Maize calreticulin localizes preferentially to plasmodesmata in root apex

The Plant Journal (1999) 19(4), 481±488

SHORT COMMUNICATION

Maize calreticulin localizes preferentially to plasmodesmata in root apex FrantisÏek BalusÏka1,3,*, Jozef SÏamaj1,4, Richard Napier2 and Dieter Volkmann1 1 Institute of Botany, Rheinische Friedrich-Wilhelms University Bonn, Department of Plant Cell Biology, Kirschallee 1, D-53115 Bonn, Germany, 2 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK, 3 Institute of Botany, Slovak Academy of Sciences, DuÂbravska cesta 14, SK-84223, Bratislava, Slovakia, and 4 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-95007 Nitra, Slovakia Summary Using a polyclonal antibody raised against calreticulin puri®ed and sequenced from maize, we performed an immunocytological study to characterize putative domain-speci®c subcellular distributions of endoplasmic reticulum (ER)-resident calreticulin in meristematic cells of maize root tip. At the light microscopy level, calreticulin was immunolocalized preferentially at cellular peripheries, in addition to nuclear envelopes and cytoplasmic structures. Punctate labelling at the longitudinal walls and continuous labelling at the transverse walls was characteristic. Immunogold electron microscopy revealed plasmodesmata as the most prominently labelled cell periphery structure. In order to further probe the ER-domain-speci®c distribution of maize calreticulin at plasmodesmata, root apices were exposed to mannitol-induced osmotic stress. Plasmolysis was associated with prominent accumulations of calreticulin at callose-enriched plasmodesmata and pit ®elds while the contracting protoplasts were depleted of calreticulin. In contrast, other ER-resident proteins recognized by HDEL peptide and BiP antibodies localized exclusively to contracted protoplasts. This ®nding reveals that, in plasmolysed cells, calreticulin enriched ER domains at plasmodesmata and pit ®elds are depleted of other ERresident proteins containing the HDEL retention peptide.

Received 11 January 1999; revised 10 June 1999; accepted 17 June 1999. *For correspondence (fax +49 2287 32677; e-mail [email protected]).

ã 1999 Blackwell Science Ltd

Introduction Calreticulin is a highly conserved Ca2+-sequestering protein which typically resides within the lumen of endoplasmic reticulum (ER) due to its ef®cient ER retention via KDEL and HDEL peptides (Napier et al., 1995; Pelham, 1989). The wide distribution of calreticulin throughout the multi-cellular eukaryotic kingdom, its constitutive presence in every cell type investigated and its highly conserved nature, all strongly indicate that calreticulin exerts important functions. Calreticulin was characterized as a protein essential for cardiac development, and disruption of its gene resulted in embryonic mice lethality (Mesaeli et al., 1999). Besides its well-known roles in calcium signalling and storage (e.g. Camacho and Lechleiter, 1995; Mery et al., 1996), calreticulin has recently been accepted to act as a chaperone in the quality control of glycoproteins passing through the ER (e.g. Zhang et al., 1998; for a review see Helenius et al., 1997). Furthermore, other less well-understood roles of calreticulin have also been suggested (Krause and Michalak, 1997). For instance, calreticulin binds directly to and regulates integrins at the plasma membrane (Coppolino et al., 1995, 1997; Dedhar, 1994; Rojiani et al., 1991). Therefore, we must seriously consider the existence of extra-ER calreticulin (reviewed by Crofts and Denecke, 1998). The ®rst indications that plants contain calreticulin-like proteins came from puri®ed Ca2+-binding proteins of spinach leaves which showed similarities to calreticulin sequences (Menegazzi et al., 1993). Subsequently, Chen et al. (1994) identi®ed cDNA clones encoding barley calreticulin, and Navazio et al. (1995) con®rmed the calreticulin identity for Ca2+-binding proteins from spinach leaves. Shortly afterwards, calreticulin homologues were puri®ed and sequenced from maize (Napier et al., 1995) and tobacco (Denecke et al., 1995), while calreticulin was reported to represent the major Ca2+binding protein in pea (Hassan et al., 1995). More recently, abundant accumulations of calreticulin were found in ¯oral tissues of Arabidopsis (Nelson et al., 1997) as well as in the primitive gymnosperm Ginkgo biloba (Nardi et al., 1998). Plant calreticulin is phosphorylated by protein kinase CK2 whereas animal calreticulin does not show this property (Baldan et al., 1996). In pathogen-induced elicitor signalling, calreticulin was identi®ed as one of the oligogalacturonide-modulated phosphoproteins in 481

