Green Fluorescent Protein Targeted to the Nucleus, a Transgenic Phenotype Useful for Studies in Plant Biology

Nuclear Dynamics inArabidopsis thaliana

“…NLS-GFP-GUS is expressed and targeted to the nuclei of dicots (tobacco and Arabidopsis; Grebenok et al, 1997a,b;Chytilova et al, 1999) Collings, personal communication), and lower plants (Physcomitrella patens; K. Schumaker, personal communication). The level of biosynthesis and nuclear accumulation of the chimeric GFP molecule is sufficient so that observation of the living plant tissues with the confocal microscope can be done without excessive illumination, which can lead to photodamage.…”

“…This impedes the observation of nuclei within living cells with standard bright-field or fluorescence microscopy. Although other forms of light microscopy can be used to examine nuclei, particularly phase contrast and differential interference contrast microscopy, which rely on optical properties other than absorbance and fluorescence, this is practical only in nonpigmented cells and in tissues having simple three-dimensional organization.We have developed an alternative method for observation of nuclei in living plant cells that uses confocal laser scanning fluorescence microscopy (Chytilova et al, 1999). Based on the transgenic expression of the Green Fluorescent Protein (GFP) of Aequorea victoria, it involves biosynthesis of a chimeric protein, comprising a codon-optimized GFP coding sequence translationally fused to an effective nuclear localization signal (NLS) and to the complete coding region of ␤-glucuronidase (GUS) from Escherichia coli (Grebenok et al, 1997a,b).…”

“…Based on the transgenic expression of the Green Fluorescent Protein (GFP) of Aequorea victoria, it involves biosynthesis of a chimeric protein, comprising a codon-optimized GFP coding sequence translationally fused to an effective nuclear localization signal (NLS) and to the complete coding region of ␤-glucuronidase (GUS) from Escherichia coli (Grebenok et al, 1997a,b). This large (Ͼ100 kDa) protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm, where it can be conveniently observed with confocal microscopy.NLS-GFP-GUS is expressed and targeted to the nuclei of dicots (tobacco and Arabidopsis; Grebenok et al, 1997a,b;Chytilova et al, 1999) Collings, personal communication), and lower plants (Physcomitrella patens; K. Schumaker, personal communication). The level of biosynthesis and nuclear accumulation of the chimeric GFP molecule is sufficient so that observation of the living plant tissues with the confocal microscope can be done without excessive illumination, which can lead to photodamage.…”

New insights into cytomixis: specific cellular features and prevalence in higher plants

“…For example, the root growth is accompanied by migration of the nuclei from epidermal cells into the middle part of root hair (Chytilova et al 1999) or nuclei of the filamentous fungi migrate along the growing hypha to uniformly spread over its space (Suelmann et al 1997). The nuclear migration in these cases is provided for by the interaction of cytoskeleton and motor proteins (Suelmann et al 1997;Fischer 1999;Liu et al 2003).…”

Cytometry and plant sciences: A personal retrospective

“…Alternatively, nuclear transcripts can be used for production of fluorescent targets that can be hybridized to microarrays (80,93). Given the ability to produce transgenic plants expressing nucleartargeted GFP within specific cell types, such as phloem companion cells and guard cells (94), it soon should be possible to categorize the gene expression profiles of any and all plant cell types. We are confident that these methods are applicable to animal cells.…”