Skip to main content
Log in

Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses

  • Research Articles
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris. Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed. cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized. The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5- mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids. The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation. The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Agarwal ML, Cullis CA: The ubiquitin-encoding multi-gene family of flax, Linum usitatissimum. Gene 99: 69–75 (1991).

    Google Scholar 

  2. Baker RT, Board PG: The human ubiquitin gene family: structure of a gene and pseudogenes from the Ub B subfamily. Nucl Acids Res 15: 443–463 (1987).

    Google Scholar 

  3. Berg JM: Potential metal-binding domains in nucleic acid binding proteins. Science 232: 485–487 (1985).

    Google Scholar 

  4. Binet MN, Steinmetz A, Tessier LH: The primary structure of sunflower (Helianthus annuus) ubiquitin. Nucl Acids Res 17: 2119 (1989).

    Google Scholar 

  5. Binet MN, Weil JH, Tessier LH: Structure and expression of sunflower ubiquitin genes. Plant Mol Biol 17: 395–407 (1991).

    Google Scholar 

  6. Bond U, Schlesinger MJ: Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol Cell Biol 5: 949–956 (1985).

    Google Scholar 

  7. Burke TJ, Callis J, Vierstra RD: Characterization of a polyubiquitin gene from Arabidopsis thaliana Mol Gen Genet 213: 435–443 (1988).

    Google Scholar 

  8. Callis J, Pollmann L, Shanklin J, Wettern M, Vierstra RD: Sequence of a cDNA from Chlamydomonas reinhardtii encoding a ubiquitin 52 amino acid extension protein. Nucl Acids Res 17: 8377 (1989).

    Google Scholar 

  9. Callis J, Raasch JA, Vierstra RD: Ubiquitin extension proteins of Arabidopsis thaliana. Structure, localization, and expression of their promoters in transgenic tobacco. J Biol Chem 265: 12486–12493 (1990).

    Google Scholar 

  10. Carr JP, Klessig DF: The pathogenesis-related proteins of plants. In: Setlow JK (ed) Genetic Engineering: Principles and Experimental Procedures, vol 11, pp. 65–109. New York and London: Plenum Press (1989).

    Google Scholar 

  11. Christensen AH, Quail PH: Sequence analysis and transcriptional regulation by heat shock of polyubiquitin transcripts from maize. Plant Mol Biol 12: 619–632 (1989).

    Google Scholar 

  12. Ciechanover A, Schwartz AL: How are substrates recognized by the ubiquitin-mediated proteolytic system? Trends Biochem Sci 14: 483–488 (1989).

    Google Scholar 

  13. Ciechanover A, Digiuseppe JA, Schwartz AL, Brodeur GM: Degradation of MYCN oncoprotein by the ubiquitin system. In: Evans AE, Dangio GJ, Knudson AG, Seeger RC (eds) Advances in Neuroblastoma Research 3. Progress in Clinical and Biological Research vol 366, pp. 37–44, Wiley-Liss, New York (1990).

    Google Scholar 

  14. Criqui MC, Plesse B, Durr A, Marbach J, Parmentier Y, Jamet E, Fleck J: Characterization of genes expressed in mesophyll protoplasts of Nicotiana sylvestris before the re-initiation of the DNA replicational activity. Mech Dev, in press (1992).

  15. Davie JR, Lin R, Allis CD: Timing of appearance of ubiquitinated histones in developing new macronuclei of Tetrahymena thermophila. Biochem Cell Biol 69: 66–71 (1991).

    Google Scholar 

  16. Denhardt DT: A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 26: 641–646 (1966).

    Google Scholar 

  17. Dingwall C, Laskey RA: Protein import into the cell nucleus. Annu Rev Cell Biol 2: 366–390 (1986).

    Google Scholar 

  18. Dunigan DD, Dietzgen RG, Schoelz JE, Zaitlin M: Tobacco mosaic virus particles contain ubiquitinated coat protein subunits. Virology 165: 310–312 (1988).

