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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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CPRF2 is imported  into the nucleus of evacuolated parsley protoplasts in  response to irradiation with  UV-containing white light.  Autoradiograms of EMSSAs  with a radioactive-labeled  C-box probe are shown. For  EMSSA 50 μg per lane of cytosolic (panel I) and 20 μg  per lane of nuclear (panel II)  extracts isolated from dark-cultivated cells were used  that were either further kept  in continuous darkness (A)  or irradiated for 30 min with  UV-containing white light  (B) before compartment separation. For CPRF–antiserum interaction tests, the extracts were incubated for 10  min on ice with 1 μl of serum  and the radioactive-labeled  C-box probe before loading  the samples on the gel (A  and B, lanes 3 and 4, and 6 and 7, respectively). In lanes 2 and 6, no  serum was added (−). DNA–CPRF2 complexes are marked (F2).  Arrows, positions of supershifted DNA–CPRF2 complex. For further abbreviations see Fig. 1.
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Figure 2: CPRF2 is imported into the nucleus of evacuolated parsley protoplasts in response to irradiation with UV-containing white light. Autoradiograms of EMSSAs with a radioactive-labeled C-box probe are shown. For EMSSA 50 μg per lane of cytosolic (panel I) and 20 μg per lane of nuclear (panel II) extracts isolated from dark-cultivated cells were used that were either further kept in continuous darkness (A) or irradiated for 30 min with UV-containing white light (B) before compartment separation. For CPRF–antiserum interaction tests, the extracts were incubated for 10 min on ice with 1 μl of serum and the radioactive-labeled C-box probe before loading the samples on the gel (A and B, lanes 3 and 4, and 6 and 7, respectively). In lanes 2 and 6, no serum was added (−). DNA–CPRF2 complexes are marked (F2). Arrows, positions of supershifted DNA–CPRF2 complex. For further abbreviations see Fig. 1.

Mentions: To test the possibility that CPRF2 is the target for a light-modulated nuclear import we performed an additional set of EMSSA with cytoplasmic and nuclear extracts from evacuolated parsley protoplasts that were either irradiated for 30 min with UV-containing white light exciting all plant photoreceptor systems or kept in darkness for the same time period before isolation of the compartments. To detect CPRF2 more clearly we switched from the G-box to the C-box as DNA probe. CPRF2 has a very high affinity to this sequence whereas the binding activities of CPRF1 and CPRF4 are low, reducing the signals of these two bZIP factors in EMSSA (Izawa et al., 1993; Foster et al., 1994). Furthermore, the C-box allows a better resolution of C-box–binding factors in EMSSA (Fig. 2) (Wellmer, F., S. Kircher, A. Rügner, H. Frohnmeyer, E. Schäfer, and K. Harter, manuscript submitted for publication). Using the highly specific CPRF2 antiserum we could not detect any CPRF2-dependent DNA-binding activity in the nuclear extract of dark-kept parsley cells demonstrating again the absence of the factor from this compartment under dark conditions (Fig. 2 A, lane 6). However, a supershifted CPRF2 signal can be observed in the corresponding cytosol (Fig. 2 A, lane 3). Irradiation of the cells caused the appearance of a supershifted CPRF2-containing DNA–protein complex in the nucleus (Fig. 2 B, lane 6). The use of the corresponding preimmunoserum showed no effect (Fig. 2, A and B, lanes 4 and 7). Note, that the amount of nuclear proteins used in the assays is 2.5 times lower than that of cytoplasmic one. In evacuolated parsley protoplasts, about 45%, each, of total protein was determined to be localized in the cytosol and the nucleus (Harter et al., 1994a). From this, we conclude that the amount of nucleus-imported CPRF2 is at least 50% of the total pool. We could not detect any changes in the intracellular distribution pattern of supershifted DNA–protein complexes when we used the antisera raised against rCPRF1 and rCPRF4 in EMSSA (data not shown). In conclusion, these results strongly indicate that CPRF2 is in fact the bZIP factor that is translocated from the cytosol into the nucleus in response to light. Note that there are additional C-box–binding activities in the cytoplasmic as well as in the nuclear extract that were not detected previously using the G-box as DNA probe (Fig. 2). Moreover, the pattern of these activities derived from unknown C-box binding proteins changed in the nuclear extract after light irradiation (Fig. 2).


