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Plant Physiology 147:1800-1804 (2008)
© 2008 American Society of Plant Biologists

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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 single-stranded DNA-binding proteins and has been described to have functions in the nucleus (Desveaux et al., 2002Go; Yoo et al., 2007Go). 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., 2005Go). 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., 2005Go; Krause et al., 2005Go). The three proteins share the putative DNA-binding domain KGKAAL, which is highly conserved in all Why proteins identified so far (Desveaux et al., 2005Go).

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 pathogenesis-related gene PR-10a of potato (Solanum tuberosum; Desveaux et al., 2000Go). 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., 2000Go). Crystallographic analyses revealed that StWhy1 forms homotetrameres that have high preference for 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., 2002Go).

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., 2007Go).

Though the Why1 protein fulfills different functions in the nucleus, computer-based analyses predicted its targeting to plastids (Desveaux et al., 2005Go; Krause et al., 2005Go; Schwacke et al., 2007Go). 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., 2005Go). Although AtWhy3 was shown to be targeted to plastids, too, AtWhy2 was shown to be targeted to mitochondria (Krause et al., 2005Go). 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., 2008Go).

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, 1988Go; Krupinska, 1992Go). 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).


Figure 1
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  		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.

 
To investigate the subcellular localization of the native Why1, three antibodies were raised. Two were 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 ({alpha}-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
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  		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 {alpha}-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.

 
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 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).


Figure 3
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  		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 {alpha}-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 µm. B, Fluorescence micrograph after DAPI staining of the same specimen as shown in A; scale bar is 10 µm. C, Electron micrograph depicting a section of a chloroplast after immunogold labeling silver enhancement; scale bar is 1 µm. D and E, Electron micrographs depicting sections of a nucleus (n) and a chloroplast (p), respectively, after immunogold labeling; scale bars are 1 µm (D) and 0.1 µm (E).

 
Previously it has been shown that the AtWhy1-GFP fusion protein was exclusively targeted to the plastids (Krause et al., 2005Go). 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)Go, 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 molecular masses of 48 and 42 kD, respectively, are small enough to enter the nucleus.


Figure 4
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  		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 HA-YFPc155 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 + AtWhy1-YFPc155. B, {Delta}PTP-AtWhy1-YFPn173 + {Delta}PTP-AtWhy1-YFPc155. C, {Delta}PTP-HvWhy1-YFPn173 + {Delta}PTP-HvWhy1-YFPc155. D, AtWhy1-GFP. E, AtWhy1-YFPn173 + empty vector. Scale bars are 200 µm (A and E) and 100 µm (B–D).

 
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., 2005Go; 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, western-blot 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., 2002Go) and the proposed function of Why1 as single-stranded DNA-binding factor involved in regulation of transcription (Desveaux et al., 2004Go, 2005Go). Nevertheless, Why1 has been detected in the proteome of the transcriptionally active chromosome fraction prepared from chloroplasts of Arabidopsis (Pfalz et al., 2005Go). 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, 2001Go). Its protein composition is rather complex (Krause and Krupinska, 2000Go; Pfalz et al., 2005Go) and in addition to the subunits of the RNA polymerases contains proteins involved in posttranscriptional processes of plastid gene expression (Krause, 1999Go). 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, 2005Go; Nott et al., 2006Go). 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., 1993Go) and the Executer1 protein (Wagner et al., 2004Go) 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.


Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Test of specificity of the {alpha}-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.


   FOOTNOTES
 
1 This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft grant no. Kr1350/9). Back

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 (kkrupinska{at}bot.uni-kiel.de).

[W] The online version of this article contains Web-only data. Back

www.plantphysiol.org/cgi/doi/10.1104/pp.108.122796

* Corresponding author; e-mail kkrupinska{at}bot.uni-kiel.de.


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