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Scientific Correspondence
Single-Stranded DNA-Binding Protein Whirly1 in Barley
Leaves Is Located in Plastids and the Nucleus of the
Same Cell1[W]
Evelyn Grabowski, Ying Miao, Maria Mulisch, and Karin Krupinska*
Institute of Botany (E.G., Y.M., K.K.), and Central Microscopy, Center of Biology (M.M., K.K.),
Christian-Albrechts-University of Kiel, 24098 Kiel, Germany
This article concerns the intriguing protein Whirly1
(Why1) that belongs to a small family of singlestranded DNA-binding proteins and has been described to have functions in the nucleus (Desveaux
et al., 2002; Yoo et al., 2007). In contrast, in vitro import
assays with isolated organelles and transient expression of a fusion construct with the gfp gene revealed
that the protein is translocated into plastids (Krause
et al., 2005). In this article, specific antibodies directed
toward the Why1 protein of barley (Hordeum vulgare)
were used to analyze the subcellular location of the
native protein.
The single-stranded DNA-binding factor Why1 belongs to a small protein family found mainly in land
plants. Although most plant species have two Why
proteins, Arabidopsis (Arabidopsis thaliana) has three of
them (Desveaux et al., 2005; Krause et al., 2005). The
three proteins share the putative DNA-binding domain
KGKAAL, which is highly conserved in all Why proteins identified so far (Desveaux et al., 2005).
The first member of the Why family to be identified
was p24, which was later renamed StWhy1. StWhy1
was described as the DNA-binding component of
the transcriptional activator PBF-2, which mediates
elicitor-induced gene expression of the pathogenesisrelated gene PR-10a of potato (Solanum tuberosum;
Desveaux et al., 2000). Unlike other transcriptional activators that have been identified, StWhy1 was shown
to bind to single-stranded DNA. Electrophoretic mobility shift assays indicated that StWhy1 binds to the
inverted repeat sequence of the elicitor response element (ERE) of the PR10a gene of potato (Desveaux et al.,
2000). Crystallographic analyses revealed that StWhy1
forms homotetrameres that have high preference for
1
This work was supported by the German Research Foundation
(Deutsche Forschungsgemeinschaft grant no. Kr1350/9).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Karin Krupinska ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.122796
1800
single-stranded DNA. It has been proposed accordingly that the protein may bind to melted promoter regions and may thus modulate transcription
(Desveaux et al., 2002).
Recent results obtained with Arabidopsis showed
that Why1 also binds to single-stranded telomeric
DNA and appears to modulate telomere length homeostasis by inhibiting the action of telomerase (Yoo
et al., 2007).
Though the Why1 protein fulfills different functions
in the nucleus, computer-based analyses predicted its
targeting to plastids (Desveaux et al., 2005; Krause
et al., 2005; Schwacke et al., 2007). Indeed, AtWhy1 is
imported into plastids, as demonstrated by in vitro
import assays with isolated organelles and by transient expression of a fusion construct of the AtWhy1
gene and the gfp gene (Krause et al., 2005). Although
AtWhy3 was shown to be targeted to plastids, too,
AtWhy2 was shown to be targeted to mitochondria
(Krause et al., 2005). Recently, it has been shown that
AtWhy2 is associated with mitochondrial DNA and
causes the development of dysfunctional mitochondria when it is overexpressed (Marechal et al., 2008).
To examine whether the localization of the Why1
protein may change during chloroplast development,
primary foliage leaves of barley were used to study its
subcellular localization. Due to their basal meristem,
leaves of barley contain proplastids at the base and
gradually advancing stages of chloroplast development up to the leaf tip (Mullet, 1988; Krupinska, 1992).
A complementary DNA (cDNA) of the barley Why1
gene (HvWhy1) was gained from an EST clone with the
GenBank accession number BF6136. It encodes almost
the complete mature HvWhy1 protein as shown by
comparison with the corresponding sequences from
potato (St AF233342) and Arabidopsis (At 1g14410;
Fig. 1). The HvWhy1 sequence has the conserved
KGKAAL domain, a putative nuclear localization signal and a putative autoregulatory domain (Fig. 1). The
recombinant protein obtained by overexpression of
the cDNA in Escherichia coli was found to bind to the
ERE GTCAAAA as well as to the characteristic heptanucleotide TTTAGGG of plant telomeres (data not
shown).
