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. 1998;37(5):885–896. doi: 10.1023/A:1006051832213

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

Jagdeep S Sandhu 2, Carl I Webster 3, John C Gray 1,✉
PMCID: PMC7089012  PMID: 9678583

Abstract

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the β-glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, β-glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.

Keywords: enhancer, patatin, plastocyanin, potato, tobacco

References

  • 1.Allen G., Hall EGJ, Childs LC, Weissinger AK, Spiker S, Thompson WF. Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell. 1993;5:603–613. doi: 10.1105/tpc.5.6.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Avramova Z, Bennetzen JL. Isolation of matrices from maize leaf nuclei: identification of a matrix-binding site adjacent to the Adh1 gene. Plant Mol Biol. 1993;22:1135–1143. doi: 10.1007/BF00028982. [DOI] [PubMed] [Google Scholar]
  • 3.Banerji J, Rusconi S, Schaffner W. Expression of a b-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981;27:299–308. doi: 10.1016/0092-8674(81)90413-x. [DOI] [PubMed] [Google Scholar]
  • 4.Benfey PN, Chua N-H. The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science. 1990;250:959–966. doi: 10.1126/science.250.4983.959. [DOI] [PubMed] [Google Scholar]
  • 5.Benfey P, Ren L, Chua N-H. The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J. 1989;8:2195–2202. doi: 10.1002/j.1460-2075.1989.tb08342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Bevan M. Binary Agrobacterium vectors for plant transformation. Nucl Acids Res. 1984;12:8711–8721. doi: 10.1093/nar/12.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bichler J, Herrmann RG. Analysis of the promoters of the single-copy genes for plastocyanin and subunit d of the chloroplast ATP synthase from spinach. Eur J Biochem. 1990;190:415–426. doi: 10.1111/j.1432-1033.1990.tb15591.x. [DOI] [PubMed] [Google Scholar]
  • 8.Blundy KS, Blundy MAC, Carter D, Wilson F, Park WD, Burrell MM. The expression of class 1 patatin gene fusions in transgenic potato varies with both gene and cultivar. Plant Mol Biol. 1991;16:153–160. doi: 10.1007/BF00017925. [DOI] [PubMed] [Google Scholar]
  • 9.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  • 10.Breyne P, Van Montagu M, Depicker N, Gheysen G. Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell. 1992;4:463–471. doi: 10.1105/tpc.4.4.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Breyne P, Van Montagu M, Gheysen G. The role of scaffold attachment regions in the structural and functional organization of plant chromatin. Transgenic Res. 1994;3:195–202. doi: 10.1007/BF01973987. [DOI] [PubMed] [Google Scholar]
  • 12.Bustos MM, Guiltinan MJ, Jordano J, Begum D, Kalkan FA, Hall TC. Regulation of b-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean b-phaseolin gene. Plant Cell. 1989;1:839–853. doi: 10.1105/tpc.1.9.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Castresana C, Garcia-Luque L, Alonso E, Malik VS, Cashmore AR. Both positive and negative elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia. EMBO J. 1988;7:1929–1936. doi: 10.1002/j.1460-2075.1988.tb03030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chen Z-L, Pan N-S, Beachy RN. A DNA sequence element that confers seed-specific enhancement to a constitutive promoter. EMBO J. 1988;7:297–302. doi: 10.1002/j.1460-2075.1988.tb02812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Czarnecka E, Ingersoll JC, Gurley WB. AT-rich promoter elements of soybean heat shock gene Gmhsp 17.5E bind two sets of nuclear proteins in vitro. Plant Mol Biol. 1992;19:985–1000. doi: 10.1007/BF00040530. [DOI] [PubMed] [Google Scholar]
  • 16.Dean C, Favreau M, Bond-Nutter D, Bedbrook J, Dunsmuir P. Sequences 50 to translation start regulate expression of petunia rbcS genes. Plant Cell. 1989;1:209–215. doi: 10.1105/tpc.1.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ellis JG, Llewellyn DJ, Dennis ES, Peacock WJ. Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. EMBO J. 1987;6:11–16. doi: 10.1002/j.1460-2075.1987.tb04711.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Fluhr R, Kuhlemeier C, Nagy F, Chua N-H. Organ-specific and light-induced expression of plant genes. Science. 1986;232:1106–1111. doi: 10.1126/science.232.4754.1106. [DOI] [PubMed] [Google Scholar]
  • 19.Grierson C, Du J-S, Zabala MT, Beggs K, Goldsworth M, Bevan M. Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant J. 1994;5:815–826. doi: 10.1046/j.1365-313x.1994.5060815.x. [DOI] [PubMed] [Google Scholar]
  • 20.Grosschedl R, Giese K, Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994;10:94–100. doi: 10.1016/0168-9525(94)90232-1. [DOI] [PubMed] [Google Scholar]
