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Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products

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Summary

The putative maize transcription factor genes R and C1 are required for expression of reporter genes with promoters from the Bz1 and A1 genes, which encode enzymes required for anthocyanin biosynthesis in maize. Bz2 is another anthocyanin biosynthetic gene; we show that expression of a reporter gene from the Bz2 promoter also requires R and C1 when the fusion construct is introduced into maize kernels by particle gun bombardment. When electroporated into maize protoplasts from a suspension cell line not synthesizing anthocyanins, reporter genes with Bz2, Bz1, and A1 promoters are expressed only when both R and C1 expression plasmids are co-electroporated. Electroporation of R and C1 expression plasmids also induces the endogenous genes required for anthocyanin synthesis, resulting in pink protoplasts within 24 h. RNase protection analysis demonstrates that accumulation of mRNA from the endogenous Bzl and Bz2 genes absolutely requires introduced R and C1. In time-course experiments there is a delay of 3–6 h before the Bz2 promoter is activated, supporting the proposed role for R- and C1-encoded proteins in transcriptional control. An excess of R relative to C1 suppresses expression of A1, Bz1, and Bz2 promoters, suggesting an interaction between the R and C1 proteins.

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References

  • Bradford MM (1976) 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

    Article  CAS  PubMed  Google Scholar 

  • Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1: 1183–1200

    Google Scholar 

  • Chandler VL, Radicella JP, Robbins TP, Chen J, Turks D (1989) Two regulatory genes of the maize anthocyanin pathway are homologous: Isolation of B utilizing R genomic sequences. Plant Cell 1:1175–1183

    Google Scholar 

  • Christie PJ, Walbot V (1992) Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: Enhancement of transcript abundance and anthocyanin pigmentation in maize (B73N) seedlings. Plant Physiol, in press

  • Coe EH Jr, Neuffer MG (1988) The genetics of corn. In: Sprague GF, Dudley JW (ed) Corn and corn improvement, third edition. Am Soc Agronomy, Madison, Wisconsin, pp 81–258

    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

    Google Scholar 

  • de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737

    Google Scholar 

  • Dellaporta SL, Greenblatt I, Kermicle JL, Hicks JB, Wessler SR (1988) Molecular cloning of the maize R-nj allele by transposon tagging with Ac. In: Gustafson JP, Appels R (ed) Chromosome structure and function: Impact of new concepts. 18th Stadler Genetics Symposium. Plenum Press, New York, pp 263–282

    Google Scholar 

  • Dooner HK (1983) Coordinate genetic regulation of flavonoid biosynthetic enzymes in maize. Mol Gen Genet 189:136–141

    Google Scholar 

  • Dooner HK, Robbins TP, Jorgensen RA (1991) Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet 25:173–199

    Article  CAS  PubMed  Google Scholar 

  • Fedoroff NV, Furtek DB, Nelson OE Jr (1984) Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable element Activator (Ac). Proc Natl Acad Sci USA 81:3825–3829

    Google Scholar 

  • Ferl RJ, Nick HS (1987) In vivo detection of regulatory factor binding sites in the 5′ flanking region of maize Adhl. J Biol Chem 262:7947–7950

    Google Scholar 

  • Fromm M, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82:5824–5828

    Google Scholar 

  • Fromm M, Callis J, Taylor LP, Walbot V (1987) Electroporation of DNA and RNA into plant protoplasts. Methods Enzymol 153:351–366

    Google Scholar 

  • Gallie DR, Lucas WJ, Walbot V (1989) Visualizing mRNA expression in plant protoplasts: Factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301–311

    Google Scholar 

  • Giuliano G, Pichersky E, Malik VS, Timko MP, Scolnik PA, Cashmore AR (1988) An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene. Proc Natl Acad Sci 85:7089–7093

    CAS  PubMed  Google Scholar 

  • Goff SA, Klein TM, Roth BA, Fromm ME, Cone KC, Radicella JP, Chandler VL (1990) Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. EMBO J 9:2517–2522

    Google Scholar 

  • Hattori T, Vasil V, Rosenkrans L, Hannah LC, McCarty DR, Vasil IK (1992) The Viviparous-1 gene and abscisic acid activate the C1 regulatory gene for anthocyanin biosynthesis during seed maturation in maize. Genes Dev 6:609–618

