Fluorescent protein applications in plants.Methods Cell Biol. 2008; 85:153-77.MC
Study of plant cell biology has benefited tremendously from the use of fluorescent proteins (FPs). Development of well-established techniques in genetics, by transient expression or by Agrobacterium-mediated plant cell transformation, makes it possible to readily create material for imaging molecules tagged with FPs. Confocal microscopy of FPs is routine and, in highly scattering tissues, multiphoton microscopy improves deep imaging. The abundance of autofluorescent compounds in plants in some cases potentially interferes with FP signals, but spectral imaging is an effective tool in unmixing overlapping signals. This approach allows separate detection of DsRed and chlorophyll, DsRed and GFP, and green fluorescent protein (GFP) and yellow fluorescent protein (YFP). FPs have been targeted to most plant organelles. Free (untargeted) FPs in plant cells are not only cytoplasmic, but also go into the nucleus due to their small size. FP fluorescence is potentially unstable in acidic vacuoles. FPs have been targeted to novel compartments, including protein storage vacuoles in seeds. Endoplasmic reticulum (ER)-targeted GFP has identified novel inclusion bodies that are surprisingly dynamic. FP-tagged Rab GTPases have allowed documentation of the dynamics of membrane trafficking. Investigation of virus infections has progressed significantly with the aid of FP-tagged virus proteins. Advanced techniques are giving plant scientists the ability to quantitatively analyze the behavior of FP-tagged proteins. Fluorescence lifetime microscopy is becoming the method of choice for fluorescence resonance energy transfer (FRET) analysis of FP-tagged proteins. Fluorescence correlation spectroscopy (FCS) of FPs provides information on molecular diffusion and intermolecular interactions. Use of FPs in elucidating the behavior of plant cells has a bright future.