To investigate the subcellular localization of the
native Why1, three antibodies were raised. Two were
Plant Physiology, August 2008, Vol. 147, pp. 1800–1804, www.plantphysiol.org Ó 2008 American Society of Plant Biologists
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Whirly1 Is Dually Located in Plastids and Nuclei
The specificity of the immunoreactions was further
tested by comparison of the immunodetection pictures
before and after preincubation of the antibody with
either oligopeptide2 or bovine serum albumin as specific or unspecific competitor, respectively. In both
cases total foliar protein extracts as well as protein
extracts from purified chloroplasts and nuclei were
electrophoretically separated and blotted. When the
oligopeptide was added, the signal of the 25-kD
Figure 1. The amino acid sequence of Why1. The HvWhy1 sequence is
compared to the sequences of StWhy1 and AtWhy1. An alignment of
the mature protein sequence of Why1 from barley with the complete
protein sequences of Why1 from potato and Arabidopsis was generated
using the T-Coffee program (www.bioinformatics.nl/tools/t_coffee.
html). Hv, Barley; At, Arabidopsis; St, potato; Cons, consensus sequence *; PTP, chloroP-predicted target peptide; PTD, putative transactivation domain (underlined); pNLS, potential nuclear localization
signal; PAD, putative autoregulatory domain. The arrowheads indicate
the position of the chloroP-predicted target peptide cleavage sites. Why
domain, conserved ssDNA-binding domain is shaded in gray. Oligopeptides for antibodies in barley are boxed.
raised toward different oligopeptides picked from the
HvWhy1 amino acid sequence (sequences of both
peptides are indicated in Fig. 1) and one antibody
directed toward the recombinant HvWhy1 protein
was obtained by overexpression of the cDNA in E.
coli. All antibodies detected a protein with a molecular
mass of about 25 kD in leaf extracts at different stages
of development (data not shown). The antibody directed toward peptide 2 (a-HvWhy1-P2) was suited
best for immunoblot analysis and was used for immunological analysis of protein extracts prepared
from isolated plastids and nuclei. To examine putative
development-related changes in the distribution of the
protein, plastids and nuclei were prepared from segments I, II, and III (Fig. 2A) of the barley primary
foliage leaves. Immunoblot analysis showed that the
Why1 protein is located in plastids as well as in nuclei
(Fig. 2B). The protein was detectable in plastids from
all three leaf segments. During chloroplast development, the level increased transiently being highest in
leaf segment II (Fig. 2B). Furthermore, the analysis
showed that the protein has the same molecular mass
in both plastids and in nuclei (Fig. 2B). To control the
purity of isolated plastid and nuclei fractions immunological reactions were performed with antibodies
directed toward apoprotein A of cytochrome b559 and
histone H2B, respectively.
Figure 2. Immunological detection of HvWhy1 in protein fractions
derived from plastids and nuclei, respectively. A, Scheme depicting the
segments I, II, and III excised from barley primary foliage leaves
collected at 5 or 7 d after sowing. B, Plastidal and nuclear proteins were
prepared from the same three leaf segments (I, II, and III). The blot was
immunodecorated with the a-HvWhy1-P2 antibody (Fig. 1), followed
by immunodecoration with an antibody specific for the cytochrome
b559 apoprotein A (9.5 kD) and an antibody specific for histone H2B
(14 kD), respectively. C, Immunoblot analysis of plastid, stroma, and
thylakoid membrane proteins. To show equal loading, a part of the
Coomassie Blue-stained gel is shown.
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Grabowski et al.
HvWhy1 protein was not detectable, whereas it was
still detectable when the same amount of bovine
serum albumin was used instead of the oligopeptide
(Supplemental Fig. S1). To gain insight into the distribution of Why1 within the plastid, stroma and membrane fractions were prepared from chloroplasts. By
immunoblot analysis the major part of Why1 was
detected in the stroma while a minor part was found in
the membrane fraction (Fig. 2C).
