title: A new set of reversibly photoswitchable fluorescent proteins for use in transgenic plants.
abstract: Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the 'positively switchable' PADRON C-s with the 'negatively switchable' rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.
authors: Molecular Cell Physiology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors: Martina
authors: M
authors: Lummer
authors: Fabian
authors: F
authors: Humpert
authors: Matthias
authors: M
authors: Wiedenlübbert
authors: Markus
authors: M
authors: Sauer
authors: Mark
authors: M
authors: Schüttpelz
authors: Dorothee
authors: D
authors: Staiger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D005453:Fluorescence; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000235:genetics; D030821:Plants, Genetically Modified; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins
keywords: Arabidopsis; PADRON-s.; bsDRONPA-s; codon usage; reversibly photoswitchable fluorescent protein; rsFastLIME-s