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Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants

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Abstract

Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is ‘brighter’ because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.

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References

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

    Article  PubMed  Google Scholar 

  2. Blum H, Beier H, Gross HJ: Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93–99 (1987).

    Google Scholar 

  3. Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D: Green fluorescent protein as a marker for gene expression. Science 263: 802–805 (1994).

    PubMed  Google Scholar 

  4. Christou P, McCabe D, Martinell B, Swain W: Soybean genetic engineering: commercial production of transgenic plants. Trends Biotechnol 8: 145–151 (1990).

    Article  Google Scholar 

  5. Corbett AH, Koepp DM, Schlenstedt G, Lee MS, Hoper AK, Silver PA: Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import. J CellBiol 130: 1017–1026 (1995).

    Google Scholar 

  6. Cormack BP, Valdivia RH, Falkow S: FACS-optimizedmutants of the green fluorescent protein (GFP). Gene 173: 33–38 (1996).

    Article  PubMed  Google Scholar 

  7. Crameri A, Whitehorn E, Tate E, Stemmer P: 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).

    PubMed  Google Scholar 

  9. Cutler M, Ward WW: Spectral analysis and proposed model for GFP dimerization. In: Hastings JW, Kricka LJ, Stanley PE (eds) Bioluminescence and Chemiluminescence; Molecular Reporting with Photons, pp. 403–406. John Wiley, New York (1997).

    Google Scholar 

  10. Davis SJ, Vierstra RD: Soluble derivatives of green fluorescent protein (GFP) for use in Arabidopsis thaliana. Weeds World 3: 43–48 (1996) at http://genomewww. stanford.edu/Arabidopsis/ww/Vol3ii/sol-modGFP.html.

    Google Scholar 

  11. Galbraith DW, Lambert GM, Grebenok RJ, Sheen J: Methods in Plant Cell Biology. Academic Press, San Diego (1995).

    Google Scholar 

  12. Haseloff J, Amos B: GFP in plants. Trends Genet 11: 328–329 (1995).

    PubMed  Google Scholar 

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

    Google Scholar 

  14. Heim R, Cubitt A, Tsien R: Improved green fluorescence. Nature 373: 663–664 (1995).

    Article  Google Scholar 

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

    Google Scholar 

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

    PubMed  Google Scholar 

  17. Heinlein M, Epel BL, Padgett HS, Beachy RN: Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270: 1983–1985 (1995).

    Google Scholar 

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

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  20. Kaether C, Gerdes HH: Visualization of protein transport along the secretory pathway using green fluorescent protein. FEBS Lett 369: 267–271 (1995).

    PubMed  Google Scholar 

  21. Marks MD, Esch JJ: Trichome formation in Arabidopsis as a genetic model for studying cell expansion. Curr Top Plant Biochem Physiol 11: 131–142 (1992).

    Google Scholar 

  22. 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 

  23. Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ: Crystal structure of the Aequoria victoria green fluorescent protein. Science 273: 1392–1395 (1996).

    PubMed  Google Scholar 

  24. Ow DW, Wood KV, DeLuca M, deWet JR, Helinski DR, Howell SH: Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234: 856–859 (1986).

    Google Scholar 

  25. Pang S, DeBoer DL, Wan Y, Ye G, Layton JG, Neher MK, Armstrong CL, E. FJ, Hinchee MAW, Fromm ME: An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol 112: 893–900 (1996).

    Article  PubMed  Google Scholar 

  26. Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C: Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono-and dicotyledonous plant cells. Proc Natl Acad Sci USA 93: 5888–5893 (1996).

    Google Scholar 

  27. Rizzuto R, Brini M, DeGiorgi F, Rossi R, Heim R, Tsien RY, Pozzan T: Double labeling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol 6: 183–188 (1996).

    PubMed  Google Scholar 

  28. Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW: Green-fluorescent protein as a new vital marker in plant cells. Plant J 8: 777–784 (1995).

    Article  PubMed  Google Scholar 

  29. Wang SX, Hazelrigg T: Implications for bcd mRNA localization from spacial distribution of exu protein in Drosophila oogenesis. Nature 369: 400–403 (1994).

    PubMed  Google Scholar 

  30. Yang F, Moss LG, Phillips GN: The molecular structure of green fluorescent protein. Nature Biotechnol 14: 1246–1220 (1996).

    Article  Google Scholar 

  31. Yokoe H, Meyer T: Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nature Biotechnol 14: 1252–1256 (1996).

    Google Scholar 

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Authors and Affiliations

  1. Department of Horticulture and Laboratory of Genetics, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, 53706, U.S.A.

    Seth J. Davis & Richard D. Vierstra

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  1. Seth J. Davis
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  2. Richard D. Vierstra
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Davis, S.J., Vierstra, R.D. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol Biol 36, 521–528 (1998). https://doi.org/10.1023/A:1005991617182

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