Plant Molecular Biology

, Volume 32, Issue 4, pp 599–609 | Cite as

Structure and regulation of the maize Bronze2 promoter

  • John P. Bodeau
  • Virginia Walbot
Research Article


The maize Bronze2 (Bz2) gene encodes a glutathione S-transferase that is required for anthocyanin pigment accumulation. Two classes of regulatory proteins, R and C1, are required for transcriptional activation of Bz2 and several additional structural genes. Functional domains of the Bz2 promoter were identified using Bz2 promoterdriven luciferase reporter genes electroporated into maize protoplasts together with R and C1 expression plasmids. Complete regulation was conferred by 224 nt of the Bz2 promoter. Within this region at least two separable regions are independently capable of conferring regulation by R and C1. Predicted regulatory elements corresponding to two classes of sequence motifs, the Myb-box homologous ‘C1-motif’, TAACTG/CAGTTA, and the G-box and E-box homologous ‘R-motif’, CACGTG, were shown to be important for full R and C1 activation of the Bz2 promoter. Expression of reconstructed Bz2 genes with mutated promoters was quantified using RNase protection, and this analysis confirmed results obtained using reporter genes.

Key words

anthocyanin Bronze2 gene transcriptional regulation Zea mays 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Biedenkapp H, Borgmeyer U, Sippel AE, Klempnauer KH: Viral Myb oncogene encodes a sequence-specific DNA-binding activity. Nature 335: 835–837 (1988).Google Scholar
  2. 2.
    Bodeau JP: Molecular and genetic regulation of Bronze-2 and other maize anthocyanin genes. Ph.D. dissertation, Stanford University (1994).Google Scholar
  3. 3.
    Bodeau JP, Walbot V: Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products. Mol Gen Genet 233: 379–387 (1992).Google Scholar
  4. 4.
    Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).Google Scholar
  5. 5.
    Callis J, Fromm M, Walbot V: Introns increase gene expression in cultured maize cells. Genes Devel 1: 1183–1200 (1987).Google Scholar
  6. 6.
    Carle-Urioste JC, Ko CH, Benito M, Walbot V: In vivo analysis of intron processing using splicing-dependent reporter gene assays. Plant Mol Biol 26: 1785–1795 (1994).Google Scholar
  7. 7.
    Chandler VL, Radicella JP, Robbins TP, Chen J, Turks D: Two regulatory genes of the maize anthocyanin pathway are homologous: isolation of B utilizing R genomic sequences. Plant Cell 1: 1175–1183 (1989).Google Scholar
  8. 8.
    Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159 (1987).Google Scholar
  9. 9.
    Christie PJ, Alfenito MR, Walbot V: Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194: 541–549 (1994).Google Scholar
  10. 10.
    Coe EH, Neuffer MG, Hoisington DA: The genetics of corn. In: Sprague GF, Dudley JW (eds) Corn and Corn Improvement, 3rd ed., pp. 81–258. American Society of Agronomy, Madison, WI (1988).Google Scholar
  11. 11.
    Cone KC, Cocciolone SM, Burr B, Burr B: Maize anthocyanin regulatory gene pl is a duplicate of c1 that functions in the plant. Plant Cell 5: 1795–1805 (1993).Google Scholar
  12. 12.
    Deng WP, Nickoloff JA: Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200: 81–88 (1992).Google Scholar
  13. 13.
    DeVetten NC, Ferl RJ: Transcriptional regulation of environmentally inducible genes in plants by an evolutionary conserved family of G-box binding factors. Int J Biochem 26: 1055–1068 (1994).Google Scholar
  14. 14.
    Dooner HK, Robbins TP, Jorgensen RA: Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet 25: 173–200 (1991).Google Scholar
  15. 15.
    Goff SA, Cone KC, Chandler VL: 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 Devel 6: 864–875 (1992).Google Scholar
  16. 16.
    Grotewold E, Athma P, Peterson T: Alternatively spliced products of the maize P gene encode proteins with homology to the DNA-binding domain of myb-like transcription factors. Proc Natl Acad Sci USA 88: 4587–4591 (1991).Google Scholar
  17. 17.
