–102PubMedCrossRefGoogle Scholar
  2. Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol 48:958–970PubMedCrossRefGoogle Scholar
  3. Baudry A, Caboche M, Lepiniec L (2006) TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana. Plant J 46:768–779PubMedCrossRefGoogle Scholar
  4. Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394PubMedCrossRefGoogle Scholar
  5. Burr FA, Burr B, Scheffler BE, Blewitt M, Wienand U, Matz EC (1996) The maize repressor-like gene intensifier1 shares homology with the r1/b1 multigene family of transcription factors and exhibits missplicing. Plant Cell 8:1249–1259PubMedCrossRefGoogle Scholar
  6. Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EG, Hall RD, Bovy AG, Luo J, Martin C (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26:1301–1308PubMedCrossRefGoogle Scholar
  7. 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 147:2041–2053PubMedCrossRefGoogle Scholar
  8. Feller A, Hernandez JM, Grotewold E (2006) An ACT-like domain participates in the dimerization of several plant basic-helix-loop-helix transcription factors. J Biol Chem 281:28964–28974PubMedCrossRefGoogle Scholar
  9. Fukasawa-Akada T, Kung SD, Watson JC (1996) Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana. Plant Mol Biol 30:711–722PubMedCrossRefGoogle Scholar
  10. Goff SA, Cone KC, Chandler VL (1992) Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins. Genes Dev 6:864–875PubMedCrossRefGoogle Scholar
  11. Gong ZZ, Yamagishi E, Yamazaki M, Saito K (1999) 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 41:33–44PubMedCrossRefGoogle Scholar
  12. Goodrich J, Carpenter R, Coen ES (1992) A common gene regulates pigmentation pattern in diverse plant species. Cell 68:955–964PubMedCrossRefGoogle Scholar
  13. Grant GA (2006) The ACT domain: a small molecule binding domain and its role as a common regulatory element. J Biol Chem 281:33825–33829PubMedCrossRefGoogle Scholar
  14. Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780PubMedCrossRefGoogle Scholar
  15. Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20:735–747PubMedCrossRefGoogle Scholar
  16. Hellens RP, Moreau C, Lin-Wang K, Schwinn KE, Thomson SJ, Fiers MW, Frew TJ, Murray SR, Hofer JM, Jacobs JM, Davies KM, Allan AC, Bendahmane A, Coyne CJ, Timmerman-Vaughan GM, Ellis TH (2010) Identification of Mendel’s white flower character. PLoS One 5:e13230PubMedCrossRefGoogle Scholar
  17. Hichri I, Heppel SC, Pillet J, Leon C, Czemmel S, Delrot S, Lauvergeat V, Bogs J (2010) The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Mol Plant 3:509–523PubMedCrossRefGoogle Scholar
  18. Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V (2011) Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot. doi:10.1093/jxb/exq442
  19. Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J 34:733–739PubMedCrossRefGoogle Scholar
  20. Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242PubMedCrossRefGoogle Scholar
  21. Lee D, Douglas CJ (1996) Two divergent members of a tobacco 4-coumarate: coenzyme A ligase (4CL) gene family cDNA structure, gene inheritance and expression, and properties of recombinant proteins. Plant Physiol 112:193–205PubMedCrossRefGoogle Scholar
  22. Lim KY, Matyasek R, Kovarik A, Leitch AR (2004) Genome evolution in allotetraploid Nicotiana. Biol J Linn Soc 82:599–606CrossRefGoogle Scholar
  23. Lloyd AM, Walbot V, Davis RW (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258:1773–1775PubMedCrossRefGoogle Scholar
  24. 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 86:7092–7096PubMedCrossRefGoogle Scholar
  25. Matsui K, Ohme-Takagi M (2010) Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain. Plant J 61:570–578PubMedCrossRefGoogle Scholar
  26. Matus JT, Poupin MJ, Canon P, Bordeu E, Alcalde JA, Arce-Johnson P (2010) Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.). Plant Mol Biol 72:607–620PubMedCrossRefGoogle Scholar
