Skip to main content
Log in

Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital bifunctional reporters in plants

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

By fusing the genes encoding green fluorescent protein (GFP) and β-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

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. Baulcombe DC, Chapman S, Santa Cruz S: Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7: 1045–1053 (1995).

    Google Scholar 

  2. Benes V, Homstomsky Z, Arnold L, Paces V: M13 and pUC vectors with new unique restriction sites for cloning. Gene 130: 151–152 (1993).

    Google Scholar 

  3. Benfey PN, Ren L, Chua NH: The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue specific expression patterns. EMBO J 8: 2195–2202 (1989).

    Google Scholar 

  4. Chalfie M, Tu Y, Euskirchen G, Ward WW, Parsher PC: Green fluorescent protein as a marker for gene expression. Science 263: 802–805 (1994).

    Google Scholar 

  5. Chapman S, Kavanagh T, Baulcombe D: Potato virus X as a vector for gene expression. Plant J 2: 549–557 (1992).

    Google Scholar 

  6. Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J: Engineered GFP as a vital reporter in plants. Curr Biol 6: 325–330 (1996).

    Google Scholar 

  7. Crameri A, Whiteborn EA, Tate E, Stemmer WPC: Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol 14: 315–319 (1996).

    Google Scholar 

  8. Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY: Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20: 448–455 (1995).

    Google Scholar 

  9. Datla RSS, Hammerlindl JK, Pelcher LE, Crosby WL, Selvaraj G:Abifunctional fusion between β-glucuronidase and neomycin phosphotransferase: a broadspectrum marker for plants. Gene 101: 239–246 (1991).

    Google Scholar 

  10. den Dulk-Ras A, Hooykaas PJJ: Electroporation of Agrobacterium tumefaciens. In Nikelhoff JA (ed) Plant Cell Electroporation and Electrofusion Protocols, pp. 63–72. Humana Press Inc. Totowa (1995).

    Google Scholar 

  11. Ditta G, Stanfield S, Corbin D, Helsinki DR: Broad host range DNA cloning system for Gramnegative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 347–351 (1980).

    Google Scholar 

  12. Gadella TWJ: Fluorescence lifetime imaging microscopy. Eur Microscopy & Analysis 47: 9–11 (1997).

    Google Scholar 

  13. Gerdes HH, Kaether C: Green fluorescent protein: applications in cell biology. FEBS Lett 389: 44–47 (1996).

    Google Scholar 

  14. Hajdukiewicz P, Svab Z, Maliga P: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25: 989–994 (1994).

    Google Scholar 

  15. Handberg K, Stiller J, Thyljaer T, Stougaard J: Transgenic plants: Agrobacteriummediated transformation of the diploid legume Lotus japonicus. Cell Biology: Alaboratory handbook, pp. 119–127. Academic Press, Inc. (1994).

  16. Handberg K, Stougaard J: Lotus japonicus, an autogamous, diploid legume species for classical molecular genetics. Plant J 2: 487–496 (1992).

    Google Scholar 

  17. Haseloff J, Siemering KR, Prasher DG, Hodge S: Removal of a cryptic intron and subcellular localization of green fluorescent protein are required tomark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94: 2122–2127 (1997).

    Google Scholar 

  18. Heim R, Cubitt AB, Tsien RY: Improved green fluorescence. Nature 373: 663–664 (1995).

    Google Scholar 

  19. Heim R, Prasher DG, Tsien RY: Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91: 12501–12504 (1994).

    Google Scholar 

  20. Heim R, Tsien RY: Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6: 178–182 (1996).

    Google Scholar 

  21. Hood EE, Gelvin SB, Melchers LS, Hoekema A: New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res: 208–218 (1993).

  22. Hu W, Cheng CL: Expression of Aequorea green fluorescent protein in plant cells. FEBS Lett 369: 331–334 (1995).

    Google Scholar 

  23. Inouye S, Tsuji FI: Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett 341: 277–280 (1994).

    Google Scholar 

  24. Jefferson RA, Burgess SM, Hirsch D: β-Glucuronidase from Escherichia coli as a gene fusion marker. Proc Natl Acad Sci USA 83: 8447–8451 (1986).

    Google Scholar 

  25. Jefferson RA, Kavanagh T, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    Google Scholar 

  26. Jones JDG, Shlumukov L, Carland F, English J, Scofield SR, Bishop GJ, Harrison K: Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgenic Res 1: 285–297 (1992).

    Google Scholar 

  27. Laemmli UK: Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).

    Google Scholar 

  28. Lazo GR, Stein PA, Ludwig RA: A DNA transformationcompetent Arabidopsis genomic library in Agrobacterium. Bio/Technology 9: 963–967 (1991).

    Google Scholar 

  29. LópezLara IM, van den Berg JDJ, Thomas-Oates JE, Glushka J, Lugtenberg BJJ, Spaink HP: Structural identification of the lipochitin oligosaccharide nodulation signals of Rhizobium loti. Mol Microbiol 15: 627–638 (1995).

    Google Scholar 

  30. Marsh JL, Erfle M, Wykes EJ: The pIC plasmids and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32: 481–485 (1984).

