Abstract
Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of β-d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)7CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.
This is a preview of subscription content, access via your institution.
References
An C, Ichinose Y, Yamada T, Tanaka Y, Shiraishi T, Oku H: Organization of the genes encoding chalcone synthase in Pisum sativum. Plant Mol Biol 21: 789–803 (1993).
Arias JA, Dixon RA, Lamb CJ: Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system. Plant Cell 5: 485–496 (1993).
Armstrong GA, Weisshaar B, Hahlbrock K: Homodimeric and heterodimeric leucine zipper proteins and nuclear factors from parsley recognize diverse promoter elements with ACGT cores. Plant Cell 4: 525–537 (1992).
Batschauer A, Ehmann B, Schafer E: Cloning and characterization of a chalcone synthase gene from mustard and its light-dependent expression. Plant Mol Biol 16: 175–185 (1991).
Bell JN, Ryder TB, Wingate VPM, Bailey JA, Lamb CJ: Differential accumulation of plant defense gene transcripts in a compatible and incompatible plant-pathogen interaction. Mol Cell Biol 6: 1615–1623 (1986).
Benfey PN, Chua N-H: The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250: 959–966 (1990).
Benfey PN, Ren L, Chua N-H: Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J 9: 1677–1684 (1990).
Block A, Dang JL, Hahlbrock K, Schulze LP: Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter. Proc Natl Acad Sci USA 87: 5387–5391 (1990).
Chory J: Light signals in leaf and chloroplast development: photoreceptors and downstream responses in search of a transduction pathway. New Biol 3: 538–548 (1991).
Coen ES, Carpenter R, Martin C: Transposable elements generate novel spatial patterns of gene expression in Antirrhinum majus. Cell 47: 285–296 (1986).
Dellaporta SL, Wood J, Hicks JB: A plant DNA mini preparation: version II. Plant Mol Biol Rep 1: 19–21 (1983).
Dewick PH: Isoflavonoids. In: Harborne JB (ed) The Flavonoids, pp. 125–209. Chapman and Hall, London (1988).
Dixon RA, Harrison MJ: Activation, structure and organization of genes involved in microbial defense in plants. Adv Genet 28: 165–234 (1990).
Dixon RA, Lamb CJ: Molecular communication in plant: microbial pathogen interaction. Annu Rev Plant Physiol Plant Mol Biol 41: 339–367 (1990).
Doerner PW, Stermer BA, Schmid J, Dixon RA, Lamb CJ: Plant defense gene promoter-reporter gene fusions in transgenic plants: tools for identification of novel inducer. Bio/technology 8: 845–848 (1990).
Donald GK, Cashmore AR: Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A. EMBO J 9: 1717–1726 (1990).
Drews GN, Beals TP, Bui AQ, Goldberg RB: Regional and cell-specific gene expression patterns during petal development. Plant Cell 4: 1383–1404 (1992).
Dron M, Clouse SD, Dixon RA, Lawton MA, Lamb CJ: Glutathione and fungal elicitor regulation of plant defense gene promoter in electroporated protoplasts. Proc Natl Acad Sci USA 85: 6738–6742 (1988).
Feinbaum RL, Ausubel FM: Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol Cell Biol 8: 1985–1992 (1988).
Franken P, Niesbach-Klosgen U, Weydemann U, Marechal-Drouard L, Saedler H, Wienand U: The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated: evidence for translational control of Whp expression by the anthocyanin intensifying gene. EMBO J 10: 2605–2612 (1991).
Fritze K, Staiger D, Czaja I, Walden R, Schell J, Wing D: Developmental and UV light regulation of the snapdragon chalcone synthase promoter. Plant Cell 3: 893–905 (1991).
Grotewold E, Drummond BJ, 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).
Hahlbrock K, Scheel D: Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40: 347–364 (1989).
Hatton D, Sablowski R, Yung M-H, Smith C, Schuch W, Bevan M: Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco. Plant J 7: 859–876 (1995).
Harrison MJ, Choudhary AD, Dubery I, Lamb CJ, Dixon RA: Stress responses in alfalfa (Medicago sativa L.). 8. Cis- elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts. Plant Mol Biol 16: 877–890 (1991).
Hauffe KD, Lee SP, Subramamiam R, Douglas CJ: Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco. Plant J 4: 235–253 (1993).
Herrmann A, Schulz W, Hahlbrock K: Two alleles of the single-copy chalcone synthase gene in parsley differ by a transposonlike element. Mol Gen Genet 212: 93–98 (1988).
