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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 distribution of CPRF2 is regulated by phytochrome. Confocal images of immunostained parsley cells  probed with the preimmunoserum (A) and the CPRF2-specific  antiserum (B–K). Before fixation cells had been kept either in  darkness (B) or irradiated for 2 h with continuous UV-A (C),  blue (D), red (E) or far-red light (F) or pulse-irradiated for 5 min  with red light (G), RG9 light (H), or red light followed by a 5-min  RG9 pulse (I). In J, cells incubated for 2 h in 0.1 mg/ml of PMG  elicitor from Phytophtera megasperma and in K, cells treated by a  30-min heat shock (37°C) followed by 90 min of cultivation at 25°C  in the dark are shown. After treatments cells were fixed and probed  with a CPRF2-specific antiserum. nu, position of the nuclei.
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Figure 3: Intracellular distribution of CPRF2 is regulated by phytochrome. Confocal images of immunostained parsley cells probed with the preimmunoserum (A) and the CPRF2-specific antiserum (B–K). Before fixation cells had been kept either in darkness (B) or irradiated for 2 h with continuous UV-A (C), blue (D), red (E) or far-red light (F) or pulse-irradiated for 5 min with red light (G), RG9 light (H), or red light followed by a 5-min RG9 pulse (I). In J, cells incubated for 2 h in 0.1 mg/ml of PMG elicitor from Phytophtera megasperma and in K, cells treated by a 30-min heat shock (37°C) followed by 90 min of cultivation at 25°C in the dark are shown. After treatments cells were fixed and probed with a CPRF2-specific antiserum. nu, position of the nuclei.

Mentions: To confirm our biochemical data and to elucidate the role of the different plant photoreceptors in the induction of CPRF2 nuclear import, we analyzed the intracellular distribution of the factor under different light conditions by an immunolocalization assay combined with confocal microscopy. For this purpose, dark-grown parsley cells were irradiated for 2 h with light of different wavelengths or further kept in darkness, respectively. After treatment cells were fixed and then stained with anti-CPRF2 or preimmunoserum and FITC-labeled secondary antibodies. The rapid fixation protocol enables a semiquantitative analysis of the intracellular distribution of CPRF2 even if the cells disintegrate during the procedure as a result of the strong intracellular osmotic pressure (see Materials and Methods). Before scanning the cells the positioning of nuclei was confirmed by transmission microscopy. Whereas the preimmunoserum showed a very weak and constitutive background fluorescence (Fig. 3 A), the endogenous CPRF2 is detected in the cytosol of dark-incubated cells, but not in the nucleus (Fig. 3 B). However, after irradiation of the cells with continuous UV-A, blue, red, or far-red light, CPRF2 appeared in the nucleus and, dependent on the light quality, became less pronounced in the cytosol (Fig. 3, C–F). This shows that CPRF2 was actively moving from the cytosol into the nucleus. The efficiency of the nuclear translocation of CPRF2 seems to be dependent on the light quality with red light being most effective followed by blue, far-red, and UV-A light. These differences in the efficiency of the translocation response to the tested light qualities point to phyA and phyB to be the main photoreceptors involved in this photoresponse. To further test this hypothesis we treated dark-grown parsley cells with pulses of red and long wavelength far-red (RG9) light and then transferred them back into darkness for another 2 h before fixation. As shown in Fig. 3 G a red light pulse of 5 min induced an almost complete translocation of CPRF2 from the cytosol to the nucleus, whereas a 5-min RG9 pulse led to no effect (Fig. 3 H). If the red light pulse was followed by a RG9 pulse, the import of CPRF2 into the nucleus was less pronounced (Fig. 3 I) compared with the red light pulse alone (Fig. 3 G). The reversion of the red light effect by the RG9 pulse was incomplete, though, if one compares the cytoplasmic staining of CPRF2 in Fig. 3 I with that of Fig. 3 H. To test whether, in addition to light, other exogenic factors can induce the translocation of CPRF2, dark-grown cells were treated either with elicitor from Phytophtera megaspermum f. sp. glycinea (PMG; Somssich et al., 1986) or with heat (Walter, 1989). Neither the PMG elicitor nor the heat shock treatment produced any effect on the intracellular distribution of CPRF2 (Fig. 3, J and K). In conclusion, our irradiation data indicate a control of nuclear transport of CPRF2 by both phyA and phyB (see Discussion).


