The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants
<p>Abstract</p> <p>Background</p> <p>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-overlapp...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2007-06-01
|
| Series: | BMC Plant Biology |
| Online Access: | http://www.biomedcentral.com/1471-2229/7/28 |
| _version_ | 1818490663174406144 |
|---|---|
| author | Binder Stefan Forner Joachim |
| author_facet | Binder Stefan Forner Joachim |
| author_sort | Binder Stefan |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-12-10T17:19:55Z |
| format | Article |
| id | doaj.art-dee10aedf8a44349890be4a3fe99edff |
| institution | Directory Open Access Journal |
| issn | 1471-2229 |
| language | English |
| last_indexed | 2024-12-10T17:19:55Z |
| publishDate | 2007-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Plant Biology |
| spelling | doaj.art-dee10aedf8a44349890be4a3fe99edff2022-12-22T01:40:00ZengBMCBMC Plant Biology1471-22292007-06-01712810.1186/1471-2229-7-28The red fluorescent protein eqFP611: application in subcellular localization studies in higher plantsBinder StefanForner Joachim<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1471-2229/7/28 |
| spellingShingle | Binder Stefan Forner Joachim The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants BMC Plant Biology |
| title | The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants |
| title_full | The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants |
| title_fullStr | The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants |
| title_full_unstemmed | The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants |
| title_short | The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants |
| title_sort | red fluorescent protein eqfp611 application in subcellular localization studies in higher plants |
| url | http://www.biomedcentral.com/1471-2229/7/28 |
| work_keys_str_mv | AT binderstefan theredfluorescentproteineqfp611applicationinsubcellularlocalizationstudiesinhigherplants AT fornerjoachim theredfluorescentproteineqfp611applicationinsubcellularlocalizationstudiesinhigherplants AT binderstefan redfluorescentproteineqfp611applicationinsubcellularlocalizationstudiesinhigherplants AT fornerjoachim redfluorescentproteineqfp611applicationinsubcellularlocalizationstudiesinhigherplants |