Abstract
We have previously shown by RNA gel blot analyses that the tobacco polyubiquitin-encoding gene Ubi.U4 is expressed in a complex pattern during plant development (Genschik et al., 1994). In order to study its tissue-specific expression, we cloned the fragment containing the −263 bp proximal promoter of the gene, the leader intron and the first ubiquitin monomer in front of the reporter GUS gene. Histochemical analyses for GUS activity during tobacco plant development revealed that the gene is expressed at variable amounts in many plant tissues with high levels in metabolically active and/or dividing cells and in the vascular tissues of the plant. We also analysed the expression pattern of constructs in which either the intron or the intron together with the first ubiquitin monomer were deleted. Our results indicate that the ubiquitin leader intron is not only a quantitative determinant of gene expression but may also influence the tissue-specific expression pattern.
Similar content being viewed by others
References
Almoguera, C., Coca, M.A. and Jordano, J. 1995. Differential accumulation of sunflower tetraubiquitin mRNAs during zygotic embryogenesis and developmental regulation of their heat-shock response. Plant Physiol. 107: 765–773.
Atanassova, R., Favet, N., Martz, F., Chabbert, B., Tollier, M.T., Monties, B., Fritig, B. and Legrand, M. 1995. Altered lignin composition in transgenic tobacco expressing Omethyltransferase sequences in sense and antisense orientation. Plant J. 8: 465–477.
Bachmair, A., Becker, F., Masterson, R.V. and Schell, J. 1990. Perturbation of the ubiquitin system causes leaf curling, vascular tissue alterations and necrotic lesions in a higher plant. EMBO J. 9: 4543–4549.
Belknap, W.R. and Garbarino, J.E. 1996. The role of ubiquitin in plant senescence and stress responses. Trends Plant Sci. 1: 331–335.
Binet, M.-N., Weil, J.-H. and Tessier, L.-H. 1991. Structure and expression of sunflower ubiquitin genes. Plant Mol. Biol. 17: 395–407.
Bolle, C., Sopory, S., Lübberstedt, T., Herrmann, R.G. and Oelmüller, R. 1994. Segments encoding 5′-untranslated leaders of genes for thylakoid proteins contain cis-elements essential for transcription. Plant J. 6: 513–523.
Butt, T.R., Jonnalagadda, S., Monia, B.P., Sternberg, E.J., Marsh, J.A., Stadel, J.M., Ecker, D.J. and Crooke, S.T. 1989. Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proc. Natl. Acad. Sci. USA 86: 2540–2544.
Callis, J. and Bedinger, P. 1994. Developmentally regulated loss of ubiquitin and ubiquitinated proteins during pollen maturation in maize. Proc. Natl. Acad. Sci. USA 91: 6074–6077.
Callis, J, Raasch, J.A. and Vierstra, R.D. 1990. Ubiquitin extension proteins of Arabidopsis thaliana. Structure, localization, and expression of their promoters in transgenic tobacco. J. Biol. Chem. 265: 12486–12493.
Callis, J., Carpenter, T., Sun, C.-W. and Vierstra, R.D. 1995. Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia. Genetics 139: 921–939.
Caspar, T. and Quail, P.H. 1993. Promoter and leader regions involved in the expression of the Arabidopsis ferredoxin A gene. Plant J. 3: 161–174.
Chaboute, M.E., Chaubet, N., Philipps, G., Ehling, M. and Gigot, C. 1987. Genomic organization and nucleotide sequences of two histone H3 and two histone H4 genes of Arabidopsis thaliana. Plant Mol. Biol. 8: 179–191.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675–689.
Cornejo, M.J., Luth, D., Blankenship, K.M., Andersen, O.D. and Blechl, A.E. 1993. Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol. Biol. 23: 567–581.
Dietrich, R.A., Radke, S.E. and Harada, J.J. 1992. Downstream DNA sequences are required to activate a gene expressed in the root cortex of embryos and seedlings. Plant Cell 4: 1371–1382.
Ecker, D.J., Stadel, J.M., Butt, T.R., Marsh, J.A., Monia, B.P., Powers, D.A., Gorman, J.A., Clark, P.E., Warren, F., Shatzman, A. and Crooke, S.T. 1989. Increasing gene expression in yeast by fusion to ubiquitin. J. Biol. Chem. 264: 7715–7719.
Feinberg, A.P. and Vogelstein, B. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13.
Finley, D., Özkaynak, E. and Varshavsky, A. 1987. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48: 1035–1046.
Fu, H.Y. and Park, W.D. 1995. Sink and vascular-associated sucrose synthase functions are encoded by different gene classes in potato. Plant Cell 7: 1369–1385.
Fu, H.Y., Kim, S.Y. and Park, W.D. 1995a. High-level tuber expression and sucrose inducibility of a potato sus4 sucrose synthase gene require 5′ and 3′ flanking sequences and the leader intron. Plant Cell 7: 1387–1394.