    Google Scholar 

  19. Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).

    Google Scholar 

  20. Finley D, Özkaynak E, Varshavsky A: The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48: 1035–1046 (1987).

    Google Scholar 

  21. Finley D, Bartel B, Varshavsky A: The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338: 394–400 (1989).

    Google Scholar 

  22. Fleck J, Durr A, Lett MC, Hirth L: Changes in protein synthesis during the initial stage of life of tobacco protoplasts. Planta 145: 279–285 (1979).

    Google Scholar 

  23. Fleck J, Durr A, Fritsch C, Vernet T, Hirth L: Osmoticshock ‘stress proteins’ in protoplasts of Nicotiana sylvestris. Plant Sci Lett 26: 159–165 (1982).

    Google Scholar 

  24. Gausing K, Barkardottir R: Structure and expression of ubiquitin genes in higher plants. Eur J Biochem 158: 57–62 (1986).

    Google Scholar 

  25. Genschik P, Parmentier Y, Criqui MC, Fleck J: Sequence of a ubiquitin carboxyl extension protein of Nicotiana tabacum. Nucl Acids Res 18: 4007 (1990).

    Google Scholar 

  26. Genschik P, Criqui MC, Parmentier Y, Marbach J, Durr A, Fleck J, Jamet E: Isolation and characterization of a cDNA encoding a 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Nicotiana sylvestris. Plant Mol Biol 20: 337–341 (1992).

    Google Scholar 

  27. Glotzer M, Murray AW, Kirschner MW: Cyclin is degraded by the ubiquitin pathway. Nature 349: 132–138 (1991).

    Google Scholar 

  28. Hazelwood D, Zaitlin M: Ubiquitinated conjugates are found in preparation of several plant viruses. Virology 177: 352–356 (1990).

    Google Scholar 

  29. Heller R: Recherches sur la nutrition minérale des tissus végétaux cultivés in vitro Ann Sci Nat Bot Biol Vég 14: 1–223 (1953).

    Google Scholar 

  30. Hochstrasser M, Varshavsky A: In vivo degradation of a transcriptional regulator: the yeast α2 repressor. Cell 61: 697–708 (1990).

    Google Scholar 

  31. Jabben M, Shanklin J, Vierstra RD: Ubiquitin-phytochrome conjugates: pool dynamics during in vivo phytochrome degradation. J Biol Chem 264: 4998–5005 (1989).

    Google Scholar 

  32. Jamet E, Durr A, Fleck J: Absence of some trucated genes in the amphidiploid Nicotiana tabacum. Gene 59: 213–221 (1987).

    Google Scholar 

  33. Jamet E, Durr A, Parmentier Y, Criqui MC, Fleck J: Is ubiquitin involved in the dedifferentiation of higher plants cells? Cell Diff Dev 29: 37–46 (1990).

    Google Scholar 

  34. Larkin PJ: Purification and viability determinations of plants protoplasts. Planta 128: 213–216 (1976).

    Google Scholar 

  35. Latchman DS, Estridge JK, Kemp LM: Transcriptional induction of the ubiquitin gene during herpes simplex virus infection is dependent upon the viral immediateearly protein ICP4. Nucl Acids Res 15: 7283–7293 (1987).

    Google Scholar 

  36. Lee H, Simon JA, Lis JT: Structure and expression of ubiquitin genes in Drosophila melanogaster Mol Cell Biol 8: 4727–4735 (1988).

    Google Scholar 

  37. Lindquist S: The heat shock response. Annu Rev Biochem 45: 39–72.

  38. Logemann J, Mayer JE, Schell J, Willmitzer L: Differential expression of genes in potato tubers after wounding. Proc Natl Acad Sci USA 85: 1136–1140 (1988).

    Google Scholar 

  39. Malik AN, McLean PM, Roberts A, Barnett PS, Demaine AG, Banga JP, McGregor AM: A simple high yield method for the preparation of lambda gt 10 DNA suitable for subcloning, amplification and direct sequencing. Nucl Acids Res 13: 4031–4032 (1990).

