Institut für Biologie II/Botanik, Universität Freiburg, 79104 Freiburg, Germany
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Abstract |
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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.
Key words: light regulation; phytochrome; bZIP transcription factors; nucleocytoplasmic partitioning; retention domain ![]() |
Introduction |
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LIGHT plays a central role in the morphogenesis of plants
(Kendrick and Kronenberg, 1994). They have evolved
a set of photoreceptor systems that include phytochromes, blue/UV-A, and UV-B receptors to percept the
quality, quantity, direction, and duration of environmental light conditions to adapt optimal growth (Frankhauser and
Chory, 1997). Many of the developmental changes occuring
during photomorphogenesis are controlled by members of
the phytochrome (phy)1 photoreceptor family encoded by
five different genes in Arabidopsis thaliana (PHYA to
PHYE). Phytochromes are photoreversible chromoproteins that are synthesized in their physiologically inactive,
red light-absorbing forms (Pr) and converted to their active, far-red light-absorbing forms (Pfr) upon irradiation.
The red/far-red photoreversibility (reversible Pfr/Pr conversion) of a given response (low fluence response) and
the responsiveness to continuous far-red light (high irradiance response) are two well-established operational criteria for the involvement of phyB and phyA, respectively, in light
perception and signaling (Quail et al., 1995
; Whitelam and
Devlin, 1997
).
Regulatory expression of genes is essential for plant adaptation during photomorphogenesis (Frankhauser and
Chory, 1997). Transcriptional regulation of gene expression is largely mediated through sequence-specific DNA-binding proteins that interact with cis-acting elements located in the promoter regions of the corresponding genes.
The binding of transcription factors to relevant cis-acting elements alters the activity of the general transcription
machinery stimulating or suppressing the expression of
genes (for review see Tjian and Maniatis, 1994). Several
cis-acting elements were identified within plant promotors
(Terzaghi and Cashmore, 1995). One of the best characterized elements is the hexameric G-box (CACGTG)
found in the promotors of light-regulated genes such as
ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit from tomato and chalcon synthase (CHS) from parsley (for review see Menkens et al., 1995
; Frankhauser and
Chory, 1997). The G-box often functions together with
other cis-elements as, for example, has been demonstrated
for the light-regulated elements of the parsley CHS promotor (Schulze-Lefert et al., 1989a
; Block et al., 1990
).
The proteins that bind to the G-box belong to the basic-region leucine-zipper (bZIP) transcription factor family
(Foster et al., 1994
). This domain comprises two structural
motifs: a stretch rich in basic residues mediating DNA-binding (Weiss et al., 1990
; O'Neil et al., 1991
; O'Shea et al.,
1991
; Ellenberger et al., 1992
) adjacent to a leucine-zipper
responsible for homo- and heterodimer formation (Rasmussen et al., 1991
). The basic stretch also contains the nuclear localization sequence (NLS) as shown exemplarily
for opaque 2 (Varagona et al., 1992
) and TGA-1 (van der
Krol and Chua, 1991
). In addition, with few exceptions, all
plant bZIPs that have been cloned so far, such as the
G-box-binding factors (GBF) from Arabidopsis and the
CPRFs from parsley, contain a proline-rich NH2-terminal
region that functions as a transcriptional activation or repression domain in animal and plant cells (Weisshaar et al., 1991
; Schindler et al., 1992a
,b; Feldbrügge et al., 1994
). The specificity of homodimeric bZIP proteins for a G-box-like
element depends on sequences flanking the ACGT core
(for review see Foster et al., 1994
; Meshi and Iwabuchi,
1995
). For instance, the GBFs from Arabidopsis and
CPRF1 and CPRF4 from parsley bind with high affinity to
the classical G-box (CACGTG). In contrast, the bZIP factors TGA1, OBF4, and bA19 from Arabidopsis bind to
GACGT(T/C) motifs but not to the G-box itself. Proteins
with intermediate characteristics for DNA-binding specificity as for example maize opaque2 and parsley CPRF2
are known as well.