Fu, H.Y., Kim, S.Y., Park, W.D. 1995b. A potato sus3 sucrose synthase gene contains a context-dependent 3′ element and a leader intron with both positive and negative tissue-specific effects. Plant Cell 7: 1395–1403.
Garbarino, J.E. and Belknap, W.R. 1994. Isolation of a ubiquitinribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants. Plant Mol. Biol. 24: 119–127.
Garbarino, J.E., Rockhold, D.R. and Belknap, W.R. 1992. Expression of stress-responsive ubiquitin genes in potato tubers. Plant Mol. Biol. 20: 235–244.
Garbarino, J.E., Osumi, T. and Belknap, W.R. 1995. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109: 1371–1378.
Genschik, P., Parmentier, Y., Durr, A., Marbach, J., Criqui, M.-C., Jamet, E. and Fleck J. 1992. Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses. Plant Mol. Biol. 20: 897–910.
Genschik, P., Marbach, J., Uze, M., Feuerman, M., Plesse, B. and Fleck, J. 1994. Structure and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of Nicotiana tabacum. Gene 148: 195–202.
Genschik P., Criqui, M.C., Parmentier, Y., Derevier, A. and Fleck, J. 1998. Cell cycle-dependent proteolysis in plants: identification of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132. Plant Cell 10: 2063–2075.
Gidekel, M., Jimenez, B. and Herrera-Estrella, L. 1996. The first intron of the Arabidopsis thaliana gene coding for elongation factor 1 beta contains an enhancer-like element. Gene 170: 201–206.
Görlach, J., Raesecke, H.R., Abel, G., Wehrli, R., Amrhein, N. and Schmid, J. 1995. Organ-specific differences in the ratio of alternatively spliced chorismate synthase (LeCS2) transcripts in tomato. Plant J. 8: 451–456.
Hemerly, A.S., Ferreira, P., de Almeida Engler, J., Van Montagu, M., Engler, G. and Inzé, D. 1993. cdc2a expression in Ara-bidopsis is linked with competence for cell division. Plant Cell 5: 1711–1723.
Hershko, A. and Ciechanover, A. 1998. The ubiquitin system. Annu. Rev. Biochem. 67: 425–479.
Hondred, D. and Vierstra, R.D. 1992. Novel applications of the ubiquitin-dependent proteolytic pathway in plant genetic engi-neering. Curr. Opin. Biotechnol. 3: 147–151.
Hondred, D., Walker, J.M., Mathews, D.E. and Vierstra, R.D. 1999. Use of ubiquitin fusions to augment protein expression in transgenic plants. Plant Physiol. 119: 713–724.
Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. 1985. A simple and general method for transferring genes into plants. Science 1229–1231.
Hoyt, M.A. 1997. Eliminating all obstacles: regulated proteolysis in the eukaryotic cell cycle. Cell 91: 149–151.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.
Kawalleck, P., Somssich, I.E., Feldbrügge, M., Hahlbrock, K. and Weisshaar, B. 1993. Polyubiquitin gene expression and structural properties of the ubi 4–2 gene in Petroselinum crispum.Plant Mol. Biol. 21: 673–684
King, R.W., Deshaies, R.J., Peters, J.-M. and Kirschner, M.W. 1996. How proteolysis drives the cell cycle. Science 274: 1652–1659.
Kiss, T., Kis, M. and Solymosy, F. 1989. Nucleotide sequence of a 25S rRNA gene from tomato. Nucl. Acids Res. 17: 796.
Kopriva, S., Cossu, R. and Bauwe, H. 1995. Alternative splicing results in two different transcripts for H-protein of the glycine cleavage system in the C4 species Flaveria trinervia. Plant J. 8: 435–441.
Koziel, M.G., Carozzi, N.B. and Desai, N. 1996. Optimizing expression of transgenes with an emphasis on post-transcriptional events. Plant Mol. Biol. 32: 393–405.
Kulikauskas, R., Hou, A., Muschietti, J. and McCormick, S. 1995. Comparaisons of diverse plant species reveal that only grasses show drastically reduced levels of ubiquitin monomer in mature pollen. Sex. Plant Reprod. 8: 326–332.
Maliga, P., Breznovits, A.Sz. and Marton, L. 1973. Streptomycinresistant plants from callus culture of haploid tobacco. Nature New Biol. 244: 29–30.
Marillonnet, S. and Wessler, S.R. 1997. Retrotransposon insertion into the maize waxy gene results in tissue-specific RNA processing. Plant Cell 9: 967–978.
Marshall, J.S., Stubbs, J.D., Chitty, J.A., Surin, B. and Taylor, W.C. 1997. Expression of the C4 Me1 gene from Flaveria bidentis requires an interaction between 5′ and 3′ sequences. Plant Cell 9: 1515–1525.
