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The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants.

Forner J, Binder S - BMC Plant Biol. (2007)

Bottom Line: The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed.In plants, eqFP611 is a suitable fluorescent reporter protein.Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molekulare Botanik, Universität Ulm, Ulm, Germany. joachim.forner@uni-ulm.de

ABSTRACT

Background: Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.

Results: When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.

Conclusion: In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

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Constitutive expression of mitochondrially targeted eqFP611. Protoplasts derived from stably transformed N. tabacum plants constitutively expressing eqFP611 targeted to mitochondria (pIVD145-eqFP611-pBI121). (A) Image taken through MitoTracker filter set. Scale bar: 10 μm. (B) Plasmid used for transformation. The IFP expression cassette is identical with that in Figure 2, but has been inserted into pBI121. Kanr: kanamycin resistance cassette (NOS promoter, neomycin phosphotransferase II, NOS terminator). RB: right border, LB: left border. Vector backbone: pBI121.
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Figure 8: Constitutive expression of mitochondrially targeted eqFP611. Protoplasts derived from stably transformed N. tabacum plants constitutively expressing eqFP611 targeted to mitochondria (pIVD145-eqFP611-pBI121). (A) Image taken through MitoTracker filter set. Scale bar: 10 μm. (B) Plasmid used for transformation. The IFP expression cassette is identical with that in Figure 2, but has been inserted into pBI121. Kanr: kanamycin resistance cassette (NOS promoter, neomycin phosphotransferase II, NOS terminator). RB: right border, LB: left border. Vector backbone: pBI121.

Mentions: A third way to use eqFP611 as a mitochondrial marker in plant cells is the generation of transgenic plants constitutively expressing mitochondrially targeted eqFP611. To create such plants, the RFP-expression cassette of pIVD145-eqFP611 was cloned into pBI121. The resulting plasmid pIVD145-eqFP611-pBI121 was stably transformed into tobacco by leaf disc transformation. Several independent plant lines were regenerated from transgenic calli and screened for bright red fluorescence in mitochondria. Red fluorescent mitochondria were observed in all T0 transformants, but expression levels varied between individual plants. In addition, segregation was observed in the next generation. Thus, only the offspring of the most strongly fluorescent T1 plant was used for propagation (Fig. 8). The transgenic plants completed their life cycle like wild-type plants and the red fluorescence in mitochondria was stably transmitted up to the T3 generation, the last generation analyzed. No phenotypic differences were observed between the transgenic and wild-type plants. Thus, eqFP611 obviously causes no cytotoxic or other detrimental effects even upon constitutive expression over several generations.


The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants.

Forner J, Binder S - BMC Plant Biol. (2007)

Constitutive expression of mitochondrially targeted eqFP611. Protoplasts derived from stably transformed N. tabacum plants constitutively expressing eqFP611 targeted to mitochondria (pIVD145-eqFP611-pBI121). (A) Image taken through MitoTracker filter set. Scale bar: 10 μm. (B) Plasmid used for transformation. The IFP expression cassette is identical with that in Figure 2, but has been inserted into pBI121. Kanr: kanamycin resistance cassette (NOS promoter, neomycin phosphotransferase II, NOS terminator). RB: right border, LB: left border. Vector backbone: pBI121.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904219&req=5

Figure 8: Constitutive expression of mitochondrially targeted eqFP611. Protoplasts derived from stably transformed N. tabacum plants constitutively expressing eqFP611 targeted to mitochondria (pIVD145-eqFP611-pBI121). (A) Image taken through MitoTracker filter set. Scale bar: 10 μm. (B) Plasmid used for transformation. The IFP expression cassette is identical with that in Figure 2, but has been inserted into pBI121. Kanr: kanamycin resistance cassette (NOS promoter, neomycin phosphotransferase II, NOS terminator). RB: right border, LB: left border. Vector backbone: pBI121.
Mentions: A third way to use eqFP611 as a mitochondrial marker in plant cells is the generation of transgenic plants constitutively expressing mitochondrially targeted eqFP611. To create such plants, the RFP-expression cassette of pIVD145-eqFP611 was cloned into pBI121. The resulting plasmid pIVD145-eqFP611-pBI121 was stably transformed into tobacco by leaf disc transformation. Several independent plant lines were regenerated from transgenic calli and screened for bright red fluorescence in mitochondria. Red fluorescent mitochondria were observed in all T0 transformants, but expression levels varied between individual plants. In addition, segregation was observed in the next generation. Thus, only the offspring of the most strongly fluorescent T1 plant was used for propagation (Fig. 8). The transgenic plants completed their life cycle like wild-type plants and the red fluorescence in mitochondria was stably transmitted up to the T3 generation, the last generation analyzed. No phenotypic differences were observed between the transgenic and wild-type plants. Thus, eqFP611 obviously causes no cytotoxic or other detrimental effects even upon constitutive expression over several generations.

Bottom Line: The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed.In plants, eqFP611 is a suitable fluorescent reporter protein.Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molekulare Botanik, Universität Ulm, Ulm, Germany. joachim.forner@uni-ulm.de

ABSTRACT

Background: Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.

Results: When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.

Conclusion: In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

Show MeSH
Related in: MedlinePlus