The capacity for heterodimer formation as well as for
heterotypic interaction with members of other transcription factor families (Armstrong et al., 1992; Schindler et al.,
1992b
; Büttner and Singh, 1997
; Vicente-Carbajosa et al.,
1997) provides a wealth of possible transcription factor
complexes with potentially distinct binding activities and
activation abilities resulting in signal-specific regulation of
a particular target gene. The regulation of the formation
and activity of such transcription factor complexes and,
thus, the ability to transactivate a certain gene is accomplished by several mechanisms on different regulatory levels. (a) Although most plant bZIP proteins are constitutively expressed, some are restricted to a particular tissue
or differentially regulated by exogenic or endogenic signals (Schindler et al., 1992a
; Menkens and Cashmore,
1994
; Kircher et al., 1998
). (b) The DNA-binding activity
as well as the transactivation ability of bZIPs can be regulated by posttranslational modifications as, for instance, phosphorylation (Klimczak et al., 1992
, 1995
; Harter et al.,
1994a
). (c) The control of nuclear localization is also
known to regulate transcription factor activity. The cytosolic retention in absence of the appropriate signal can be
achieved by anchoring proteins or by retention factors (for
reviews see Jans and Hübner, 1996
; Ghosh et al., 1998
).
Cytoplasmic localization of some plant bZIP proteins was
recently demonstrated for G-box binding proteins in dark-grown parsley cells (Harter et al., 1994a
) and for Arabidopsis GBF1 and GBF2 in Arabidopsis and transiently
transformed soybean protoplasts (Terzaghi et al., 1997
).
After irradiation with white light at least one of the cytosolic parsley bZIP factors is imported into the nucleus in
vitro (Harter et al., 1994a
). Similarly, during cultivation of
transiently transformed soybean cells under blue light, the
pool of GBF2, but not of GBF1 protein is now found in
the nuclear compartment (Terzaghi et al., 1997
).
Here we report that the bZIP factors CPRF1, CPRF2, and CPRF4 are differentially distributed within dark-cultivated parsley cell with CPRF1 localized in the nucleus, CPRF2 found in the cytosol, and CPRF4 present in both compartments. Using three different in vivo assays we are able to show that CPRF2 is transported from the cytosol into the nucleus upon light irradiation. Furthermore, immunolocalization studies reveal that phyA via high irradiance response and phyB via a low fluence response are the main photoreceptors involved in the nuclear translocation response of CPRF2. Mapping of the retention domains within the CPRF2 sequence reveals two functionally independent motifs responsible for cytoplasmic localization and let us propose two alternative hypothesis for the molecular mechanism of CPRF2 retention in darkness.
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Materials and Methods |
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Light Sources
UV-containing white light, UV-A light, and blue light sources were used
as described in Frohnmeyer et al. (1992). Standard red, far-red, and RG9
light conditions were used as described in Schäfer (1977)
. If not otherwise
indicated, all experiments were carried out under dim-green safelight according to Schäfer (1977)
.
Suspension Culture, Preparation of Protoplasts, and Isolation of Cytosolic and Nuclear Extracts
Protoplasts were prepared from a dark-grown parsley (Petroselinum
crispum) cell culture 6 d after subculturing (Dangl et al., 1987). Preparation of parsley protoplasts and evacuolated protoplasts was performed as
described by Frohnmeyer et al. (1994)
. Isolation of cytosolic and nuclear
extracts from evacuolated protoplasts was performed as described previously (Harter et al., 1994a
). For technical reasons we used a newly established cell culture for immunostaining. A large part of these cells contained several small vacuoles instead of a big central one reducing the
proportion of disintegrated cells which typically occurs during fixation of
plant cells.
