Light-dependent Translocation of a Phytochrome B-GFP Fusion Protein to the Nucleus in Transgenic Arabidopsis

Abstract

Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859–868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.

Light-Induced Nuclear Translocation of Endogenous Pea Phytochrome A Visualized by Immunocytochemical Procedures

“…In accordance with this, structural/functional analyses of mutated PhyA and PhyB suggest common signal transduction mechanisms for those two major phytochrome species Ni et al, 1998). It is intriguing that the kinetics of the light-induced nuclear import of PHYA ( Figure 4) and those of PhyB (Yamaguchi et al, 1999) are very similar, although the former are elicited with FR light and the latter with R light. This prompts speculation as to whether another common mechanism regulates the nucleo/ cytoplasmic partitioning of these phytochromes.…”

“…On the other hand, R light induces nuclear translocation of both PHYA and PhyB-GFP, but nuclear accumulation of PHYA (Figure 4) reaches a maximum earlier than does that of PHYB (Kircher et al, 1999b;Yamaguchi et al, 1999). During the time in which PhyB-GFP accumulates in the nuclei in cR light, PHYA becomes undetectable (Figure 4) as a result of degradation ( Figure 6 and Table 2).…”

“…A biologically active Arabidopsis PhyB-GFP fusion protein localized to the nucleus in transgenic Arabidopsis under cR light (Yamaguchi et al, 1999). R light pulses induced nuclear localization of biologically active tobacco PhyB-GFP in transgenic tobacco, but this was reversible by exposure to FR light (Kircher et al, 1999b).…”

“…Histochemical staining of GUS activity indicated nuclear localization of the fusion protein. More recently, a phenotypic analysis of transgenic Arabidopsis plants expressing a fusion of full-length Arabidopsis PHYB and GFP showed that the fusion protein was both spectrally and biologically functional in vivo and accumulated in the nucleus in a lightdependent manner (Yamaguchi et al, 1999). A parsley PhyA apoprotein (PHYA)-GFP fusion transiently expressed in parsley protoplasts was constitutively cytosolic (Kircher et al, 1999a), whereas both biologically active rice PhyA-GFP and tobacco PhyB-GFP expressed in transgenic tobacco plants showed nuclear import dependent on the quality of the light (Kircher et al, 1999b).…”

“…In tissues, this regulation may affect the transmission of the light signal from the site of photoperception to the responsive organ. An analysis of the way in which photoreceptors are redistributed within the cell in response to light treatment may provide information about the early steps in signal transduction (Sakamoto and Nagatani, 1996;Yamaguchi et al, 1999). The distribution of phytochrome in plants has been analyzed previously by various methods, including spectrophotometry (Furuya and Hillman, 1964), microbeam irradiation (Haupt, 1970), immunochemistry (Pratt, 1994, and techniques using reporter genes (Komeda et al, 1991; Adam et al, 1994 Adam et al, , 1996Somers and Quail, 1995).…”