    Google Scholar 

  31. Millar AJ, Carre IA, Strayer CA, Chua NH, Kay SA: Circadian clockmutants in Arabidopsis indentified by luciferase imaging. Science 267: 1161–1163 (1995).

    Google Scholar 

  32. Millar AJ, Short SR, Chua NH, Kay SA: A novel circadian phenotype based on firefly luciferase gene expression in transgenic plants. Plant Cell 4: 1075–1087 (1992).

    Google Scholar 

  33. Niedz RP, Sussman MR, Satterlee JS: Green fluorescent protein: an in vivo reporter of plant gene expression. Plant Cell Rep 14: 403–406 (1995).

    Google Scholar 

  34. Oparka KJ, Roberts AG, Prior DAM, Chapman S, Baulcombe D, Santa Cruz S: Imaging the green fluorescent protein in plants – viruses carry the torch. Protoplasma 189: 133–141 (1995).

    Google Scholar 

  35. Pang SZ, Deboer DL, Wan Y, Ye G, Layton JG, Neher MK, Armstrong CL, Fry JE, Hinche MAW, Fromm ME: An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol 112: 893–900 (1996).

    Google Scholar 

  36. Plautz JD, Day RN, Dailey GM, Welsh SB, Hall JC, Halpain S, Kay SA: Green fluorescent protein and its derivates as versatile marker for gene expression in living Drosophila melanogaster, plant and mammalian cells. Gene 173: 83–87 (1996).

    Google Scholar 

  37. Reichel C, Marthur J, Eckes P, Langekemper K, Koncz C, Schell J, Reiss B, Maas C: Enhanced green fluorescent protein by the expression of an Aequorea victoria green fluorescent protein mutant in monoand dicotelydonous plant cells. Proc Natl Acad Sci USA 93: 5888–5893 (1996).

    Google Scholar 

  38. Rigler R, Mets Ñ, Widengren J, Kask P: Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Europ Biophys J 22: 169–176 (1997).

    Google Scholar 

  39. Rouwendal GJA, Mendes O, Wolberts EJH, de Boer DA: Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant Mol Biol 33: 989–999 (1997).

    Google Scholar 

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

    Google Scholar 

  41. Schlaman HRM, Hooykaas PJJ: Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacteriummediated plant transformation. Plant J 11: 1377–1385 (1997).

    Google Scholar 

  42. Scholthof HB, Scholthof KBG, Jackson AO: Plant virus expression vectors for transient expression of foreign proteins in plants. Ann Rev Phytopathol 34: 299–323 (1996).

    Google Scholar 

  43. Scopsi L, Larsson LI: Increased sensitivity in peroxidase immunochemistry. A comparative study of a number of peroxidase visualization methods employing a model system. Histochemistry 84: 221–230 (1986).

    Google Scholar 

  44. Shiga Y, Tanaka-Matakatsu M, Hayashi S: A nuclear GFP/β-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Develop Growth Differ 38: 99–106 (1996).

    Google Scholar 

  45. Sijmons PC, Dekker BMM, Schrammeijer B, Verwoerd TC, van den Elzen P, Hoekema A: Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 6: 217–221 (1990).

    Google Scholar 

  46. Thompson NL.: Fluorescence correlation spectroscopy. In Lakowicz JR (ed) Topics in Fluorescence Spectroscopy, pp. 337–410. Plenum Press, New York (1995).

    Google Scholar 

  47. Timmons L, Becker J, Barthmaier P, Fyrberg C, Shearn A, Fyrberg E: Green fluorescent protein/β-galactosidase double reporters for visualizing Drosophila gene expression patterns. Dev Genet 20: 338–347 (1997).

    Google Scholar 

  48. Valvekens D, Van Montagu M, Van Lijsebettens M: Agrobacterium tumefaciensmediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85: 5536–5540 (1988).

    Google Scholar 

  49. van Engelen FA, Molthoff JW, Conner AJ, Nap JP, Pereira A, Stiekema WJ: pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res 4: 288–290 (1995).

    Google Scholar 

  50. Vancanneyt G, Schmidt R, O'Conner-Sanchez A, Willmitzer L, Rocha-Sosa M: Construction of an introncontaining markergene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacteriummediated plant transformation. Mol Gen Genet 220: 245–250 (1990).

    Google Scholar 

  51. Vergunst AC, De Waal EC, Hooykaas PJJ: Root transformation by Agrobacterium tumefaciens, pp 227–235. In Salinas J, Martinez-Zapaler J (eds) Arabidopsis protocols. Human Press, Totowa (1998).

    Google Scholar 

  52. Viera J, Messing J: New pUC-derived cloning vectors with different selectable markers and DNA repication origins. Gene 100: 189–194 (1990).

    Google Scholar 

  53. Zernicka-Goetz M, Pines J, McLean Hunter S, Dixon JPC, Siemering KR, Haseloff J, Evans MJ: Following cell fate in living mouse embryo. Development 124: 1133–1137 (1997).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Quaedvlieg, N.E., Schlaman, H.R., Admiraal, P.C. et al. Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital bifunctional reporters in plants. Plant Mol Biol 37, 715–727 (1998). https://doi.org/10.1023/A:1006068129255

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006068129255

Navigation