Plasmid Construction
All primers used in this study are shown in Table II. The green fluorescent
protein (GFP) expression cassette was removed from the pBI121-GFP
construct (Haseloff et al., 1997) by HindIII/EcoRI digest and transferred
into pUC18 (GFP-pUC18). Afterwards a 5' BamHI followed by an in-frame SmaI restriction site were introduced into GFP coding region by
PCR using the primers P1 and P2. The resulting plasmid (mAV4) was
used for the construction of COOH-terminal fusions of the CPRF1 and
CPRF2 coding regions with GFP. For this purpose, BamHI-compatible
restriction sites were introduced at the 5' ends and SmaI sites at the 3'
ends into the full-length coding regions of common plant regulatory factors (CPRFs) by PCR using the gene-specific primers P3 to P6. For the
construction of the 5' deletions of CPRF2 the 5' primers P7 (amino acid
[aa]80CPRF2), P8 (aa159CPRF2), and P9 (aa178CPRF2) were used in
combination with primer P6 introducing a BamHI-compatible site and a
start ATG in front of the first codon. The
CPRF2-GFP construct was produced as follows: primers P5 and P10 were used to produce a PCR
product encoding aa 1-177 that contains a BamHI restriction site at its 5'
end and a KpnI site at its 3' end. Primers P11 and P6 were used to polymerize an additional PCR product encoding amino acid 193-403 that contains a KpnI site at its 5' end and a SmaI site at its 3' end. After cutting
with BamHI and KpnI the PCR products were ligated into mAV4. The
NLS-GFP and PHYA-GFP constructs were produced as follows: primer
P12, P13, and P14 and P15, respectively, were used to polymerize 5'
BamHI and 3' SmaI-containing PCR products that encode either the NLS
of CPRF4 (aa306-332; according to Van der Krol and Chua, 1991 and
Varagona et al., 1992
) or phyA. The BamHI and SmaI-cut PCR products
were ligated into mAV4 as described above. After the BamHI-compatible sites all 5' primers also introduce the short sequence stretch 5'-AACA-3'
allowing efficient translation of RNA in plant cells.
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Transient Transformation of Protoplasts by Electroporation
Electroporation of protoplasts was performed in 0.4-cm cuvettes for 5 s at
320 V and 125 µF (
, pulse time = 10 ms) using a Gene Pulser II
(Bio-Rad) at room temperature. For transformation, 1 vol of protoplast
suspension was diluted with 5 vol of electrode buffer (10 mM Tris/Hepes,
pH 7.2, 15 mM MgCl2, 0.5 M sucrose). The suspension was centrifuged for
5 min at 500 g and the floating protoplasts were removed. 5 × 106 protoplasts were mixed with 75 µg of plasmid DNA in a final volume of 800 µl
electrode buffer. After electroporation, protoplasts were diluted in 5 ml
hemagglutinin and 0.4 M sucrose medium, transferred to Petri dishes, and
then used for irradiation experiments.
Immunostaining and Confocal Microscopy
Immunofluorescence labeling of CPRF2 was performed following a protocol described in Petráek et al. (1998)
. Parsley cells were settled on glass
slides that had been coated with Meyer's adhesive (1% wt/vol sodium salicylate in 1:1 vol/vol egg-white/glycerol). Cells were fixed for 30 min in
freshly prepared paraformaldehyde (3.7% wt/vol), solved in MS buffer
(50 mM Pipes, pH 6.9, 1 mM MgSO 4, 5 mM EGTA, 1% vol/vol glycerol,
0.25% vol/vol Triton X-100), and then washed twice for 5 min in MS
buffer. Before antibody incubation, the cells were incubated with a mixture of 1% wt/vol macerozyme (Yakuruto) and 0.1% wt/vol pectolyase
(Yakuruto) in MS buffer for 5 min at 30°C. Afterwards the slides were
blocked with 5% vol/vol goat normal serum () in TBST (20 mM
Tris-HCl, pH 7.3, 150 mM NaCl, 0.25% vol/vol Triton X-100) at 25°C and
incubated with primary antibodies (diluted 1:300 in TBST). The slides
were kept in a moist chamber at 37°C for 1 h, washed three times with TBST, and then incubated with anti mouse-IgG antibody conjugated to
FITC () diluted 1:100 in TBST. The cells were washed three times
with TBST, sealed with a glass slide, and then stored at 4°C until microscopy. Each experiment was performed at least three times.
