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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 contains two separable domains responsible for  cytoplasmic retention in the dark. Confocal microscopy of parsley protoplasts transiently transformed with GFP fusion constructs expressing either 5′-deletions (A–C) or an internal deletion (D) of CPRF2. After transformation, protoplasts were kept  in darkness for 16 h before microscopical analysis. The relative  size and composition of the expressed GFP fusion protein is  shown schematically below each photograph. aa80CPRF2, NH2-terminal deletion of CPRF2 starting with aa 80; aa159CPRF2,  NH2-terminal deletion of CPRF2 starting with aa 159;  aa178CPRF2, NH2-terminal deletion of CPRF2 starting with aa  178; ΔCPRF2, internal deletion of CPRF2 (aa178–192); NLS/ bZIP, nuclear localization sequence-containing basic-region leucine-zipper domain; GFP, green fluorescent protein. Arrows, positions of the nuclei.
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Figure 5: CPRF2 contains two separable domains responsible for cytoplasmic retention in the dark. Confocal microscopy of parsley protoplasts transiently transformed with GFP fusion constructs expressing either 5′-deletions (A–C) or an internal deletion (D) of CPRF2. After transformation, protoplasts were kept in darkness for 16 h before microscopical analysis. The relative size and composition of the expressed GFP fusion protein is shown schematically below each photograph. aa80CPRF2, NH2-terminal deletion of CPRF2 starting with aa 80; aa159CPRF2, NH2-terminal deletion of CPRF2 starting with aa 159; aa178CPRF2, NH2-terminal deletion of CPRF2 starting with aa 178; ΔCPRF2, internal deletion of CPRF2 (aa178–192); NLS/ bZIP, nuclear localization sequence-containing basic-region leucine-zipper domain; GFP, green fluorescent protein. Arrows, positions of the nuclei.

Mentions: For our mapping purposes we now produced a set of CPRF2 constructs bearing NH2-terminal deletions fused to the GFP gene. These constructs were again transiently transformed into parsley protoplasts and the intracellular distribution of the corresponding fusion proteins determined by confocal microscopy after 16 h of cultivation in darkness. As shown in Fig. 5 A the fusion protein, where the first NH2-terminal 80 aa (aa80CPRF2–GFP) have been deleted, is observed in both the cytosol and the nucleus like it was shown for full-length CPRF2–GFP (compare to Fig. 4 D). The removal of additional 79 aa (aa15CPRF2– GFP) resulted in the loss of the cytoplasmic pool of the truncated CPRF2 (Fig. 5 B). The fusion peptide that misses the whole NH2 terminus up to aa 178 (aa178CPRF2–GFP) is exclusively found in the nuclear compartment as well (Fig. 5 C). In addition to these NH2-terminal deletions we determined the intracellular distribution of a fusion peptide from which an acidic amino acid stretch was removed (ΔCPRF2– GFP). This stretch (aa 178-DHSDDDDELEGETET-aa 192) is localized NH2-terminal of the NLS-containing basic DNA-binding motif of CPRF2. As shown in Fig. 5 D the fusion protein encoded by the ΔCPRF2–GFP construct is only observed in the nuclear compartment of dark-cultivated parsley protoplasts.


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 contains two separable domains responsible for  cytoplasmic retention in the dark. Confocal microscopy of parsley protoplasts transiently transformed with GFP fusion constructs expressing either 5′-deletions (A–C) or an internal deletion (D) of CPRF2. After transformation, protoplasts were kept  in darkness for 16 h before microscopical analysis. The relative  size and composition of the expressed GFP fusion protein is  shown schematically below each photograph. aa80CPRF2, NH2-terminal deletion of CPRF2 starting with aa 80; aa159CPRF2,  NH2-terminal deletion of CPRF2 starting with aa 159;  aa178CPRF2, NH2-terminal deletion of CPRF2 starting with aa  178; ΔCPRF2, internal deletion of CPRF2 (aa178–192); NLS/ bZIP, nuclear localization sequence-containing basic-region leucine-zipper domain; GFP, green fluorescent protein. Arrows, positions of the nuclei.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: CPRF2 contains two separable domains responsible for cytoplasmic retention in the dark. Confocal microscopy of parsley protoplasts transiently transformed with GFP fusion constructs expressing either 5′-deletions (A–C) or an internal deletion (D) of CPRF2. After transformation, protoplasts were kept in darkness for 16 h before microscopical analysis. The relative size and composition of the expressed GFP fusion protein is shown schematically below each photograph. aa80CPRF2, NH2-terminal deletion of CPRF2 starting with aa 80; aa159CPRF2, NH2-terminal deletion of CPRF2 starting with aa 159; aa178CPRF2, NH2-terminal deletion of CPRF2 starting with aa 178; ΔCPRF2, internal deletion of CPRF2 (aa178–192); NLS/ bZIP, nuclear localization sequence-containing basic-region leucine-zipper domain; GFP, green fluorescent protein. Arrows, positions of the nuclei.
Mentions: For our mapping purposes we now produced a set of CPRF2 constructs bearing NH2-terminal deletions fused to the GFP gene. These constructs were again transiently transformed into parsley protoplasts and the intracellular distribution of the corresponding fusion proteins determined by confocal microscopy after 16 h of cultivation in darkness. As shown in Fig. 5 A the fusion protein, where the first NH2-terminal 80 aa (aa80CPRF2–GFP) have been deleted, is observed in both the cytosol and the nucleus like it was shown for full-length CPRF2–GFP (compare to Fig. 4 D). The removal of additional 79 aa (aa15CPRF2– GFP) resulted in the loss of the cytoplasmic pool of the truncated CPRF2 (Fig. 5 B). The fusion peptide that misses the whole NH2 terminus up to aa 178 (aa178CPRF2–GFP) is exclusively found in the nuclear compartment as well (Fig. 5 C). In addition to these NH2-terminal deletions we determined the intracellular distribution of a fusion peptide from which an acidic amino acid stretch was removed (ΔCPRF2– GFP). This stretch (aa 178-DHSDDDDELEGETET-aa 192) is localized NH2-terminal of the NLS-containing basic DNA-binding motif of CPRF2. As shown in Fig. 5 D the fusion protein encoded by the ΔCPRF2–GFP construct is only observed in the nuclear compartment of dark-cultivated parsley protoplasts.

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