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Nuclear import of the parsley bZIP transcription factor CPRF2 is regulated by phytochrome photoreceptors.

Kircher S, Wellmer F, Nick P, Rügner A, Schäfer E, Harter K - J. Cell Biol. (1999)

Bottom Line: To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum).Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell.We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie II/Botanik, Universität Freiburg, 79104 Freiburg, Germany.

ABSTRACT
In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

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Intracellular sorting of GFP fusion proteins within  parsley protoplasts. Confocal sections of parsley protoplasts transiently transformed with fusion constructs expressing phyA–GFP  (A), NLS–GFP (B), CPRF1–GFP (C), and CPRF2–GFP (D and  E). After transformation by electroporation protoplasts were  kept for 16 h in darkness (A–D) or continuously irradiated with  UV-containing white light (E) for the same time period before  microscopical analysis. Arrows, positions of the nuclei.
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Figure 4: Intracellular sorting of GFP fusion proteins within parsley protoplasts. Confocal sections of parsley protoplasts transiently transformed with fusion constructs expressing phyA–GFP (A), NLS–GFP (B), CPRF1–GFP (C), and CPRF2–GFP (D and E). After transformation by electroporation protoplasts were kept for 16 h in darkness (A–D) or continuously irradiated with UV-containing white light (E) for the same time period before microscopical analysis. Arrows, positions of the nuclei.

Mentions: The observation that CPRF2 is found in the cytosol of dark-grown parsley cells prompted us to map the amino acid stretch of the CPRF2 molecule that could be responsible for cytoplasmic retention. For this purpose, we translationally fused the cDNA coding for CPRF2 to a 35 S promotor-driven GFP gene (Haseloff et al., 1997) resulting in a GFP fusion protein upon transient transformation into parsley protoplasts. A parsley PHYA–GFP, a nuclear targeting NLS–GFP, and a CPRF1–GFP fusion construct were used as controls for cytoplasmic (phyA–GFP; Speth et al., 1986, 1987) and nuclear localization, respectively (NLS–GFP, CPRF1–GFP; van der Krol and Chua, 1991; Varagona et al., 1992) (data in Fig. 1). After transformation, the protoplasts that were derived from dark-grown parsley cells were incubated for 16 h in darkness or irradiated with continuous UV-containing white light. The intracellular distribution of the GFP fusion proteins was then analyzed by confocal microscopy. Before scanning the cells the positioning of the nuclei was confirmed by transmission microscopy. As shown in Fig. 4 A, phyA–GFP was exclusively localized in the cytosol from dark-cultivated protoplasts. The NLS–GFP as well as the CPRF1–GFP fusion, in contrast, were always confined to the nucleus (Fig. 4, B and C). Irradiation of the protoplasts had no influence on the GFP fluorescence intensity indicating that the used light sources do not induce any bleaching effect. Furthermore, we observed no changes in the subcellular distribution of the control fusion proteins in response to light treatment (data not shown). In contrast, the CPRF2–GFP is observed in both compartments of dark-kept protoplasts (Fig. 4 D). Irradiation of the protoplasts expressing CPRF2–GFP resulted in the disappearance of the fusion protein from the cytosol indicating its nuclear import (Fig. 4 E). Due to a strong nuclear overreflection signal, a clear additional accumulation of CPRF2–GFP inside the nucleus could not be observed. However, as shown previously (Kircher et al., 1998), the total amount of CPRF2 is not reduced in response to irradiation excluding a compartment-specific degradation as reason for the disappearance of the bZIP factor from the cytosol.


Nuclear import of the parsley bZIP transcription factor CPRF2 is regulated by phytochrome photoreceptors.

Kircher S, Wellmer F, Nick P, Rügner A, Schäfer E, Harter K - J. Cell Biol. (1999)

Intracellular sorting of GFP fusion proteins within  parsley protoplasts. Confocal sections of parsley protoplasts transiently transformed with fusion constructs expressing phyA–GFP  (A), NLS–GFP (B), CPRF1–GFP (C), and CPRF2–GFP (D and  E). After transformation by electroporation protoplasts were  kept for 16 h in darkness (A–D) or continuously irradiated with  UV-containing white light (E) for the same time period before  microscopical analysis. Arrows, positions of the nuclei.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132893&req=5

Figure 4: Intracellular sorting of GFP fusion proteins within parsley protoplasts. Confocal sections of parsley protoplasts transiently transformed with fusion constructs expressing phyA–GFP (A), NLS–GFP (B), CPRF1–GFP (C), and CPRF2–GFP (D and E). After transformation by electroporation protoplasts were kept for 16 h in darkness (A–D) or continuously irradiated with UV-containing white light (E) for the same time period before microscopical analysis. Arrows, positions of the nuclei.
Mentions: The observation that CPRF2 is found in the cytosol of dark-grown parsley cells prompted us to map the amino acid stretch of the CPRF2 molecule that could be responsible for cytoplasmic retention. For this purpose, we translationally fused the cDNA coding for CPRF2 to a 35 S promotor-driven GFP gene (Haseloff et al., 1997) resulting in a GFP fusion protein upon transient transformation into parsley protoplasts. A parsley PHYA–GFP, a nuclear targeting NLS–GFP, and a CPRF1–GFP fusion construct were used as controls for cytoplasmic (phyA–GFP; Speth et al., 1986, 1987) and nuclear localization, respectively (NLS–GFP, CPRF1–GFP; van der Krol and Chua, 1991; Varagona et al., 1992) (data in Fig. 1). After transformation, the protoplasts that were derived from dark-grown parsley cells were incubated for 16 h in darkness or irradiated with continuous UV-containing white light. The intracellular distribution of the GFP fusion proteins was then analyzed by confocal microscopy. Before scanning the cells the positioning of the nuclei was confirmed by transmission microscopy. As shown in Fig. 4 A, phyA–GFP was exclusively localized in the cytosol from dark-cultivated protoplasts. The NLS–GFP as well as the CPRF1–GFP fusion, in contrast, were always confined to the nucleus (Fig. 4, B and C). Irradiation of the protoplasts had no influence on the GFP fluorescence intensity indicating that the used light sources do not induce any bleaching effect. Furthermore, we observed no changes in the subcellular distribution of the control fusion proteins in response to light treatment (data not shown). In contrast, the CPRF2–GFP is observed in both compartments of dark-kept protoplasts (Fig. 4 D). Irradiation of the protoplasts expressing CPRF2–GFP resulted in the disappearance of the fusion protein from the cytosol indicating its nuclear import (Fig. 4 E). Due to a strong nuclear overreflection signal, a clear additional accumulation of CPRF2–GFP inside the nucleus could not be observed. However, as shown previously (Kircher et al., 1998), the total amount of CPRF2 is not reduced in response to irradiation excluding a compartment-specific degradation as reason for the disappearance of the bZIP factor from the cytosol.

Bottom Line: To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum).Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell.We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie II/Botanik, Universität Freiburg, 79104 Freiburg, Germany.

ABSTRACT
In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

Show MeSH
Related in: MedlinePlus