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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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Analysis of the cytoplasmic and nuclear distribution of G-box–binding activities in dark-kept evacuolated  parsley protoplasts. (A) Western blot analysis of cytoplasmic (cytosol) and nuclear (nucleus) extracts probed with  histone 2A/2B (panel I) and  tubulin (panel II) antibodies.  In (I) 25 μg of protein per  lane and in (II) 10 μg of protein per lane were loaded. In  B and C autoradiograms of  EMSSAs with 50 μg per lane  of cytoplasmic and 20 μg per  lane of nuclear extract are  shown. For CPRF/antiserum  interaction test the extracts  were incubated for 10 min on  ice with 1 μl of serum and  the radioactive-labeled G-box  probe before loading the samples on the gel (A and B, lanes  2–7). In B a 12- (top) and a  24-h (bottom) exposure of  the identical shift gel is  shown. In lanes 1, the binding reaction mix contained  neither a serum nor protein  (free probe). CPRF-containing DNA–protein complexes  are marked (F1, CPRF1; F2,  CPRF2; F4, CPRF4). Arrow,  positions of supershifted  DNA–CPRF complex.
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Figure 1: Analysis of the cytoplasmic and nuclear distribution of G-box–binding activities in dark-kept evacuolated parsley protoplasts. (A) Western blot analysis of cytoplasmic (cytosol) and nuclear (nucleus) extracts probed with histone 2A/2B (panel I) and tubulin (panel II) antibodies. In (I) 25 μg of protein per lane and in (II) 10 μg of protein per lane were loaded. In B and C autoradiograms of EMSSAs with 50 μg per lane of cytoplasmic and 20 μg per lane of nuclear extract are shown. For CPRF/antiserum interaction test the extracts were incubated for 10 min on ice with 1 μl of serum and the radioactive-labeled G-box probe before loading the samples on the gel (A and B, lanes 2–7). In B a 12- (top) and a 24-h (bottom) exposure of the identical shift gel is shown. In lanes 1, the binding reaction mix contained neither a serum nor protein (free probe). CPRF-containing DNA–protein complexes are marked (F1, CPRF1; F2, CPRF2; F4, CPRF4). Arrow, positions of supershifted DNA–CPRF complex.

Mentions: Recently, the involvement of bZIP proteins in G-box– binding activity in the cytosol of evacuolated dark-cultivated parsley protoplasts was demonstrated (Harter et al., 1994a). At least one of these bZIP factors was translocated into the nucleus in response to light, indicating that an inducible nuclear transport of a transcription factor is part of the light-modulated signal transduction network (Harter et al., 1994a). To identify this bZIP factor we used the CPRF antisera described in Table I in EMSSA using a G-box–containing sequence as DNA probe that allows detection of a wide spectrum of plant bZIP proteins (Foster et al., 1994; Menkens et al., 1995). EMSSA allows a sensitive monitoring of DNA-binding activities in combination with specific detection of the binding protein in crude cell extracts (Feldbrügge et al., 1994; Harter et al., 1994a). Dependent on the epitopes recognized within the aa sequence of its antigen, a polyclonal antiserum added to the binding reaction can inhibit the DNA/protein interaction resulting in the disappearance of a shifted band and/or can induce a supershifted complex with lower electrophoretic mobility (Harter et al., 1994a). The cytoplasmic and nuclear extracts for EMSSA were prepared from evacuolated parsley protoplast according to Harter et al. (1994b). These extracts were not contaminated by nuclear and cytoplasmic proteins, respectively, as demonstrated by Western blot analysis using histone 2A/2B- and tubulin-specific antibodies (Fig. 1 A). As shown in Fig. 1, B and C, lanes 3, 5, and 7, two major DNA–protein complexes could be detected in the cytoplasmic and one in the nuclear extract of dark-cultivated evacuolated parsley protoplasts in the presence of the preimmunosera. Addition of the CPRF2-specific antiserum to the DNA/cytosol-binding reaction caused the disappearance of the upper DNA–protein complex and a supershifted band (Fig. 1 B, lane 4). However, an EMSSA of the nuclear extract showed no disappearing CPRF2-containing complex (Fig. 1 C, lane 4). These data demonstrate that CPRF2 is present in the cytoplasmic compartment but not inside the nuclei of dark-cultivated, evacuolated parsley protoplasts. The use of the antiserum raised against rCPRF4 resulted in the disappearance of the lower cytoplasmic DNA–protein complex (Fig. 1 B, lane 6). An appearance of a supershifted DNA–protein complex as well as a weakening of a shifted band was also observed in nuclear extracts (Fig. 1 C, lane 6). In contrast, we obtained only a weak antibody activity in the cytoplasmic but a strong in the nuclear extracts when we tested the serum produced against rCPRF1 (Fig. 1, B and C, lanes 2). Since there is cross-reactivity of the CPRF1 antiserum with CPRF4 (see Table I), we conclude that the weak cytoplasmic antibody activity observed with the CPRF1 serum was due to cross-interaction with CPRF4, whereas the strong effect in the nuclear extract was due to true interaction with CPRF1.