The fixed cells were visualized under a confocal laser microscope (model DM RBE, Leica), using a two-channel scan with an argon-krypton laser at 488 and 568 nm excitation, a beam splitter at 575 nm, and a 580- and 590-nm emission filter. To eliminate filter leakage, in some experiments, the cells were viewed by subsequent one-channel scans with identical scanning intervals. For this constellation, the FITC signal was analyzed using excitation at 488 nm, a beam splitter at 510 nm, and a 515-nm emission filter. Since our confocal microscope has no 4',6-diamidino-2-phenylindole exciting laser, positioning of nuclei was assayed by transmission microscopy.
Antiserum Production, ELISA, Protein Assay, and Western Blot Analysis
Construction of expression plasmids, expression in Escherichia coli, purification of (His)6-tagged recombinant CPRF1, CPRF2, and CPRF4, and refolding of the bZIP factors as well as the production of antisera in mice
are described in Kircher et al. (1998). For ELISA, 50 µl of purified antigen (concentration: 5 µg/ml) were bound for 2 h to M129 B high-affinity
plate (Dynatech). After washing with PBST (8 mM Na2HPO4, 1.8 mM
KH2PO4, pH 7.4, 0.14 M NaCl, 2.7 mM KCl, 0.05% vol/vol Tween 20) the
plate was blocked for 2 h with PBST containing 3% wt/vol bovine serum albumin. After washing twice with PBST 50 µl of PBST containing
a 1:1,000 dilution of antiserum was added and the plate incubated for 1 h
at room temperature. The plate was washed twice with PBST. Afterwards, 50 µl of PBST containing a 1:1,200 dilution of horseradish peroxidase- labeled secondary antibody was added, and the plate again incubated for
1 h at room temperature. After washing twice with PBST the reaction was
developed for 5 min in orthophenyldiamin/H2O2 solution, stopped by addition of 50 µl of 2 M H2SO4 (Harlow and Lane, 1988
), and then the optical density was quantified at 490 nm. Protein assay and Western blot analysis were performed as described in Harter et al. (1994b)
. The polyclonal
histone 2A/2B antibody was diluted 1:10,000 for use (Harter et al., 1994b
).
The monoclonal tubulin antibody (clone DM 1A; SERVA) was diluted
1:1,000 for use. The secondary anti-rabbit and anti-mouse sera were from
and diluted according to the manufacturer's instruction.
Electrophoretic Mobility Supershift Assay
For electrophoretic mobility supershift assay (EMSSA), a monomeric DNA
probe (5'-AATTCTCCCTTATTCCACGTGGCCATCCGG-3') according to the G-box of the parsley CHS promotor (Schulze-Lefert et al., 1989a,b)
or a monomeric C-box (5'-AATTCTCCCTTATCTGACGTCAGCATCCGG-3') with a core-sequence (underline) according to Izawa et al. (1993)
was used. Preparation of radioactive-labeled probes as well as experimental conditions for EMSSA were performed according to Harter et al.
(1994a)
. For the assay 50 µg of cytoplasmic or 20 µg of nuclear protein in
a total volume of 10 µl was incubated for 10 min on ice with 1 µl of CPRF
antisera or the corresponding preimmunsera in the binding reaction mixture (Harter et al., 1994a
).