    Google Scholar 

  • Inghirami G, Grignani F, Sternas L, Lombardi L, Knowles DM, Dalla-Favera R (1990) Down-regulation of LFA-1 adhesion receptors by c-myc oncogene in human B lymphoblastoid cells. Science 250:682–686

    Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451

    CAS  PubMed  Google Scholar 

  • Klein TM, Gradziel T, Fromm ME, Sanford JC (1988) Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. Bio/Technology 6:559–563

    Google Scholar 

  • Klein TM, Roth BA, Fromm ME (1989) Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. Proc Natl Acad Sci USA 86:6681–6685

    Google Scholar 

  • Lipphardt S, Brettschneider R, Kreuzaler F, Schell J, Dangl JL (1988) UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric Antirrhinum majus chalcone synthase gene. EMBO J 7:4027–4033

    Google Scholar 

  • Ludwig SE, Bowen B, Beach L, Wessler SR (1990) A regulatory gene as a novel visible marker for maize transformation. Science 247:449–450

    Google Scholar 

  • Ludwig SR, Habera LF, Dellaporta SL, Wessler S (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

    Google Scholar 

  • Luehrsen KR, Walbot V (1991) Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol Gen Genet 225:81–93

    Google Scholar 

  • Luehrsen KR, de Wet JR, Walbot V (1992) Transient expression analysis in plants using the firefly luciferase reporter gene. Methods Enzymol, in press

  • Marcotte WR, Russell SH, Quatrano RS (1989) Abscisic acidresponsive sequences from the Em gene of wheat. Plant Cell 1:969–976

    Article  CAS  PubMed  Google Scholar 

  • McCarty DR, Carson CB, Stinard PS, Robertson DS (1989) Molecular analysis of viviparous-1: An abscisic acid-insensitive mutant of maize. Plant Cell 1:523–532

    Google Scholar 

  • McLaughlin M, Walbot V (1987) Cloning of a mutable bz2 allele of maize by transposon tagging and differential hybridization. Genetics 117:771–776

    Google Scholar 

  • Menssen A, Hohmann S, Martin W, Schnable PS, Peterson PA, Saedler H, Gierl A (1990) The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene. EMBO J 9:3051–3057

    CAS  PubMed  Google Scholar 

  • Murre C, McCaw PS, Vaessin H, Candy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB, Weintraub H, Baltimore D (1989) Interactions between heterologous helixloop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58:537–544

    Article  CAS  PubMed  Google Scholar 

  • Nash J, Luehrsen KR, Walbot V (1990) Bronze-2 gene of maize: reconstruction of a wild-type allele and analysis of transcription and splicing. Plant Cell 2:1039–1049

    Google Scholar 

  • O'Reilly C, Shepherd NS, Pereira A, Schwarz-Sommer Z, Bertram I, Robertson DS, Peterson PA, Saedler H (1985) Molecular cloning of the al locus of Zea mays using the transposable elements En and Mul. EMBO J 4:877–882

    Google Scholar 

  • Oard JH, Paige DF, Simmonds JA, Gradziel TM (1990) Transient gene expression in maize, rice, and wheat cells using an airgun apparatus. Plant Physiol 92:334–339

    Google Scholar 

  • Paz-Ares J, Wienand U, Peterson P, Saedler H (1986) Molecular cloning of the c locus of Zea mays: A locus regulating the anthocyanin pathway. EMBO J 5:829–833

    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

    Google Scholar 

  • Perrot GH, Cone KC (1989) Nucleotide sequence of the maize R-S gene. Nucleic Acids Res 17:8003

    Google Scholar 

  • Planckaert F, Walbot V (1989) Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus. Plant Cell Rep 8:144–147

    Google Scholar 

  • Roth BA, Goff SA, Klein TM, Fromm ME (1991) 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

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Taylor LP, Briggs WR (1990) Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings. Plant Cell 2:115–127

    Google Scholar 

  • Wienand U, Weydemann U, Niesbach-Kloesgen U, Peterson PA, Saedler H (1986) Molecular cloning of the C2 locus of Zea mays, the gene coding for chalcone synthase. Mol Gen Genet 203:202–207

    Google Scholar 

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Communicated by H. Saedler

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Bodeau, J.P., Walbot, V. Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products. Molec. Gen. Genet. 233, 379–387 (1992). https://doi.org/10.1007/BF00265434

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