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Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis.

Yamaguchi R, Nakamura M, Mochizuki N, Kay SA, Nagatani A - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

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.

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Fluorescence microscopic images of different parts of  light-grown PBG-5 seedlings. Samples were stained with Hoechst  No. 33342 and viewed under epifluorescence optics with blue  (left) and UV (middle) excitation. DIC images of the same sample are shown (right). (a–c) PBG-5 leaf epidermis, ×100 objective. Bar, 10 μm. (d–f) PBG-5 root cells, ×40 objective. Bar, 25 μm.  (g–i) PBG-root hair, ×100 objective. Bar, 10 μm.
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Figure 5: Fluorescence microscopic images of different parts of light-grown PBG-5 seedlings. Samples were stained with Hoechst No. 33342 and viewed under epifluorescence optics with blue (left) and UV (middle) excitation. DIC images of the same sample are shown (right). (a–c) PBG-5 leaf epidermis, ×100 objective. Bar, 10 μm. (d–f) PBG-5 root cells, ×40 objective. Bar, 25 μm. (g–i) PBG-root hair, ×100 objective. Bar, 10 μm.

Mentions: Intracellular localization of the phyB-GFP fusion protein in the PBG-5 seedlings was examined. Epidermal layers including cortex were peeled from the light-grown seedlings and observed under a fluorescence microscope. At lower magnification, bright green spots of GFP fluorescence were observed (Fig. 4, a–c). Positions of the spots matched well with those of the nuclei revealed by the Hoechst staining. Similar fluorescence images were obtained for another transgenic line, PBG-7 (data not shown). Interestingly, observation at higher magnification revealed that the phyB-GFP fluorescence was speckled within the nuclear region (Fig. 4, d–f). The apparent size of each speckle appeared to be <1 μm. Although speckles were observed in all of the nuclei, the number per nucleus varied. In most cases, one nucleus contained 5–10 speckles. The intracellular localization of phyB-GFP was then examined in other parts of the seedling. As shown in Fig. 5, nuclear fluorescence was confirmed in leaf (Fig. 5, a–c), root (Fig. 5, d–f), and root hair cells (Fig. 5, g–i). Furthermore, the speckles were observed in all of the cell types examined.


Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis.

Yamaguchi R, Nakamura M, Mochizuki N, Kay SA, Nagatani A - J. Cell Biol. (1999)

Fluorescence microscopic images of different parts of  light-grown PBG-5 seedlings. Samples were stained with Hoechst  No. 33342 and viewed under epifluorescence optics with blue  (left) and UV (middle) excitation. DIC images of the same sample are shown (right). (a–c) PBG-5 leaf epidermis, ×100 objective. Bar, 10 μm. (d–f) PBG-5 root cells, ×40 objective. Bar, 25 μm.  (g–i) PBG-root hair, ×100 objective. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2185089&req=5

Figure 5: Fluorescence microscopic images of different parts of light-grown PBG-5 seedlings. Samples were stained with Hoechst No. 33342 and viewed under epifluorescence optics with blue (left) and UV (middle) excitation. DIC images of the same sample are shown (right). (a–c) PBG-5 leaf epidermis, ×100 objective. Bar, 10 μm. (d–f) PBG-5 root cells, ×40 objective. Bar, 25 μm. (g–i) PBG-root hair, ×100 objective. Bar, 10 μm.
Mentions: Intracellular localization of the phyB-GFP fusion protein in the PBG-5 seedlings was examined. Epidermal layers including cortex were peeled from the light-grown seedlings and observed under a fluorescence microscope. At lower magnification, bright green spots of GFP fluorescence were observed (Fig. 4, a–c). Positions of the spots matched well with those of the nuclei revealed by the Hoechst staining. Similar fluorescence images were obtained for another transgenic line, PBG-7 (data not shown). Interestingly, observation at higher magnification revealed that the phyB-GFP fluorescence was speckled within the nuclear region (Fig. 4, d–f). The apparent size of each speckle appeared to be <1 μm. Although speckles were observed in all of the nuclei, the number per nucleus varied. In most cases, one nucleus contained 5–10 speckles. The intracellular localization of phyB-GFP was then examined in other parts of the seedling. As shown in Fig. 5, nuclear fluorescence was confirmed in leaf (Fig. 5, a–c), root (Fig. 5, d–f), and root hair cells (Fig. 5, g–i). Furthermore, the speckles were observed in all of the cell types examined.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

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.

Show MeSH