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Results |
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Characterization of CPRF-specific Antisera
To determine the intracellular distribution of members of
the CPRF family from parsley we first had to produce
CPRF-specific antisera and to test them for specifity
against the refolded antigens. Therefore, recombinant (r)
His-tagged CPRF1, CPRF2, and CPRF4 were overexpressed in E. coli, purified, and then used for antisera production (Kircher et al., 1998). As shown by ELISA, using
refolded, functionally intact antigen, the antisera raised
against rCPRF1 and rCPRF4 showed cross-reactivity not
only with other CPRFs (Table I) but also with GBFs from
Arabidopsis (data not shown). In contrast, the antiserum
raised against rCPRF2 recognized only its antigen and did
not cross-react with rCPRF1, rCPRF4 (Table I), or the GBFs from Arabidopsis (data not shown). As shown previously (Kircher et al., 1998
), Western blot analysis of
crude extracts from parsley cells demonstrated that the
rCPRF2 antiserum detects a single band of 46 kD representing endogenous CPRF2, whereas the sera produced
against rCPRF1 and rCPRF4 recognize two different proteins of 42 and 44 kD representing endogenous CPRF1
and CPRF4, respectively. These data are in good agreement with our ELISA results indicating that the serum
produced against rCPRF2 is highly specific for its native
antigen, whereas we had to expect a certain amount of
cross-reactivity between CPRF1 and CPRF4 when we use
the antisera raised against rCPRF1 and rCPRF4.
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CPRF1, CPRF2, and CPRF4 Show Differential Intracellular Distribution in Dark-cultivated Parsley Cells
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.
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Taken together, our data strongly indicate that the three
members of the CPRF family exhibit a differential intracellular distribution in dark-kept cells with CPRF1 exclusively localized in the nucleus, CPRF4 found in both compartments, and CPRF2 retained in the cytosol. Furthermore,
the absence from the nuclear compartment suggests that
CPRF2 could be the bZIP factor that is translocated into
the nucleus in response to light as described previously (Harter et al., 1994a). We therefore focused our further analysis on CPRF2 using its highly specific antiserum as a tool.
CPRF2 Is Transported In Vivo into the Nucleus in Response to Light
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).
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Nuclear Import of CPRF2 Is Triggered by phyA and phyB
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).
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The NH2-terminal Domain Is Responsible for the Retention of CPRF2 in Dark-grown Cells
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.
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Taken together, our data indicate that transiently transformed parsley protoplasts are capable of maintaining the
correct intracellular sorting as demonstrated by the phyA-
GFP, NLS-GFP, and CPRF1-GFP controls. Secondly, the
observation that CPRF2-GFP is at least in part retained in
the cytosol in darkness but transported into the nucleus in
response to light indicates that the involved molecular
mechanism is also functionally maintained. Since we never
observed endogenous CPRF2 to be present in the nucleus
of dark-grown cells (see Fig. 3 A), the accumulation of
CPRF2-GFP in the nucleus of dark-kept protoplasts is
most likely caused by overtitration of the cytoplasmic retention machinery due to strong overexpression of the fusion protein. Similar effects were found for GFP fusion
proteins in other systems (Fukuda et al., 1997).
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 (CPRF2-
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
CPRF2-GFP construct is only observed in the nuclear compartment of dark-cultivated parsley protoplasts.
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Discussion |
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Differential Subcellular Localization of the CPRF Family Members
Our data show that CPRF1, CPRF2, and CPRF4 are differentially distributed within dark-cultivated parsley cells. Whereas CPRF1 is exclusively localized in the nucleus and CPRF2 is only found in the cytosol, CPRF4 is present in both cell compartments (Fig. 1). This differential distribution suggests that these three bZIP factors exhibit different functional duties within the regulatory networks of the plant cell. For instance, the constitutive localization of CPRF1 inside the nucleus may indicate that this factor is constitutively active or is posttranscriptionally regulated on the level of DNA-binding or transcriptional activity rather than on intracellular partitioning. In contrast, the more uniform distribution of CPRF4 and the absence of CPRF2 from the nucleus point to inducible redistribution of these bZIP factors as a regulatory mechanism, though the inducing signal for CPRF4 is unknown. However, we could show for CPRF2 that light is the inducing signal for its import from the cytosol into the nucleus. We observed quantitative differences in the extent of nuclear-imported CPRF2 that is most likely due to the different parsley cell systems (evacuolated protoplasts versus cell suspension versus protoplasts). However, the existence of the light- induced nuclear import of CPRF2 was confirmed by following three in vivo assays: (a) in EMSSA, CPRF2 can be detected as supershifted DNA-protein complex in the nuclear extract of irradiated evacuolated parsley protoplasts that is absent in the corresponding extract of dark-kept cell culture (Fig. 2). (b) Immunolocalization technique combined with confocal microscopy showed that, in dark-grown parsley cells, CPRF2 is only found in the cytosol and is imported into the nucleus in response to irradiation (see Fig. 3). (c) Using a transient transformation system we demonstrated that a pool of CPRF2-GFP exists in the cytosol of dark-kept parsley protoplasts that is moved into the nucleus in response to light (see Fig. 4).
