title: Bellis perennis: a useful tool for protein localization studies.
abstract: Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar "Galaxy red") which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.
authors.affiliation: Plant Physiology, Justus Liebig University Giessen, Senckenbergstr. 3, 35390 Giessen, Germany. Janina.K.Jaedicke@bio.uni-giessen.de
authors.forename: Katharina
authors.initials: K
authors.lastname: Jaedicke
authors.forename: Jutta
authors.initials: J
authors.lastname: Rösler
authors.forename: Tanja
authors.initials: T
authors.lastname: Gans
authors.forename: Jon
authors.initials: J
authors.lastname: Hughes
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000378:metabolism*; D019659:Asteraceae / Q000378:metabolism*; D019470:Biolistics / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D008164:Luminescent Proteins; D008928:Mitochondria / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism; D019441:Plant Epidermis / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein
chemical_list: D029681:Arabidopsis Proteins; D049452:Green Fluorescent Proteins; D006419:Heme Oxygenase (Decyclizing); D008164:Luminescent Proteins; D009701:Nucleoside-Diphosphate Kinase; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C121988:nucleoside diphosphate kinase 2