    Grotewold E, Drummond B, Bowen B, Peterson T: The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76: 543–553 (1994).Google Scholar
  18. 18.
    Hershberger RJ, Warren CA, Walbot V: Mutator activity in maize correlates with the presence and expression of the Mu transposable element Mu9. Proc Natl Acad Sci USA 88: 10198–10202 (1991).Google Scholar
  19. 19.
    Jefferson RA, Burgess SM, Hirsh D: β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83: 8447–8451 (1986).Google Scholar
  20. 20.
    Klein TM, Roth BA, Fromm ME: Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. Proc Natl Acad Sci USA 86: 6681–6685 (1989).Google Scholar
  21. 21.
    Littlewood TD, Evan GI: Transcription factors 2: helix-loophelix. Protein Profile 2: 621–702 (1995).Google Scholar
  22. 22.
    Loake GJ, Faktor O, Lamb CJ, Dixon RA: Combination of H-box (CCTACC(N7)CT) and G-box (CACGTG) cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid. Proc Natl Acad Sci USA 89: 9230–9234 (1992).Google Scholar
  23. 23.
    Ludwig SR, Habera LF, Dellaporta SL, Wessler SR: 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 (1989).Google Scholar
  24. 24.
    Luehrsen KR, deWet JR, Walbot V: Transient expression analysis in plants using firefly luciferase reporter gene. Meth Enzymol 216: 397–414 (1992).Google Scholar
  25. 25.
    Marrs KA, Alfenito MR, Lloyd AM, Walbot V: A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature: 397–400 (1995).Google Scholar
  26. 26.
    Nash J, Luehrsen KR, Walbot V: Bronze-2 gene of maize: reconstruction of a wild-type allele and analysis of transcription and splicing. Plant Cell 2: 1039–1049 (1990).Google Scholar
  27. 27.
    Ness SA, Marknell Å, Graf T: The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell 59: 1115–1125 (1989).Google Scholar
  28. 28.
    Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H: 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 (1987).Google Scholar
  29. 29.
    Pollak PE, Vogt T, Mo Y, Taylor LP: Chalcone synthase and flavonol accumulation in stigmas and anthers of Petunia hybrida. Plant Physiol 102: 925–932 (1993).Google Scholar
  30. 30.
    Roth BA, Goff SA, Klein TM, Fromm ME: C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites. Plant Cell 3: 317–325 (1991).Google Scholar
  31. 31.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).Google Scholar
  32. 32.
    Schmitz G, Theres K: Structural and functional analysis of the Bz2 locus of Zea mays: characterization of overlapping transcripts. Mol Gen Genet 233: 269–277 (1992).Google Scholar
  33. 33.
    Searle PF: Zinc dependent binding of a liver nuclear factor to metal response element MRE-a of the mouse metallothionein-I gene and variant sequences. Nucl Acids Res 18: 4683–4690 (1990).Google Scholar
  34. 34.
    Shieh MW, Wessler SR, Raikhel NV: Nuclear targeting of the maize R protein requires two nuclear localization sequences. Plant Physiol 101: 353–361 (1993).Google Scholar
  35. 35.
    Taylor LP, Briggs WR: Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings. Plant Cell 2: 115–127 (1990).Google Scholar
  36. 36.
    Tuerck JA, Fromm ME: Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes. Plant Cell 6: 1655–1663 (1994).Google Scholar
  37. 37.
    Walbot V, Benito M, Bodeau J, Nash J: Absciscic acid induces pink pigmentation in maize aleurone tissue in the absence of Bronze-2. Maydica 39: 19–28 (1994).Google Scholar
  38. 38.
    Wienand U, Paz-Ares J, Scheffler B, Saedler H: Molecular analysis of gene regulation in the anthocyanin pathway of Zea mays. In: Lamb CJ, Beachy RN (eds) Plant Gene Transfer, pp. 111–124. Wiley-Liss, New York (1990).Google Scholar
  39. 39.
    Yu LM, Lamb CJ, Dixon RA: Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes. Plant J 3: 805–816 (1993).Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • John P. Bodeau
    • 1
  • Virginia Walbot
    • 1
  1. 1.Department of Biological SciencesStanford UniversityStanfordUSA
  2. 2.Center for Engineering Plants for Resistance Against PathogensUniversity of CaliforniaDavisUSA

Personalised recommendations