Induction of Nuclear Import of CPRF2 Is Light-specific and Regulated by Phytochrome
The induction of nuclear transport of CPRF2 is specific for
light. Neither heat shock treatment nor treatment with the
PMG elicitor from Phytophtera megaspermum had any effect on the intracellular distribution of CPRF2 (Fig. 3, J
and K). This excludes that the nuclear import of CPRF2
directly participates in elicitor- or stress-induced signal
transduction. The irradiation of cells with continuous light
of different wavelengths suggests that the translocation response of CPRF2 is triggered by phytochrome. The strong
import response achieved by irradiation of the cells with
continuous far-red light indicate the involvement of phyA
via a high irradiance response. Although the effects of irradiation with continuous blue and UV-A light can also be
attributed to a phyA action, minor contributions of other
photoreceptor systems besides phy cannot be entirely excluded. To test the participation of phyB we treated the
cells with pulses of red and far-red light to disclose a classical phyB-dependent low fluence response. A red light
pulse induced an almost complete translocation of CPRF2,
whereas a RG9 pulse had no effect. When the RG9 pulse
was given immediately after an inducing red light pulse,
this partially "reverted" the translocation of the factor
(see Fig. 3). A partial reversion of the effect of an inducing
red light pulse by a subsequent RG9 pulse is described for
several phy-dependent photoresponses (Schäfer and Briggs, 1986) and can be explained by a very fast coupling of the
excited photoreceptor to downstream signal transducing
elements (e.g., phosphorylation). Taken together, our data
suggest that phyA and phyB trigger the nuclear import of
the plant bZIP factor CPRF2. Recently, Terzaghi et al.
(1997)
described a blue/UV light-specific cytoplasmic/nuclear
relocalization of a GUS-GBF2 fusion protein in soybean cell
cultures. Since GBF2 from Arabidopsis and CPRF2 from
parsley belong to two different subclasses of bZIP proteins with different characteristics for DNA-binding and homotypic dimerization (for review see Armstrong et al., 1992
;
Meier and Gruissem, 1994
; Meshi and Iwabuchi, 1995
), it is
an attractive hypothesis that different photoreceptors may
mediate wavelength-specific gene expression by the release of different transcription factors from cytosolic retention.
The incomplete "reversion" by RG9 of the translocation
response triggered by a red light pulse indicate that the nuclear import of CPRF2 is rapid and is initiated immediately upon onset of irradiation, whereas the following
RG9 terminates any further accumulation of CPRF2 in
the nucleus. Similar rapid nuclear transport kinetics have
been reported for several nucleophilic transcription factors retained in the cytosol in the absence of the inducing
stimulus (Zabel and Baeuerle, 1990; Carey et al., 1996
;
Ghosh et al., 1998
).