For analysis of the subcellular localization by immunohistochemical methods, segments excised from
segment II from 5-d-old primary foliage leaves and
from mature flag leaves of barley plants, respectively,
were fixed and embedded in LR White resin. Semithin
sections were incubated with an affinity-purified
antibody raised against oligopeptide1 (Fig. 1) and a
secondary gold-labeled antibody. After silver enhancement and 4#-6-diamidino-2-phenylindole (DAPI)staining, they were analyzed by light microscopy
(Fig. 3, A and B). The label was observed to be
associated with the nucleus that is clearly visible by
the DAPI stain in one of the cells (Fig. 3B). Furthermore, structures within the cytoplasm of the same cell
and of neighboring cells are gold labeled (Fig. 3B).
These structures are apparently chloroplasts, as clearly
identified by transmission electron microscopy (Fig.
3C). Immunogold labeling of HvWhy1 was analyzed
with (Fig. 3C) and without silver enhancement (Fig. 3,
D and E; Supplemental Fig. S2) by transmission electron microscopy of ultrathin sections. In the same cell,
chloroplasts and heterochromatin areas of the nucleus
are specifically labeled by gold particles (Fig. 3, D and
E; Supplemental Fig. S2). Almost no gold particles
were detected in mitochondria, cytoplasm, and the cell
wall of these specimens (Supplemental Fig. S2).
Previously it has been shown that the AtWhy1-GFP
fusion protein was exclusively targeted to the plastids
(Krause et al., 2005). Unexpectedly, the transient transformation assays did not give any evidence for localization in the nucleus. This could be due to the high
molecular mass of the fusion protein (68 kD). When
the sequence encoding the plastid target peptide was
removed from the construct, indeed GFP fluorescence
was detected in the cytoplasm and in the nucleus after
biolistic transformation of onion (Allium cepa) epidermal
cells (data not shown).
To examine whether HvWhy1 forms homooligomers when imported into the nucleus as postulated by
Desveaux et al. (2002), bimolecular fluorescence complementation assays were performed by transient
transformation of onion epidermal cells. For this purpose the HvWhy1 sequence lacking the PTP (for
chloroP-predicted target peptide) sequence was fused
once to the N-terminal part and once to the C-terminal
part of YFP (for yellow fluorescent protein), respectively. Both gene fusions were put under control of the
35S CaMV promoter. When the two constructs were
cotransformed into onion epidermal cells, fluorescence was indeed clearly detectable in the nucleus
indicating that the protein here forms homooligomers
(Fig. 4, B and C). Moreover, this result shows that the
fusion proteins with two parts of YFP having molec-
Figure 3. Immunohistochemical detection of
Why1 in plastids and nuclei. Sections from segments of barley flag leaves (A–C) and from leaf
segment II (D and E) were immunodecorated with
the a-HvWhy1-P1 antibody. Labeling was obtained in nuclei (n) and plastids (p). A, Semithin
section after immunogold labeling and silver enhancement as seen by light microscopy; scale bar
is 10 mm. B, Fluorescence micrograph after DAPI
staining of the same specimen as shown in A;
scale bar is 10 mm. C, Electron micrograph
depicting a section of a chloroplast after immunogold labeling silver enhancement; scale bar is
1 mm. D and E, Electron micrographs depicting
sections of a nucleus (n) and a chloroplast (p),
respectively, after immunogold labeling; scale
bars are 1 mm (D) and 0.1 mm (E).
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Whirly1 Is Dually Located in Plastids and Nuclei
Figure 4. HvWhy1 forms homooligomers in the nucleus. Onion epidermal
cells were transiently transformed with
constructs expressing HvWhy1 and
AtWhy1 without the PTP as well as with
the full-length AtWhy1. Constructs were
fused to either c-myc-YFPn173 or HAYFPc155 and vice versa. The AtWhy1
fused to full-length GFP and the empty
vectors were used as controls. All constructs were under the control of the
35S promoter. Fluorescence images are
shown above the bright field images
that are shown on the bottom row. A,
AtWhy1-YFPn173 1 AtWhy1-YFPc155. B,
DPTP-AtWhy1-YFPn173 1 DPTP-AtWhy1YFPc155. C, DPTP-HvWhy1-YFPn173 1
DPTP-HvWhy1-YFPc155. D, AtWhy1GFP. E, AtWhy1-YFPn173 1 empty vector. Scale bars are 200 mm (A and E) and
100 mm (B–D).
ular masses of 48 and 42 kD, respectively, are small
enough to enter the nucleus.