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)

CPRF2 is imported  into the nucleus of evacuolated parsley protoplasts in  response to irradiation with  UV-containing white light.  Autoradiograms of EMSSAs  with a radioactive-labeled  C-box probe are shown. For  EMSSA 50 μg per lane of cytosolic (panel I) and 20 μg  per lane of nuclear (panel II)  extracts isolated from dark-cultivated cells were used  that were either further kept  in continuous darkness (A)  or irradiated for 30 min with  UV-containing white light  (B) before compartment separation. For CPRF–antiserum interaction tests, the extracts were incubated for 10  min on ice with 1 μl of serum  and the radioactive-labeled  C-box probe before loading  the samples on the gel (A  and B, lanes 3 and 4, and 6 and 7, respectively). In lanes 2 and 6, no  serum was added (−). DNA–CPRF2 complexes are marked (F2).  Arrows, positions of supershifted DNA–CPRF2 complex. For further abbreviations see Fig. 1.
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Figure 2: CPRF2 is imported into the nucleus of evacuolated parsley protoplasts in response to irradiation with UV-containing white light. Autoradiograms of EMSSAs with a radioactive-labeled C-box probe are shown. For EMSSA 50 μg per lane of cytosolic (panel I) and 20 μg per lane of nuclear (panel II) extracts isolated from dark-cultivated cells were used that were either further kept in continuous darkness (A) or irradiated for 30 min with UV-containing white light (B) before compartment separation. For CPRF–antiserum interaction tests, the extracts were incubated for 10 min on ice with 1 μl of serum and the radioactive-labeled C-box probe before loading the samples on the gel (A and B, lanes 3 and 4, and 6 and 7, respectively). In lanes 2 and 6, no serum was added (−). DNA–CPRF2 complexes are marked (F2). Arrows, positions of supershifted DNA–CPRF2 complex. For further abbreviations see Fig. 1.
Mentions: To test the possibility that CPRF2 is the target for a light-modulated nuclear import we performed an additional set of EMSSA with cytoplasmic and nuclear extracts from evacuolated parsley protoplasts that were either irradiated for 30 min with UV-containing white light exciting all plant photoreceptor systems or kept in darkness for the same time period before isolation of the compartments. To detect CPRF2 more clearly we switched from the G-box to the C-box as DNA probe. CPRF2 has a very high affinity to this sequence whereas the binding activities of CPRF1 and CPRF4 are low, reducing the signals of these two bZIP factors in EMSSA (Izawa et al., 1993; Foster et al., 1994). Furthermore, the C-box allows a better resolution of C-box–binding factors in EMSSA (Fig. 2) (Wellmer, F., S. Kircher, A. Rügner, H. Frohnmeyer, E. Schäfer, and K. Harter, manuscript submitted for publication). Using the highly specific CPRF2 antiserum we could not detect any CPRF2-dependent DNA-binding activity in the nuclear extract of dark-kept parsley cells demonstrating again the absence of the factor from this compartment under dark conditions (Fig. 2 A, lane 6). However, a supershifted CPRF2 signal can be observed in the corresponding cytosol (Fig. 2 A, lane 3). Irradiation of the cells caused the appearance of a supershifted CPRF2-containing DNA–protein complex in the nucleus (Fig. 2 B, lane 6). The use of the corresponding preimmunoserum showed no effect (Fig. 2, A and B, lanes 4 and 7). Note, that the amount of nuclear proteins used in the assays is 2.5 times lower than that of cytoplasmic one. In evacuolated parsley protoplasts, about 45%, each, of total protein was determined to be localized in the cytosol and the nucleus (Harter et al., 1994a). From this, we conclude that the amount of nucleus-imported CPRF2 is at least 50% of the total pool. We could not detect any changes in the intracellular distribution pattern of supershifted DNA–protein complexes when we used the antisera raised against rCPRF1 and rCPRF4 in EMSSA (data not shown). In conclusion, these results strongly indicate that CPRF2 is in fact the bZIP factor that is translocated from the cytosol into the nucleus in response to light. Note that there are additional C-box–binding activities in the cytoplasmic as well as in the nuclear extract that were not detected previously using the G-box as DNA probe (Fig. 2). Moreover, the pattern of these activities derived from unknown C-box binding proteins changed in the nuclear extract after light irradiation (Fig. 2).

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