CPRF2 was initially isolated by Southwestern screening
with a DNA probe derived from the parsley CHS promotor (Weisshaar et al., 1991). This motif was functionally
characterized as an element mediating the light responsiveness of the CHS gene (Schulze-Lefert et al., 1989a
,b;
Block et al., 1990
; Weisshaar et al., 1991
; Frohnmeyer et al.,
1992
, 1994
) consisting of two domains (box 1 and box 2) that are necessary and sufficient for light regulation. Box 2 represents a classical G-box (Schulze-Lefert et al., 1989a
,b). CPRF2 was shown to bind to this G-box suggesting that it
is involved in the light regulation of CHS gene expression
(Weisshaar et al., 1991
; Armstrong et al., 1992
). Detailed
physiological analysis in cultured parsley cells and protoplasts demonstrated, however, that the expression of the
CHS gene is regulated by the activation of the UV photoreceptors but not by phytochrome (Ohl et al., 1989
; Frohnmeyer et al., 1992
). Since the nuclear import of CPRF2 is induced by phyA and phyB, it is very unlikely that the
G-box of the CHS promotor is the functional target of
CPRF2 in this system. However, no other genes are
known to be regulated by phytochrome in parsley cell culture and protoplasts. Future research must be directed toward an investigation on which genes are the in vivo targets of CPRF2.
Localization of Retention Domains within the CPRF2 Protein
The demonstration that the disappearance of CPRF2-
GFP from the cytosol of parsley cells in response to light is
due to active transport into the nucleus allowed us to perform a deletion analysis to map the retention domain(s).
The removal of the entire NH2 terminus up to aa 159 abolished cytosolic retention in darkness whereas the
aa80CPRF2-GFP fusion protein was still detectable in the
cytosol. A similar loss of cytosolic localization was found when a short internal stretch between aa 178 and 192 (CPRF2-GFP) was removed from the full-length protein. The deletion analysis suggests that CPRF2 contains
two separable domains for cytoplasmic retention (see Fig.
6 A). In addition, the aa stretch containing both retention
domains can be functionally transferred to heterologous bZIP factors (e.g., CPRF1) not found in the cytoplasmic
compartment of evacuolated parsley protoplasts (Kircher,
S., unpublished data). It is noteworthy that neither CPRF1
nor CPRF4 contain the sequence motifs that could be homologous to the retention domains of CPRF2.
|
As shown for the rel/NF-B and the glucocorticoid receptor family of transcription factors cytoplasmic localization is achieved by retention factors such as I
B and
HSP90, respectively (for review see Muller and Renkawitz, 1991
; Jans and Hübner, 1996
; Ghosh et al., 1998
).
Matsui et al. (1995)
recently identified a possible retention
factor of COP1 named CIP1. However, a detailed sequence
comparison of the CPRF2-specific retention domains with those of the mentioned protein families does not reveal
any sequence relationships. This indicates that the cytosolic compartmentalization of CPRF2 is probably not
achieved by homologues of I
B, HSP90, or CIP1. Similarly, the retention domain of Arabidopsis GBF1 does not
show any relationship to the CPRF2 sequences characterized here (Terzaghi et al., 1997
). However, the NH2-terminal amino acid stretch of CPRF2 between aa 80 and 159 (Fig. 6 A) exhibits a significant sequence homology (degree of 25% identity and of 53% similarity) and especially
structural homology to a well-characterized
-helical domain within the mammalian heat shock factor 2 (Sheldon and Kingston, 1993
). The corresponding domain of this
heat shock factor is discussed to mediate cytoplasmic retention under nonstress conditions by either intramolecular masking of the NLS or by acquiring a hypothetical
NLS-masking protein.