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)

Analysis of the cytoplasmic and nuclear distribution of G-box–binding activities in dark-kept evacuolated  parsley protoplasts. (A) Western blot analysis of cytoplasmic (cytosol) and nuclear (nucleus) extracts probed with  histone 2A/2B (panel I) and  tubulin (panel II) antibodies.  In (I) 25 μg of protein per  lane and in (II) 10 μg of protein per lane were loaded. In  B and C autoradiograms of  EMSSAs with 50 μg per lane  of cytoplasmic and 20 μg per  lane of nuclear extract are  shown. For CPRF/antiserum  interaction test the extracts  were incubated for 10 min on  ice with 1 μl of serum and  the radioactive-labeled G-box  probe before loading the samples on the gel (A and B, lanes  2–7). In B a 12- (top) and a  24-h (bottom) exposure of  the identical shift gel is  shown. In lanes 1, the binding reaction mix contained  neither a serum nor protein  (free probe). CPRF-containing DNA–protein complexes  are marked (F1, CPRF1; F2,  CPRF2; F4, CPRF4). Arrow,  positions of supershifted  DNA–CPRF complex.
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Figure 1: Analysis of the cytoplasmic and nuclear distribution of G-box–binding activities in dark-kept evacuolated parsley protoplasts. (A) Western blot analysis of cytoplasmic (cytosol) and nuclear (nucleus) extracts probed with histone 2A/2B (panel I) and tubulin (panel II) antibodies. In (I) 25 μg of protein per lane and in (II) 10 μg of protein per lane were loaded. In B and C autoradiograms of EMSSAs with 50 μg per lane of cytoplasmic and 20 μg per lane of nuclear extract are shown. For CPRF/antiserum interaction test the extracts were incubated for 10 min on ice with 1 μl of serum and the radioactive-labeled G-box probe before loading the samples on the gel (A and B, lanes 2–7). In B a 12- (top) and a 24-h (bottom) exposure of the identical shift gel is shown. In lanes 1, the binding reaction mix contained neither a serum nor protein (free probe). CPRF-containing DNA–protein complexes are marked (F1, CPRF1; F2, CPRF2; F4, CPRF4). Arrow, positions of supershifted DNA–CPRF complex.
Mentions: Recently, the involvement of bZIP proteins in G-box– binding activity in the cytosol of evacuolated dark-cultivated parsley protoplasts was demonstrated (Harter et al., 1994a). At least one of these bZIP factors was translocated into the nucleus in response to light, indicating that an inducible nuclear transport of a transcription factor is part of the light-modulated signal transduction network (Harter et al., 1994a). To identify this bZIP factor we used the CPRF antisera described in Table I in EMSSA using a G-box–containing sequence as DNA probe that allows detection of a wide spectrum of plant bZIP proteins (Foster et al., 1994; Menkens et al., 1995). EMSSA allows a sensitive monitoring of DNA-binding activities in combination with specific detection of the binding protein in crude cell extracts (Feldbrügge et al., 1994; Harter et al., 1994a). Dependent on the epitopes recognized within the aa sequence of its antigen, a polyclonal antiserum added to the binding reaction can inhibit the DNA/protein interaction resulting in the disappearance of a shifted band and/or can induce a supershifted complex with lower electrophoretic mobility (Harter et al., 1994a). The cytoplasmic and nuclear extracts for EMSSA were prepared from evacuolated parsley protoplast according to Harter et al. (1994b). These extracts were not contaminated by nuclear and cytoplasmic proteins, respectively, as demonstrated by Western blot analysis using histone 2A/2B- and tubulin-specific antibodies (Fig. 1 A). As shown in Fig. 1, B and C, lanes 3, 5, and 7, two major DNA–protein complexes could be detected in the cytoplasmic and one in the nuclear extract of dark-cultivated evacuolated parsley protoplasts in the presence of the preimmunosera. Addition of the CPRF2-specific antiserum to the DNA/cytosol-binding reaction caused the disappearance of the upper DNA–protein complex and a supershifted band (Fig. 1 B, lane 4). However, an EMSSA of the nuclear extract showed no disappearing CPRF2-containing complex (Fig. 1 C, lane 4). These data demonstrate that CPRF2 is present in the cytoplasmic compartment but not inside the nuclei of dark-cultivated, evacuolated parsley protoplasts. The use of the antiserum raised against rCPRF4 resulted in the disappearance of the lower cytoplasmic DNA–protein complex (Fig. 1 B, lane 6). An appearance of a supershifted DNA–protein complex as well as a weakening of a shifted band was also observed in nuclear extracts (Fig. 1 C, lane 6). In contrast, we obtained only a weak antibody activity in the cytoplasmic but a strong in the nuclear extracts when we tested the serum produced against rCPRF1 (Fig. 1, B and C, lanes 2). Since there is cross-reactivity of the CPRF1 antiserum with CPRF4 (see Table I), we conclude that the weak cytoplasmic antibody activity observed with the CPRF1 serum was due to cross-interaction with CPRF4, whereas the strong effect in the nuclear extract was due to true interaction with CPRF1.

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