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 distribution of CPRF2 is regulated by phytochrome. Confocal images of immunostained parsley cells  probed with the preimmunoserum (A) and the CPRF2-specific  antiserum (B–K). Before fixation cells had been kept either in  darkness (B) or irradiated for 2 h with continuous UV-A (C),  blue (D), red (E) or far-red light (F) or pulse-irradiated for 5 min  with red light (G), RG9 light (H), or red light followed by a 5-min  RG9 pulse (I). In J, cells incubated for 2 h in 0.1 mg/ml of PMG  elicitor from Phytophtera megasperma and in K, cells treated by a  30-min heat shock (37°C) followed by 90 min of cultivation at 25°C  in the dark are shown. After treatments cells were fixed and probed  with a CPRF2-specific antiserum. nu, position of the nuclei.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132893&req=5

Figure 3: Intracellular distribution of CPRF2 is regulated by phytochrome. Confocal images of immunostained parsley cells probed with the preimmunoserum (A) and the CPRF2-specific antiserum (B–K). Before fixation cells had been kept either in darkness (B) or irradiated for 2 h with continuous UV-A (C), blue (D), red (E) or far-red light (F) or pulse-irradiated for 5 min with red light (G), RG9 light (H), or red light followed by a 5-min RG9 pulse (I). In J, cells incubated for 2 h in 0.1 mg/ml of PMG elicitor from Phytophtera megasperma and in K, cells treated by a 30-min heat shock (37°C) followed by 90 min of cultivation at 25°C in the dark are shown. After treatments cells were fixed and probed with a CPRF2-specific antiserum. nu, position of the nuclei.
Mentions: To confirm our biochemical data and to elucidate the role of the different plant photoreceptors in the induction of CPRF2 nuclear import, we analyzed the intracellular distribution of the factor under different light conditions by an immunolocalization assay combined with confocal microscopy. For this purpose, dark-grown parsley cells were irradiated for 2 h with light of different wavelengths or further kept in darkness, respectively. After treatment cells were fixed and then stained with anti-CPRF2 or preimmunoserum and FITC-labeled secondary antibodies. The rapid fixation protocol enables a semiquantitative analysis of the intracellular distribution of CPRF2 even if the cells disintegrate during the procedure as a result of the strong intracellular osmotic pressure (see Materials and Methods). Before scanning the cells the positioning of nuclei was confirmed by transmission microscopy. Whereas the preimmunoserum showed a very weak and constitutive background fluorescence (Fig. 3 A), the endogenous CPRF2 is detected in the cytosol of dark-incubated cells, but not in the nucleus (Fig. 3 B). However, after irradiation of the cells with continuous UV-A, blue, red, or far-red light, CPRF2 appeared in the nucleus and, dependent on the light quality, became less pronounced in the cytosol (Fig. 3, C–F). This shows that CPRF2 was actively moving from the cytosol into the nucleus. The efficiency of the nuclear translocation of CPRF2 seems to be dependent on the light quality with red light being most effective followed by blue, far-red, and UV-A light. These differences in the efficiency of the translocation response to the tested light qualities point to phyA and phyB to be the main photoreceptors involved in this photoresponse. To further test this hypothesis we treated dark-grown parsley cells with pulses of red and long wavelength far-red (RG9) light and then transferred them back into darkness for another 2 h before fixation. As shown in Fig. 3 G a red light pulse of 5 min induced an almost complete translocation of CPRF2 from the cytosol to the nucleus, whereas a 5-min RG9 pulse led to no effect (Fig. 3 H). If the red light pulse was followed by a RG9 pulse, the import of CPRF2 into the nucleus was less pronounced (Fig. 3 I) compared with the red light pulse alone (Fig. 3 G). The reversion of the red light effect by the RG9 pulse was incomplete, though, if one compares the cytoplasmic staining of CPRF2 in Fig. 3 I with that of Fig. 3 H. To test whether, in addition to light, other exogenic factors can induce the translocation of CPRF2, dark-grown cells were treated either with elicitor from Phytophtera megaspermum f. sp. glycinea (PMG; Somssich et al., 1986) or with heat (Walter, 1989). Neither the PMG elicitor nor the heat shock treatment produced any effect on the intracellular distribution of CPRF2 (Fig. 3, J and K). In conclusion, our irradiation data indicate a control of nuclear transport of CPRF2 by both phyA and phyB (see Discussion).

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