Acidic motifs similar to that of CPRF2 (aa 178-192) are
found in several nucleophilic proteins like, for instance,
Nopp 140 (Meier and Blobel, 1992) and nucleolins from
plants (Bögre et al., 1996
) that shuttle between the cytosol
and the nucleus. The exact molecular mechanisms responsible for regulating NLS activity have remained enigmatic,
though. However, one possibility to modulate NLS activity is based on phosphorylation (Jans and Hübner, 1996
). For example, casein kinase II (CKII) increases the nuclear
import of the SV-40 T antigen through phosphorylation of
a CKII-site 13 amino acids NH2-terminal to the NLS (Rihs
et al., 1991
; Jans and Jans, 1994
). Interestingly, the acidic
domain of CPRF2 contains a CKII phosphorylation site 20 amino acids NH2-terminal to the NLS (see Fig. 6 A) that
might modulate NLS activity upon phosphorylation. Recent data (Wellmer, F., S. Kircher, A. Rügner, H. Frohnmeyer, E. Schäfer, and K. Harter, manuscript submitted
for publication) indicate that phytochrome activation causes
rapid phosphorylation of CPRF2 in vivo. This phosphorylation does not interfere with the DNA-binding activity of
CPRF2 and might therefore be involved in the regulation
of nuclear import.
The results presented in this work can be summarized in
two alternative models of phytochrome-regulated nuclear
import of CPRF2 (Fig. 6 B): CPRF2 is retarded in the cytosol of dark-grown parsley cells via retention domains 1 and 2. Irradiation photoconverts phyA and phyB from the
inactive Pr to the active Pfr form that in turn induces the
phosphorylation of the CKII-site within the acidic retention domain 2 of CPRF2. This modification either abolishes intracellular masking of the NLS (Fig. 6 B, I) or results
in the release of CPRF2 from a cytoplasmic anchoring protein (Fig. 6 B, II). In both cases, accession of the NLS for
the components of the nuclear import machinery results in
the nuclear import of CPRF2. Inside the nucleus CPRF2
binds to promotors of certain light-regulated genes. The
specificity to bind to a certain promotor element could be
achieved by homo- or heterotypic protein-protein interactions of CPRF2 with members of the bZIP or of other transcription factor families (see examples in Vicente-Carbajosa
et al., 1997; Büttner and Singh, 1997). One possible candidate for homotypic interaction could be the parsley homologue of the bZIP factor HY5 that has not been shown to
have transcriptional activity and likely needs a transcriptionally active bZIP as a partner for function (Ang et al.,
1998
). HY5 was shown to be sequestered and kept inactive
by interaction with the nuclear, general repressor of photomorphogenesis COP1 in dark-grown plants and released
upon irradiation (Ang et al., 1998
). The demonstration of a
potential CPRF2/HY5 heterodimerization would point to
how a photoreceptor-specific signaling pathway could interconnect with the general transcriptional repression mechanism consisting of the COP1 and HY5 gene products.
![]() |
Footnotes |
---|
Address correspondence to K. Harter, Institut für Biologie II/Botanik, Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany. Tel.: (49) 761-2032686. Fax: (49) 761-2032629. E-mail: harterkl{at}ruf.uni-freiburg.de
Received for publication 1 September 1998 and in revised form 13 November 1998.
S. Kircher was supported by the Konrad Adenauer Stiftung and F. Wellmer was supported by the Graduiertenkolleg "Molekulare Mechanismen pflanzlicher Differenzierung." The work was supported by grants to
E. Schäfer from the Deutsche Forschungsgemeinschaft (SFB 388) and
from the Human Frontier Science Program.
S. Kircher and F. Wellmer contributed equally to this work.
We are grateful to H. Frohnmeyer for methodical support, I. Abel (both from University of Freiburg, Freiburg, Germany) for technical assistance, and J. Haseloff (University of Cambridge, Cambridge, UK) for the GFP plasmid.
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Abbreviations used in this paper |
---|
aa, amino acid; bZIP, basic-region leucine-zipper domain; CHS, chalcon synthase; CKII, casein kinase II; CPRF, common plant regulatory factor; EMSSA, electrophoretic mobility supershift assay; GBF, G-box-binding factor; GFP, green fluorescent protein; NLS, nuclear localization sequence; Pfr, far-red light absorbing form of phytochrome; phy, phytochrome; Pr, red light-absorbing form of phytochrome; r, recombinant; RG9, long wavelength far-red light.
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