  27. Mol J, Grotewold E, Koes R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217CrossRefGoogle Scholar
  28. Murad L, Lim KY, Christopodulou V, Matyasek R, Lichtenstein CP, Kovarik A, Leitch AR (2002) The origin of the paternal genome of tobacco is traced back to a particular lineage within Nicotiana tomentosiformis (Solanaceae). Am J Bot 89:921–928PubMedCrossRefGoogle Scholar
  29. 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 12:1863–1878PubMedCrossRefGoogle Scholar
  30. 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 1759:308–318PubMedGoogle Scholar
  31. Pattanaik S, Xie CH, Yuan L (2008) The interaction domains of the plant Myc-like bHLH transcription factors can regulate the transactivation strength. Planta 227:707–715PubMedCrossRefGoogle Scholar
  32. Pattanaik S, Kong Q, Zaitlin D, Werkman JR, Xie CH, Patra B, Yuan L (2010a) Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231:1061–1076PubMedCrossRefGoogle Scholar
  33. Pattanaik S, Werkman JR, Kong Q, Yuan L (2010b) Site-directed mutagenesis and saturation mutagenesis for the functional study of transcription factors involved in plant secondary metabolite biosynthesis. Methods Mol Biol 643:47–57PubMedCrossRefGoogle Scholar
  34. 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 156:1349–1362PubMedGoogle Scholar
  35. Ptashne M (1988) How eukaryotic transcriptional activators work. Nature 335:683–689PubMedCrossRefGoogle Scholar
  36. 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 13:475–488PubMedCrossRefGoogle Scholar
  37. 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 11:1433–1444PubMedCrossRefGoogle Scholar
  38. Rabino I, Mancinelli AL (1986) Light, temperature, and anthocyanin production. Plant Physiol 81:922–924PubMedCrossRefGoogle Scholar
  39. Rushton PJ, Bokowiec MT, Han S, Zhang H, Brannock JF, Chen X, Laudeman TW, Timko MP (2008) Tobacco transcription factors: novel insights into transcriptional regulation in the Solanaceae. Plant Physiol 147:280–295PubMedCrossRefGoogle Scholar
  40. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  41. Spelt C, Quattrocchio F, Mol JN, Koes R (2000) Anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12:1619–1632PubMedCrossRefGoogle Scholar
  42. Spelt C, Quattrocchio F, Mol J, Koes R (2002) ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms. Plant Cell 14:2121–2135PubMedCrossRefGoogle Scholar
  43. 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 1769:139–148PubMedGoogle Scholar
  44. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  45. Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15:1749–1770PubMedCrossRefGoogle Scholar
  46. Urao T, Yamaguchi-Shinozaki K, Mitsukawa N, Shibata D, Shinozaki K (1996) Molecular cloning and characterization of a gene that encodes a MYC-related protein in Arabidopsis. Plant Mol Biol 32:571–576PubMedCrossRefGoogle Scholar
  47. Winkel-Shirley B (2001a) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493PubMedCrossRefGoogle Scholar
  48. Winkel-Shirley B (2001b) It takes a garden. How work on diverse plant species has contributed to an understanding of flavonoid metabolism. Plant Physiol 127:1399–1404PubMedCrossRefGoogle Scholar
  49. Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A (2003) A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development 130:4859–4869PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Yanhong Bai
    • 1
    • 3
  • Sitakanta Pattanaik
    • 2
    • 3
  • Barunava Patra
    • 2
    • 3
  • Joshua R. Werkman
    • 2
  • Claire H. Xie
    • 2
    • 3
  • Ling Yuan
    • 2
    • 3
  1. 1.College of AgronomyNorthwest A&F UniversityShaanxiPeople’s Republic of China
  2. 2.Department of Plant and Soil SciencesUniversity of KentuckyLexingtonUSA
  3. 3.Kentucky Tobacco Research and Development CenterUniversity of KentuckyLexingtonUSA

Personalised recommendations