Abstract
In an attempt to modulate the metabolism of polyamines in plants, Agrobacterium tumefaciens strains were produced which contained either a full-length or a 3′-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of the cauliflower mosaic virus 35S promoter. Plants of Nicotiana tabacum cv. Xanthi were used for transformation with these two strains of Agrobacterium. Transformations were confirmed by Southern hybridization and amplification by polymerase chain reaction. Two plants containing the full-length cDNA (ODC-12 and ODC-30) and two containing the truncated cDNA (12701-2 and 12701-31) were selected for further experiments. Northern blot analysis indicated that transcription was occurring and western blot analysis detected a polypeptide of ca. 50 kDa that was unique to the plants transformed with truncated ODC cDNA. In order to distinguish between the native and the mouse ODC in the transformed tissues, enzyme activity was assayed at pH optima for the two enzymes, i.e. pH 8.2 and 6.8, respectively.
Substantially higher levels of ODC activity were seen at pH 6.8 (optimum for mouse ODC) in the transformants as compared to the controls. This ODC activity was inhibited by α-difluoromethylornithine and anti-mouse ODC antisera in a manner consistent with that reported for the mouse ODC. Analysis of cellular polyamines showed significantly elevated levels (4–12-fold) of putrescine in callus derived from transformed plant tissues as compared to the controls. The modulation of polyamine biosynthesis in plants by these techniques should allow us to further analyze the role of these ubiquitous compounds in plant growth and development.
Similar content being viewed by others
References
An G, Ebert P, Mitra A, Ita S: In: Gelvin SB, Schilperoort RA (eds) Plant molecular Biology Manual, pp. A3: 1–19. Kluwer Academic Publishers, Dordrecht (1988).
Bagni N, Torrigiani P, Barbieri P: In vitro and in vivo effect of ornithine and arginine decarboxylase inhibitors in plant tissue culture. In: Bachrach U, Kaye A, Chayen R (eds) Advances in Polyamine Research, vol. 4, pp. 409–418. Raven Press, New York (1983).
Brabant M, McConlogue L, van Daalen Wetters T, Coffino P: Mouse ornithine decarboxylase gene: Cloning, structure, and expression. Proc Natl Acad Sci USA 85: 2200–2204 (1988).
Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye-binding. Anal Biochem 72: 248–254 (1976).
Burtin D, Martin-Tanguy J, Paynot M, Rossin N: Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi n.c. cultivated in vitro in a medium producing callus formation. Plant Physiol 89: 104–110 (1989).
Cohen E, Heimer YM, Malis-Arad S, Mizrahi Y: Involvement of polyamines in cell division of various plant tissues. In: Bachrach U, Kaye A, Chayen R (eds) Advances in Polyamine Research, vol. 4, pp. 443–444. Raven Press, New York (1993).
Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation. Version II. Plant Mol Biol Rep 1: 19–21 (1983).
DeScenzo RA: Expression of a murine ornithine decarboxylase cDNA in transgenic tobacco (Nicotiana tabacum var. Xanthi). Ph.D. Thesis, University of New Hampshire, Durham (1991).
Evans PT, Malmberg RL: Do polyamines have roles in plant development? Annu Rev Plant Physiol Plant Mol Biol 40: 235–269 (1989).
Fonzi WA, Sypherd PS: Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli. Mol Cell Biol 5: 161–166 (1985).
Galston AW: Polyamines as modulators of plant development. BioScience 33: 382–388 (1983).
Galston AW: Polyamines and plant response to stress. In: Bachrach U, Heimer YM (eds) The Physiology of Polyamines II, pp. 99–106. CRC Press, Boca Raton, FL (1989).
Ghoda L, van Daalen Wetters T, Macrae M, Ascherman D, Coffino P: Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 243: 1493–1495 (1989).
Glass JR, MacKrell M, Duffy JJ, Gerner EW: Ornithine decarboxylase production in vitro by using mouse cDNA. Biochem J 245: 127–132 (1987).
Hamill JD, Robins RJ, Parr AJ, Evans DM, Furze JM, Rhodes MJC: Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation. Plant Mol Biol 15: 27–38 (1990).
Horsch RB, Frye JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Hsieh JT, Denning MF, Heidel SM, Verma AK: Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient chinese hamster ovary cells. Cancer Res 50: 2239–2244 (1990).