Martinez, M.C., Jorgensen, J.E., Lawton, M.A., Lamb, C.J. and Doerner, P.W. 1992. Spatial pattern of cdc2 expression in relation to meristem activity and cell proliferation during plant development. Proc. Natl. Acad. Sci. USA 89: 7360–7364.
Mascarenhas, D., Mettler, I.J., Pierce, D.A. and Lowe, H.W. 1990. Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol. Biol. 15: 913–920.
Matsumoto, K., Wassarman, K.M. and Wolffe, A.P. 1998. Nuclear history of a pre-mRNA determines the translational activity of cytoplasmic mRNA. EMBO J. 17: 2107–2121.
Menkens, A.E., Schindler, U. and Cashmore, A.R. 1995. The Gbox: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem. Sci. 20: 506–510.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
Muschietti, J., Dircks, L., Vancanneyt, G. and McCormick, S. 1994. LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinate abnormally and cannot achieve fertilization. Plant J. 6: 321–338.
Nagata, T., Nemoto, Y. and Hasezawa, S. 1992. Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. Int. Rev. Cytol. 132: 1–30.
Norris, S.R., Meyer, S.E. and Callis, J. 1993. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol. Biol. 21: 895–906.
Özkaynak, E., Finley, D., Solomon, M.J. and Varshavsky, A. 1987. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 6: 1429–1439.
Park, J.H., Oh, S.A., Kim, Y.H., Woo, H.R. and Nam, H.G. 1998. Differential expression of senescence-associated mRNAs during leaf senescence induced by different senescence-inducing factors in Arabidopsis. Plant Mol. Biol. 37: 445–454.
Plantefol L. 1947. Hélices foliaires, point végétatif et stèle chez les dicotylédones. La notion d'anneau initial. Rév. Gén. Bot. 54: 49–80.
Plesse, B., Durr, A., Marbach, J., Genschik, P. and Fleck, J. 1997. Identification of a new cis-regulatory element in a Nicotiana tabacum polyubiquitin gene promoter. Mol. Gen. Genet. 266: 254–258.
Qin, L.-X., Richard, L., Perennes, C., Gadal, P. and Bergounioux, C. 1995. Identification of a cell cycle-related gene, cyclin, in Nicotiana tabacum (L.). Plant Physiol. 108: 425–426.
Rollfinke, I.K. and Pfitzner, U.M. 1994. Structure of a heptaubiquitin gene from tomato. Plant. Physiol. 104: 299–300.
Rollkinke, I.K., Silber, M.V. and Pfitzner, U.M. 1998. Characterization and expression of a heptaubiquitin gene from tomato. Gene 211: 267–276.
Sabin, E.A., Lee-Ng, C.T., Shuster, J.R. and Barr, P.J. 1989. High-level expression and in vivo processing of chimeric ubiquitin fusion proteins in Saccharomyces cerevisiae. Bio/technology 7: 705–709.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY.
Sieburth, L.E. and Meyerowitz, E.M. 1997. Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9: 355–365.
Simpson, G.G. and Filipowicz, W. 1996. Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery. Plant Mol. Biol. 32: 1–41.
Sun, C.W. and Callis, J. 1997. Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes. Plant J. 11: 1017–1027.
Takimoto, I., Christensen, A.H., Quail, P.H., Uchimiya, H. and Toki, S. 1994. Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants. Plant Mol. Biol. 26: 1007–1012.
Taylor, C.B. 1997. Promoter analysis: an insufficient measure of gene expression. Plant Cell 9: 273–274.
Thoma, S., Hecht, U., Kippers, A., Botella, J., de Vries, S. and Somerville, C. 1994. Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis. Plant Physiol. 105: 35–45.
Uknes, S., Dincher, S., Friedrich, L., Negrotto, D., Williams, S., Thompson-Taylor, H., Potter, S., Ward, E. and Ryals, J. 1993. Regulation of pathogenesis-related protein-1a gene expression in tobacco. Plant Cell 5: 159–169.
Varshavsky, A. 1997a. The ubiquitin system. Trends Biochem. Sci. 22: 383–387.
Varshavsky, A. 1997b. The N-end rule: functions, mysteries, uses. Proc. Natl. Acad. Sci. USA 93: 12142–12149.
Verwoerd, T.C., Dekker, B.M.M. and Hoekema, A. 1989. A small-scale procedure for the rapid isolation of plant RNAs. Nucl. Acids Res. 17: 2362.
Vierstra, R.D. 1996. Proteolysis in plants: mechanisms and functions. Plant Mol. Biol. 32: 275–302.
Woffenden, B.J., Freeman, T.B. and Beers, E.P. 1998. Proteasome inhibitors prevent tracheary element differentiation in Zinnia mesophyll cell cultures. Plant Physiol. 118: 419–430.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Plesse, B., Criqui, MC., Durr, A. et al. Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum. Plant Mol Biol 45, 655–667 (2001). https://doi.org/10.1023/A:1010671405594
Issue Date:
DOI: https://doi.org/10.1023/A:1010671405594