Because the constructs used for these experiments
lacked the PTP, no fluorescence was detected in plastids. When the full-length AtWhy1 was, however,
fused to full-length GFP, fluorescence was detected
in plastids as described previously (Krause et al., 2005;
Fig. 4D). When the full-length sequence encoding
AtWhy1 was fused instead with the N-terminal part
and the C-terminal part of YFP, respectively, no complementation of the YFP fluorescence was achieved
(Fig. 4A). When the full-length AtWhy1 was fused
with full-length GFP and CFP, both fluorescence emissions were detected in plastids, respectively, but no
overlay of fluorescence signals was observed (data not
shown). This also suggests that Why1 does not form
homooligomers when it is located in plastids.
To investigate whether under conditions not showing a fluorescence complementation (Fig. 4, A and E)
the constructs have been expressed in the cells, westernblot analysis with a GFP-specific antibody were performed. Immunoreactions confirmed that the N-terminal
as well as the C-terminal construct with HvWhy1
(Supplemental Fig. S3) were both expressed in the
cells.
Homooligomerization in the nucleus, as here shown
by bimolecular fluorescence complementation, is in
accordance with the results of the structural analysis
(Desveaux et al., 2002) and the proposed function of
Why1 as single-stranded DNA-binding factor involved in regulation of transcription (Desveaux et al.,
2004, 2005). Nevertheless, Why1 has been detected in
the proteome of the transcriptionally active chromosome fraction prepared from chloroplasts of Arabidopsis (Pfalz et al., 2005). This finding is in accordance
with the immunological detection of HvWhy1 in the
membrane fraction (Fig. 2C) and by the association
of gold particles with thylakoid membranes (Fig. 3C;
Supplemental Fig. S2). The transcriptionally active
chromosome fraction contains proteins bound to the
ptDNA organized in nucleoids being associated to the
thylakoid membrane of chloroplasts (Sato, 2001). Its
protein composition is rather complex (Krause and
Krupinska, 2000; Pfalz et al., 2005) and in addition to
the subunits of the RNA polymerases contains proteins involved in posttranscriptional processes of plastid gene expression (Krause, 1999). Why1 in plastids
could be involved in these processes.
To achieve a coordination between the nucleus and
the organelles and vice versa, anterograde and retrograde control mechanisms have developed (Beck,
2005; Nott et al., 2006). Intermediates of the tetrapyrrole biosynthesis, reactive oxygen species, plastid gene
expression products, and changes in the redox state of
the photosynthetic electron transport chain have been
proposed as plastid signals. Putative signal-transducing
components involved in plastid-to-nucleus signaling
pathways such as the GUN proteins (Susek et al., 1993)
and the Executer1 protein (Wagner et al., 2004) were
identified by genetic studies. With regard to its dual
localization in the nucleus and the plastids, Why1 is an
excellent candidate for transducing signals between
the plastids and the nucleus. Why1 as a DNA-binding
protein in the nucleus as well as in plastids might
contribute to the coordination between plastid gene
expression and transcription in the nucleus by a
still-unknown mechanism. The mechanism of Why1
distribution in the cell with regard to its putative
signal-transducing function remains to be determined.
Insight into the biological significance of its dual location is expected from investigations on transgenic
plants with altered levels of Why1 in the nucleus
and in plastids, respectively.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number BF6136.
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Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Test of specificity of the a-HvWhy1-P2 antibody
used for immunoblot analysis shown in Figure 1.
Supplemental Figure S2. Overview electron micrograph showing the
immunogold labeling of HvWhy1 in chloroplasts and in the nucleus.
Supplemental Figure S3. Immunoblot analysis of extracts from onion
tissue used for transient transformation assays described in Figure 3.
Supplemental Materials and Methods S1. A supplemental ‘‘Materials
and Methods’’ section.
ACKNOWLEDGMENTS
We thank Anke Schäfer and Marita Beese for technical assistance. We
acknowledge Kirsten Krause (University of Tromsö, Norway) for constructive comments on the manuscript.
Received May 12, 2008; accepted June 5, 2008; published August 6, 2008.
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