Holsters M, deWaele D, Deoicker A, Messens E, VanMontagu M, Schell J: Transfection and transformation of A. tumefaciens. Mol Gen Genet 163: 181–187 (1978).
Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).
Kaiser T, Batschauer A: Cis-acting elements of the CHS1 gene from white mustard controlling promoter activity and spatial patterns of expression. Plant Mol Biol 28: 231–243 (1995).
Katagiri F, Chua N-H: Plant transcription factors: present knowledge and future challenges. Trends Genet 8: 22–27 (1991).
Koes RE, Spelt CE, van denElzen PJ, Mol JNM: Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida. Gene 81: 245–257 (1989).
Koes RE, vanBlokland R, Quattrocchio F, vanTunen AJ, Mol JNM: Chalcone synthase promoters in petunia are active in pigmented and unpigmented cell types. Plant Cell 2: 379–392 (1990).
Lamb CJ, Lawton MA, Dron M, Dixon RA: Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56: 215–224 (1989).
Lawton MA, Lamb CJ: Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection. Mol Cell Biol 7: 335–341 (1987).
Lawton MA, Dean SM, Dron M, Kooter JM, Kragh KM, Harrison MJ, Yu L, Tanguay L, Dixon RA, Lamb CJ: Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1. Plant Mol Biol 16: 235–249 (1991).
Loake GJ, Faktor O, Lamb CJ, Dixon RA: Combination of H-box [CCTACC(N)7CT] 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).
Ozeki Y, Davies E, Takeda J: Structure and expression of chalcone synthase gene in carrot suspension cultured cells regulated by 2,4-D. Plant Cell Physiol 34: 1029–1037 (1993).
Rogers SG, Horsch RB, Fraley RT: Plant molecular biology: gene transcription in plants: production of transformed plants using Ti plasmid vectors. Meth Enzymol 118: 627–640 (1986).
Ryder TB, Hedrick SA, Bell JN, Liang XW, Clouse SD, Lamb CJ: Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris. Mol Gen Genet 210: 219–233 (1987).
Sablowski RWM, Moyano E, Culianez-Macia FA, Schuch W, Martin C, Bevan M: A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. EMBO J 13: 128–137 (1994).
Sambrook L, Fristsch EF, Maniatis T: Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Press. Cold Spring Harbor, NY (1989).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Schmid J, Doerner PW, Clous SD, Dixon RA, Lamb CJ: Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco. Plant Cell 2: 619–631 (1990).
Schulze-Lefert P, Dangl JL, Becker-Andre M, Hahlbrock K, Schulz W: Inducible in vivo DNA footprints define sequence necessary for UV light activation of the parsley chalcone synthase gene. EMBO J 8: 651–656 (1989).
Sommer H, Saedler H: Structure of the chalcone synthase gene of Antirrhinum majus. Mol Gen Genet 202: 429–434 (1986).
Sommer H, Bonas U, Saedler H: Transposon-induced alternation in the promoter region affect transcription of the chalcone synthase gene of Antirrhinum majus. Mol Gen Genet 211: 49–55 (1988).
Stermer BA, Schmid J, Lamb CJ, Dixon RA: Infection and stress activation of bean chalcone synthase promoters in transgenic tobacco. Mol Plant-Microbe Interact 3: 381–388 (1990).
Weisshaar B, Armstrong GA, Block A, Costa e Silva O, Hahlbrock K: Light-inducible and constitutively expressed DNA-binding proteins recognizing a plant promoter element with functional relevance in light responsiveness. EMBO J 10: 1777–1786 (1991).
Williams ME, Foster R, Chua N-H: Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4: 485–496 (1992).
Yu L, Lamb CJ, Dixon RA: Purification and biochemical characterization of two proteins which bind the H-box cis-elements implicated in transcriptional activation of plant defense genes. Plant J 3: 805–816 (1993).
Van derHoeven C, Dietz A, Landsmann J: Variability of organspecific gene expression in transgen tobacco plants. Transgen Res 3: 159–165 (1994).
van derMeer IM, Spelt CE, Mol JN, Stuitje AR: Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression. Plant Mol Biol 15: 95–109 (1990).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Faktor, O., Kooter, J.M., Dixon, R.A. et al. Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression. Plant Mol Biol 32, 849–859 (1996). https://doi.org/10.1007/BF00020482
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020482