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Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

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Abstract

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.

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References

  1. Ackermann C (1977) Pflanzen aus Agrobacterium rhizogenes-tumoren an Nicotiana tabacum. Plant Sci Lett 8: 23–30

    Google Scholar 

  2. Akiyoshi DE, Klee H, Amasino RM, Nester EW and Gordon MP (1984) T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci USA 81: 5994–5998

    Google Scholar 

  3. Akiyoshi DE, Morris RO, Hinz R, Mischke BS, Kosuge T, Garfinkel DJ, Gordon MP and Nester EW (1983) Cytokinin/auxin balance in crown gall tumours is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80: 407–411

    Google Scholar 

  4. An G, Ebert PR, Mitr A and Ha SB (1988) Binary vectors. In: Gelvin S, Schilperoot R and Verma D (eds) Plant Molecular Biology Manual 1988, pp. A3: 1–9. Dordrecht/Boston/London: Kluwer Academic Publishers

    Google Scholar 

  5. Barry GF, Rogers SG, Fraley RT and Brand L (1984) Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci USA 81: 4776–4780

    Google Scholar 

  6. Beaty JS, Powell GK, Lica L, Regier DA, MacDonald EMS, Hommes NG and Morris RO (1986) Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis. Mol Gen Genet 203: 274–280

    Google Scholar 

  7. Beinsberger SEI, Valcke RLM, Deblaere RY, Clijsters HMM, De Greef JA, Van Onkelen HA (1991) Effects of the introduction of Agrobacterium tumefaciens T-DNA ipt gene in Nicotiana tabacum L. cv. Petit Havana SR1 plant cells. Plant Cell Physiol 32: 489–496

    Google Scholar 

  8. Beinsberger SEI, Valcke RLM, Clijsters HMM, De Greef JA and Van Onckelen HA (1992) Effects of enhanced cytokinin levels in ipt transgenic tobacco. In: Kamínek M, Mok DWS and Zažímalová E (eds) Physiology and Biochemistry of Cytokinins in Plants 1992, pp. 77–82. The Hague: SPB Academic Publishing

    Google Scholar 

  9. Cardarelli M, Spanò L, Mariotti D, Mauro ML, Van Sluys MA and Constantino P (1987) The role of auxin in hairy root induction. Mol Gen Genet 208: 457–463

    Google Scholar 

  10. Crespi M, Messens E, Caplan A, van Montagu M and Desomer J (1992) Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene. EMBO 11: 795–804

    Google Scholar 

  11. Currier TC and Nester EW (1976) Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal Biochem 76: 431–441

    Google Scholar 

  12. De Framond AJ, Back EW, Chilton WS, Kayes L and Chilton MD (1986) Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet 202: 125–131

    Google Scholar 

  13. Eason JE (1993) The role of plasmids and cytokinins in pathogenicity of Rhodococcus facians. PhD Thesis, University of Otago

  14. Estruch JJ, Chriqui D, Grossmann K, Schell J and Spena A (1991) The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J 10: 2889–2965

    Google Scholar 

  15. Estruch JJ, Schell J and Spena A (1993) The protein encoded by the RolB plant oncogene hydrolyses indole glucosides. EMBO J 10: 3125–3128

    Google Scholar 

  16. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP and Nester EW (1981) Genetic analysis of crown gall: fine structure map of the T-DNA by site directed mutagenesis. Cell 27: 143–153

    Google Scholar 

  17. Goldberg SB, Flick JS and Rogers SG (1984) Nucleotide sequence of the tmr locus of Agrobacterium tumefaciens pTi T37 T-DNA. Nucleic Acids Res 12: 4665–4677

    Google Scholar 

  18. Heidekamp F, Dirkse WG, Hille J and Van Ormondt H (1983) Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene. Nucleic Acids Res 11: 6211–6223

    Google Scholar 

  19. Jameson PE and Morris RO (1989) Zeatin-like cytokinins in yeast: detection by immunological methods. J Plant Physiol 135: 385–390

    Google Scholar 

  20. Kado CI and Heskett MG (1970) Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60: 969–976

    Google Scholar 

  21. Klee HJ, Yanofsky MF and Nester EW (1985) Vectors for transformation of higher plants. Bio/Tech 3: 637–642

    Google Scholar 

  22. Lichtenstein C, Klee HJ, Montoya A, Garfinkel DJ, Fuller S, Flores C, Nester EW and Gordon MP (1984) Nucleotide sequence and transcript mapping of the tmr gene of the pTiA6NC octopine Ti-plasmid: a bacterial gene involved in plant tumourigenesis. J Mol Appl Genet 2: 354–362

    Google Scholar 

  23. MacDonald EMS and Morris RO (1985) Isolation of cytokinins by immunoaffinity chromatography and analysis by high-performance liquid chromatography radioimmunoassay. Methods in Enzymology 110: 347–358