    Google Scholar 

  40. Monia BP, Ecker DJ, Crooke ST: New perspectives on the structure and function of ubiquitin. Bio/technology 8: 209–215 (1990).

    Google Scholar 

  41. Müller-Taubenberger A, Hagmann J, Noegel A, Gerisch G: Ubiquitin gene expression in Dictyostelium is induced by heat and cold shock, cadmium, and inhibitors of protein synthesis. J Cell Sci 90: 51–58 (1988).

    Google Scholar 

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

    Google Scholar 

  43. Nagata T, Takebe I: Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 92: 301–308 (1970).

    Google Scholar 

  44. Nagy JJ, Maliga P: Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphys 78: 453–455 (1976).

    Google Scholar 

  45. Ohmachi T, Giorda R, Shaw DR, Ennis HL: Molecular organization of developmentally regulated Dictyostelium discoideum ubiquitin cDNAs. Biochemistry 28: 5226–5231 (1989).

    Google Scholar 

  46. Özkaynak E, Finley D, Varshavsky A: The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 312: 663–666 (1984).

    Google Scholar 

  47. Özkaynak E, Finley D, Solomon MJ, Varshavsky A: The yeast ubiquitin genes: a family of natural gene fusions. EMBO J 6: 1429–1439 (1987).

    Google Scholar 

  48. Pollmann L, vonKampen J, Wettern M: Ubiquitin in a lower plant: characterization of ubiquitin encoding DNA and RNA from Chlamydomonas reinhardtii. Eur J Biochem 202: 197–204 (1991).

    Google Scholar 

  49. Rechsteiner M: Ubiquitin-mediated pathways for intracellular proteolysis. Annu Rey Cell Biol. 3: 1–30 (1987).

    Google Scholar 

  50. Rickey TM, Belknap WR: Comparison of the expression of several stress-responsive genes in potato tubers. Plant Mol Biol 16: 1009–1018 (1991).

    Google Scholar 

  51. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  52. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    Google Scholar 

  53. Shanklin J, Jabben M, Vierstra RD: Red light-induced formation of ubiquitin-phytochrome conjugates: identification of possible intermediates of phytochrome degradation. Proc Natl Acad Sci USA 84: 359–363 (1987).

    Google Scholar 

  54. Shinshi H, Mohnen D, Meins FJR: Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci USA 84: 89–93 (1987).

    Google Scholar 

  55. Swindle J, Ajioka J, Eisen H, Sanwal B, Jacquemot C, Browder Z, Buck G: The genomic organization of the ubiquitin genes of Trypanosoma cruzi. EMBO J 7: 1121–1127 (1988).

    Google Scholar 

  56. VanRegenmortel MHV: Tobamoviruses. In: Kurstak E (ed) Handbook of Plant Virus Infections and Comparative Diagnosis, pp. 541–564. Elsevier/North Holland Medical Press, Amsterdam (1981).

    Google Scholar 

  57. Vervliet G, Holsters M, Teuchy H, vanMontagu M, Schell J: Characterization of different plaque forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26: 33–48: (1974).

    Google Scholar 

  58. Westphal M, Müller-Taubenberger A, Noegel A, Gerisch G: Transcript regulation and carboxyterminal extension of ubiquitin in Dictyostelium discoideum. FEBS Lett 209: 92–96 (1986).

    Google Scholar 

  59. Wiborg O, Pedersen MS, Wind A, Berlund LE, Marker KA, Vuust J: The human ubiquitin multigene family: some sequences contain multiple directly repeated ubiquitin coding sequences. EMBO J 4: 755–759 (1985).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Genschik, P., Parmentier, Y., Durr, A. et al. Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses. Plant Mol Biol 20, 897–910 (1992). https://doi.org/10.1007/BF00027161

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00027161

Key words

Navigation