  • 21.Hall G, Allen GC, Loer DS, Thompson WF, Spiker S. Nuclear scaffolds and scaffold-attachment regions in higher plants. Proc Natl Acad Sci USA. 1991;88:9320–9324. doi: 10.1073/pnas.88.20.9320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Herr W. The SV40 enhancer: transcriptional regulation through a hierarchy of combinatorial interactions. Semin Virol. 1993;4:3–13. [Google Scholar]
  • 23.Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT. A simple and general method for transferring genes into plants. Science. 1985;227:1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
  • 24.Jacobsen K, Laursen NB, Jensen EØ, Marcker A, Poulsen C, Marcker KA. HMG I-like proteins from leaf and nodule nuclei interact with different AT motifs in soybean nodulin promoters. Plant Cell. 1990;2:85–94. doi: 10.1105/tpc.2.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Jefferson RA, Goldsbrough A, Bevan MW. Transcriptional regulation of a patatin-1 gene in potato. Plant Mol Biol. 1990;14:995–1006. doi: 10.1007/BF00019396. [DOI] [PubMed] [Google Scholar]
  • 26.Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901–3907. doi: 10.1002/j.1460-2075.1987.tb02730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Jofuku KD, Okamuro JK, Goldberg RB. Interaction of an embryo DNA binding protein with a soybean lectin gene upstream region. Nature. 1987;328:734–737. doi: 10.1038/328734a0. [DOI] [PubMed] [Google Scholar]
  • 28.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. 1995;28:231–243. doi: 10.1007/BF00020243. [DOI] [PubMed] [Google Scholar]
  • 29.Kay R, Chan A, Daly M, McPherson J. Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science. 1987;236:1229–1302. doi: 10.1126/science.236.4806.1299. [DOI] [PubMed] [Google Scholar]
  • 30.Kim SY, May GD, Park WD. Nuclear protein factors binding to a class I patatin promoter region are tuber-specific and sucrose-inducible. Plant Mol Biol. 1994;26:603–615. doi: 10.1007/BF00013747. [DOI] [PubMed] [Google Scholar]
  • 31.Lam E, Kano-Murakami Y, Gilmartin P, Niner B, Chua NH. A metal-dependent DNA-binding protein interacts with a constitutive element of a light-responsive promoter. Plant Cell. 1990;2:857–866. doi: 10.1105/tpc.2.9.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Last DI, Gray JC. Plastocyanin is encoded by a single copy gene in the pea haploid genome. Plant Mol Biol. 1989;12:655–666. doi: 10.1007/BF00044156. [DOI] [PubMed] [Google Scholar]
  • 33.Last DI, Gray JC. Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants. Plant Mol Biol. 1990;14:229–238. doi: 10.1007/BF00018563. [DOI] [PubMed] [Google Scholar]
  • 34.Laursen NB, Larsen K, Knudsen JY, Hoffmann HJ, Poulsen C, Marcker KA, Jensen EØ. A protein binding AT-rich sequence in the soybean leghemoglobin c3 promoter is a general cis element that requires proximal DNA elements to stimulate transcription. Plant Cell. 1994;6:659–658. doi: 10.1105/tpc.6.5.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Liu X-J, Prat S, Willmitzer L, Frommer WB. Cis-regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter/GUS-gene fusion. Mol Gen Genet. 1990;223:401–406. doi: 10.1007/BF00264446. [DOI] [PubMed] [Google Scholar]
  • 36.Lübberstedt T, Oelmüller R, Wanner G, Herrmann RG. Interacting cis-elements in the plastocyanin promoter from spinach ensure regulated high-level expression. Mol Gen Genet. 1994;242:602–613. doi: 10.1007/BF00285284. [DOI] [PubMed] [Google Scholar]
  • 37.Maher JF, Nathans D. Multivalent DNA-binding properties of the HMG-I proteins. Proc Natl Acad Sci USA. 1996;93:6716–6720. doi: 10.1073/pnas.93.13.6716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Marsh JL, Erfle M, Wykes EJ. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984;32:481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  • 39.Mignery GA, Pikaard CS, Park WD. Molecular characterization of the patatin multigene family of potato. Gene. 1988;62:27–44. doi: 10.1016/0378-1119(88)90577-X. [DOI] [PubMed] [Google Scholar]
  • 40.Mlynárová L, Loonen A, Heldens J, Jansen R-C, Keizer P, Stiekema W-J, Nap J-P. Reduced position effect in mature transgenic plants conferred by the chicken lysozyme matrixassociated region. Plant Cell. 1994;6:417–426. doi: 10.1105/tpc.6.3.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Moreau P, Hen R, Wasylyk B, Everett R, Gaub MP, Chambon P. The SV40 72 base pair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucl Acids Res. 1981;9:6047–6068. doi: 10.1093/nar/9.22.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Nielsen PS, Gausing K. The precursor of barley plastocyanin. Sequence of cDNA clones and gene expression in different tissues. FEBS Lett. 1987;225:159–162. doi: 10.1016/0014-5793(87)81149-3. [DOI] [Google Scholar]
  • 43.Nieto-Sotelo J, Ichida A, Quail PH. PF1: an A-T hookcontaining DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter. Plant Cell. 1994;6:287–301. doi: 10.1105/tpc.6.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Pedersen TJ, Arwood LA, Spiker S, Guiltinan MJ, Thompson WF. High mobility group chromosomal proteins bind to ATrich tracts flanking plant genes. Plant Mol Biol. 1989;16:95–104. doi: 10.1007/BF00017920. [DOI] [PubMed] [Google Scholar]