    Google Scholar 

  24. McKnight TD, Lillis MT and Simpson RB (1987) Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 8: 439–445

    Google Scholar 

  25. Makarova R, Borisova T, Vlasov P, Macháĉková I, Eder J, Ĉermák V and Kefeli V (1992) Free cytokinin and IAA levels in transformed tobacco plants carrying Agrobacterium tumefaciens. gene 4. In: Kamínek M, Mok DWS and Zažímalová E (eds) Physiology and Biochemistry of Cytokinins in Plants 1992, pp. 115–117. The Hague: SPB Academic Publishing

    Google Scholar 

  26. Medford JI, Horgan R, El-Sawi Z and Klee HJ (1989) Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. The Plant Cell 1: 403–413

    Google Scholar 

  27. Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497

    Google Scholar 

  28. Nilsson O, Crozier A, Schmülling T, Sandberg G and Olsson O (1993) Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene. The Plant Journal 3: 681–689

    Google Scholar 

  29. Ondřej M, Macháčková I, Čatský J, Eder J, Hrouda M, Pospíšilová J and Synková H (1990) Potato transformation by T-DNA cytokinin synthesis gene. Biologia Plantarum (Praha) 32: 401–406

    Google Scholar 

  30. Parker CW and Letham DS (1974) Regulators of cell division in plant tissues XVIII. Metabolism of zeatin in Zea mays seedlings. Planta (Berl) 115: 337–344

    Google Scholar 

  31. Powell GK and Morris RO (1986) Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci. Nucleic Acids Res 14: 2555–2565

    Google Scholar 

  32. Richmond AE and Lang A (1957) Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125: 650–651

    Google Scholar 

  33. Riker AJ, Banfield WM, Wright WH, Keitt WH, and Sagen HE (1930) Studies on infectious hairy root of nursery apple trees. J Agr Res 41: 507–540

    Google Scholar 

  34. Sachs T and Thimann KV (1964) Release of lateral buds from apical dominance. Nature 201: 939–940

    Google Scholar 

  35. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB and Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491

    Google Scholar 

  36. Schmülling T, Beinsberger S, De Greef J, Van Onckelen H and Spena A (1989) Construction of a heat-inducible chimaeric gene to increase the cytokinin content in transgenic plant tissue. FEBS Lett 249: 401–406

    Google Scholar 

  37. Schmülling T, Fladung M, Grossmann K and Schell J (1993) Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA. The Plant Journal 3: 371–382

    Google Scholar 

  38. Shen WH, Petit A, Guern J and Tempé J (1988) Hairy roots are more sensitive to auxin than normal roots. Proc Natl Acad Sci USA 85: 3417–3421

    Google Scholar 

  39. Skoog F and Miller C (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11: 118–131

    Google Scholar 

  40. Smart CM, Scofield SR, Bevan MW and Dyer TA (1991) Delayed leaf senescence in tobacco plants transformed with tmr, a gene for cytokinin production in Agrobacterium. The Plant Cell 3: 647–656

    Google Scholar 

  41. Smigocki AC (1991) Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene. Plant Mol Biol 16: 105–115

    Google Scholar 

  42. Smigocki AC and Owens LD (1988) Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells. Proc Natl Acad Sci USA 85: 5131–5135

    Google Scholar 

  43. Smigocki AC and Owens LD (1989) Cytokinin-to-auxin ratios and morphology of shoots and tissues transformed by a chimeric isopentenyl transferase gene. Plant Physiol 91: 808–811

    Google Scholar 

  44. Summons RE, Duke CC, Eichholzer JV, Entsch B, Letham DS, MacLeod JK and Parker CW (1979) Mass spectrometric analysis of cytokinins in plant tissues. II. Quantitation of cytokinins in Zea mays kernals using deuterium labelled standards. Biomedical Mass Spectrometry 6: 407–413

    Google Scholar 

  45. Tepfer D (1984) Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype. Cell 37: 959–967

    Google Scholar 

  46. Trione EJ, Krygier BB, Banowetz GM and Kathrein JM (1985) The development of monoclonal antibodies against the cytokinin zeatin riboside. J Plant Growth Regul 4: 101–109

    Google Scholar 

  47. White FF, Taylor BH, Huffman GA, Gordon MP and Nester WE (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 164: 33–44

    Google Scholar 

  48. Yusibov VM II PC, Andrianov VM and Piruzian ES (1991) Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress. Plant Mol Biol 17: 825–836

    Google Scholar 

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Author notes

  1. Paula E. Jameson

    Present address: Department of Plant Biology and Biotechnology, Massey University, Private Bag 11222, Palmerston North, New Zealand

Authors and Affiliations

  1. Botany Department, University of Otago, P.O. Box 56, Dunedin, New Zealand

    Marian J. McKenzie & Paula E. Jameson

  2. Biochemistry Department, University of Otago, P.O. Box 56, Dunedin, New Zealand

    Russell T. M. Poulter

Authors
  1. Marian J. McKenzie
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  2. Paula E. Jameson
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  3. Russell T. M. Poulter
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McKenzie, M.J., Jameson, P.E. & Poulter, R.T.M. Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue. Plant Growth Regul 14, 217–228 (1994). https://doi.org/10.1007/BF00024796

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  • DOI: https://doi.org/10.1007/BF00024796

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