Hughes DW, Galau G: Preparation of RNA from cotton leaves and pollen. Plant Mol Biol Rep 6: 253–257 (1988).
Isomaa VV, Pajunen AEI, Bardin CW, Jänne OA: Ornithine decarboxylase in mouse kidney. J Biol Chem 258: 6735–6740 (1983).
Kahana C, Nathans D: Isolation of cloned cDNA encoding ODC. Proc Natl Acad Sci USA 81: 3645–3649 (1984).
Kahana C, Nathans D: Nucleotide sequence of murine ornithine decarboxylase mRNA. Proc Natl Acad Sci USA 82: 1673–1677 (1985).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Malmberg RL, Hiatt AC: Polyamines in plant mutants. In: Bachrach U, Heimer YM (eds) The Physiology of Polyamines II, pp. 147–159. CRC Press, Boca Raton, FL (1989).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Martin-Tanguy J, Negrel J, Paynot M, Martin C: Hydroxycinnamic acid amides, hypersensitivity, flowering and sexual organogenesis in plants. Plant Mol Biol 1: 253–269 (1987).
McCann PP, Pegg AE, Sjoerdsma A (eds): Inhibition of Polyamine Metabolism. Academic Press, New York (1987).
Mengoli M, Bagni N, Luccarini G, Ronchi VN, Serafini-Fracassini D: Daucus carota cell cultures: Polyamines and effect of polyamine biosynthesis inhibitors in the preembryogenic phase and different embryo stages. J Plant Physiol 134: 389–394 (1989).
Minocha SC: Relationship between polyamine and ethylene biosynthesis in plants and its significance for morphogenesis in cell culture. In: Zappia V, Pegg A (eds) Progress in Polyamine Research, pp. 601–616. Plenum Publishing, New York (1988).
Minocha SC, Minocha R, Robie CA: High-performance liquid chromotographic method for the determination of dansyl-polyamines. J Chromotogr 511: 177–183 (1990).
Murashige T, Skoog F: A revised medium for rapid growth and bioassays of tobacco tissue cultures. Physiol Plant 15: 473–479 (1962).
Negrel J: The biosynthesis of cinnamoylputrescines in callus tissue cultures of Nicotiana tabacum. Phytochemistry 28: 477–481 (1989).
Pegg AE: Recent advances in the biochemistry of polyamines of eukaryotes. Biochem J 234: 249–262 (1986).
Persson L: Antibodies to ornithine decarboxylase. Immunochemical cross-reactivity. Acta Chem Scandin B36: 685–688 (1982).
Pritchard ML, Seely JE, Pöso H, Jefferson LS, Pegg AE: Binding of radioactive alpha-difluoromethylornithine to rat liver ornithine decarboxylase. Biochem Biophys Res Comm 100: 1597–1603 (1981).
Rastogi R, Kaur-Sawhney VK: Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum Mill.). I. Level of polyamines and their biosynthesis in normal and mutant flowers. Plant Physiol 93: 439–445 (1990).
Robie CA, Minocha SC: Polyamines and somatic embryogenesis in carrot. I. The Effects of difluoromethyl ornithine and difluoromethylarginine. Plant Sci 65: 45–54 (1989).
Seely JE, Pegg AE: Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay. J Biol Chem 258: 2496–2500 (1983).
Seely JE, Pöso H, Pegg AE: Purification of ornithine decarboxylase from kidneys of androgen-treated mice. Biochemistry 21: 3394–3399 (1982).
Slocum RD, Flores HE: The Biochemistry and Physiology of Polyamines in Plants CRC Press, Boca Raton, FL (1991).
Slocum RD, Kaur-Sawhney R, Galston AW: The physiology and biochemistry of polyamines in plants. Arch Biochem Biophys 235: 283–303 (1984).
Smith TA: Plant polyamines—metabolism and function. In: Flores HE, Arteca RN, Shannon JC (eds) Polyamines and ethylene: Biochemistry, Physiology and Interactions, pp. 224–237. American Society of Plant Physiology (1990).
Zambryski P: Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet 22: 1–30 (1988).
Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J: Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regenerative capacity. EMBO J 2: 2143–2150 (1983).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
DeScenzo, R.A., Minocha, S.C. Modulation of cellular polyamines in tobacco by transfer and expression of mouse ornithine decarboxylase cDNA. Plant Mol Biol 22, 113–127 (1993). https://doi.org/10.1007/BF00039000
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00039000