  • 45.Perl A, Aviv D, Willmitzer L, Galun E. In vitro tuberization in transgenic potatoes harboring b-glucuronidase linked to a patatin promoter: effect of sucrose levels and photoperiod. Plant Sci. 1991;73:87–95. [Google Scholar]
  • 46.Pikaard CS, Brusca JS, Hannapel DJ, Park WD. The two classes of genes for the major potato tuber protein, patatin, are differentially expressed in tubers and roots. Nucl Acids Res. 1987;15:1979–1994. doi: 10.1093/nar/15.5.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Pwee K-H, Gray JC. The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants. Plant J. 1993;3:437–449. doi: 10.1046/j.1365-313X.1993.t01-22-00999.x. [DOI] [PubMed] [Google Scholar]
  • 48.Pwee K-H, Webster CI, Gray JC. HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin promoter. Plant Mol Biol. 1994;26:1907–1920. doi: 10.1007/BF00019502. [DOI] [PubMed] [Google Scholar]
  • 49.Rieping M, Schöffl F. Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco. Mol Gen Genet. 1992;231:226–232. doi: 10.1007/BF00279795. [DOI] [PubMed] [Google Scholar]
  • 50.Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  • 51.Schöffl F, Schroder G, Kliem M, Rieping M. A SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants. Transgenic Res. 1993;2:93–100. doi: 10.1007/BF01969382. [DOI] [PubMed] [Google Scholar]
  • 52.Shen W-J, Forde BG. Efficient transformation of Agrobacterium sp. by high voltage electroporation. Nucl Acids Res. 1989;17:8385. doi: 10.1093/nar/17.20.8385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Simpson J, Schell J, Van Montagu M, Herrera-Estrella L. Light-inducible and tissue-specific pea lhcp gene expression involves an upstream element combining enhancer-silencer like properties. Nature. 1986;323:551–554. [Google Scholar]
  • 54.Slatter RE, Dupree P, Gray JC. A scaffold-associated DNA region is located downstream of the pea plastocyanin gene. Plant Cell. 1991;3:1239–1250. doi: 10.1105/tpc.3.11.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Stockhaus J, Schell J, Willmitzer L. Identification of enhancer elements in the upstream region of the nuclear photosynthetic gene ST-LS1. Plant Cell. 1989;1:803–813. doi: 10.1105/tpc.1.8.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Terzaghi WB, Cashmore AR. Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol. 1995;46:445–474. doi: 10.1146/annurev.pp.46.060195.002305. [DOI] [Google Scholar]
  • 57.Timko MP, Kausch AP, Castresana C, Fassler J, Herrera-Estrella L, Van den Broeck G, Van Montagu M, Schell J, Cashmore AR. Light regulation of plant gene expression by an upstream enhancer-like element. Nature. 1985;318:579–582. doi: 10.1038/318579a0. [DOI] [PubMed] [Google Scholar]
  • 58.Tjaden G, Coruzzi GM. A novel AT-rich DNA binding protein that combines an HMG I-like DNA binding domain with a putative transcription domain. Plant Cell. 1994;6:107–118. doi: 10.1105/tpc.6.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.van der Geest AHM, Hall GE, Spiker S, Hall TC. The β phaseolin gene is flanked by matrixattachment regions. Plant J. 1994;6:413–423. doi: 10.1046/j.1365-313X.1994.06030413.x. [DOI] [Google Scholar]
  • 60.Vorst O, Kock P, Lever A, Weterings P, Weisbeek P, Smeekens S. The promoter of the Arabidopsis thaliana plastocyanin gene contains a far upstream enhancer-like element involved in chloroplast-dependent expression. Plant J. 1993;4:933–945. doi: 10.1046/j.1365-313X.1993.04060933.x. [DOI] [PubMed] [Google Scholar]
  • 61.Webster CI, Packman LC, Pwee K-H, Gray JC. High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter. Plant J. 1997;11:703–715. doi: 10.1046/j.1365-313X.1997.11040703.x. [DOI] [PubMed] [Google Scholar]
  • 62.Wenzler HC, Mignery GA., Fisher LM, Park WD. Analysis of a chimeric class-I patatin-GUS gene in transgenic potato plants: high-level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants. Plant Mol Biol. 1989;12:41–50. doi: 10.1007/BF00017446. [DOI] [PubMed] [Google Scholar]
  • 63.Yamaguchi-Shinozaki K, Shinozaki K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell. 1994;6:251–264. doi: 10.1105/tpc.6.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Yamamoto S, Nishihara M, Morikawa H, Yamauchi D, Minamikawa T. Promoter analysis of seed storage protein genes from Canavalia gladiat a D.C. Plant Mol Biol. 1995;27:729–741. doi: 10.1007/BF00020226. [DOI] [PubMed] [Google Scholar]
  • 65.Zhao K, Kas E, Gonzalez E, Laemmli UK. SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro. HMG-I/Y is enriched in H1-depleted chromatin. EMBO J. 1993;12:3237–3247. doi: 10.1002/j.1460-2075.1993.tb05993.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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