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A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Molecular Plant 2013年第5期6卷 1518-1530页

作者： Martina Lummer Fabian Humpert Matthias Wiedenubbert markus Sauer mark schuttpelz Dorothee Staiger Molecular Cell Physiology Bielefeld University Universitatsstr. 25 33615 Bielefeld Germany Institute for Genome Research and Systems Biology CeBiTec 33615 Bielefeld Germany Biomolecular Photonics Bielefeld University Universitatsstr. 25 33615 Bielefeld Germany Biotechnology and Biophysics Julius-Maximilians-University Wurzburg Am Hubland 97074 Wurzburg Germany

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-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent 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-localiza- tion 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 pho- toactivation 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.

关键词： reversibly photoswitchable fluorescent protein codon usage Arabidopsis rsFastLIME-s bsDRONPA-s PADRON-s.

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