482 FrantisÏek BalusÏka et al. tobacco (Droillard et al., 1997). Immuno¯uorescence studies on the subcellular localization of calreticulin in plants were performed only on cells isolated from the plant body. All of these studies reported about perinuclear ER networks continuously extending throughout the cytoplasm (Denecke et al., 1995; Napier et al., 1995). The possibility that a unique population of ER elements or their subcompartments (reviewed by Staehelin, 1997) could be enriched with calcium and calcium-binding proteins in plant tissues, as reported for some mammalian and animal cell types (Golovina and Blaustein, 1997; Meldolesi and Pozzan, 1998; Montero et al., 1997; Villa et al., 1991), has been left unexplored for higher plants. Here we have accomplished immuno¯uorescence and immunogold electron miscroscopy analysis of intact maize root apices. We have found that calreticulin does show ERdomain-speci®c distributions in cells of maize root apices. Preferential localization to plasmodesmata highlights calreticulin as the ®rst ER-resident protein identi®ed within

these gateable cell-to-cell communication structures that provide higher plants with their unique supracellular nature (Kragler et al., 1998; Lucas et al., 1993). Calreticulin might participate in structural and functional properties of walled plant cells plasmodesmata via its calcium buffering and signalling properties. Results Western blot analysis of calreticulin and HDEL peptide Immunoblotting using antibodies raised against maize calreticulin and HDEL peptide showed high speci®cities of these antibodies in maize roots (Figure 1a,b). The calreticulin and HDEL antibodies recognized single bands at about 50 kDa (calreticulin, Figure 1a) and at about 70 kDa (the most abundant HDEL protein) in soluble protein fractions. The absence of cross-reactivity with calreticulin, at the speci®c concentrations used in our study, makes the HDEL peptide antibody a valuable tool at the immuno¯uorescence level. Analysis of calreticulin and HDEL distributions by immuno¯uorescence

Figure 1. Western blots. Immunoblots of soluble protein fractions of maize roots (cv. Alarik) using calreticulin (the second lane in (a)) and HDEL-peptide (the ®rst lane in (b)) antibodies. Single bands are localized at about 50 kDa for calreticulin and approximately 70 kDa for HDEL-peptide antibodies. The ®rst lane in (a) represents calreticulin pre-immune serum. The third lane in (a) and the second lane in (b) represent controls in which the ®rst antibody was omitted.

The most characteristic feature of root tip cells was the preferential accumulation of calreticulin at cellular peripheries (Figure 2a,b). The only exceptions were root cap and epidermal cells (not shown) which showed preferentially calreticulin-positive ER networks similar to those visualized with the HDEL antibody (Figure 2h). Calreticulin was clearly enriched along the cell peripheries, even in the developmentally youngest root body cells just behind the quiescent centre, while a faint calreticulin-positive labelling was recognizable as spotlike structures in the cytoplasm and at the nuclear envelopes of all meristematic cells (Figure 2a). Typically, the cell periphery-associated calreticulin was seen along the entire transverse walls. In contrast, the longitudinal walls exhibited rather punctate labelling patterns. When

Figure 2. Immuno¯uorescence microscopy. (a) Calreticulin signal associates with cellular peripheries in the form of continuous labelling along their transverse walls (stars) and punctate labelling which decorates the longitudinal walls (asterisks). In addition, faint ¯uorescence of nuclear envelopes is apparent in all cells of the apical part of meristem (arrowheads). (b) If the plasma membrane with associated cortical cytoplasm is included within paradermal sections, then calreticulin-positive pit ®elds (star) are unequivocally recognizable. (c) Pre-immune serum does not produce any signal as shown here on the example of elongating inner cortex cells (compare with (d)). (d) In the elongation region, pit ®elds (arrowheads) and peripheries of inner cortex cells are strongly labelled. (e) Calreticulin remains associated with pit ®elds even in plasmolysed cortex cells, while contracting protoplasts (asterisks) are depleted of calreticulin. (f,g) Double labelling of callose (f) and calreticulin (g) in plasmolysing cells of the root cortex reveals their tight co-localizations at plasmodesmata and pit ®elds (arrows). (h) In control roots, HDEL proteins are distributed throughout the cytoplasm as shown on the example of epidermis (e) cells. (i,j) Plasmolysing epidermis (e) and underlying outer cortex cells retain their HDEL (i) and BiP (j) labellings exclusively within contracting protoplasts. Bar = 7 mm.

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 481±488

Maize calreticulin localizes to plasmodesmata in root apex 483 the plasma membrane with associated peripheral cytoplasm was included within a paradermal section

Calreticulin remains enriched at pit ®elds even in plasmolysed cells

through root cells, pit ®elds of longitudinal walls were clearly recognizable (Figure 2b,d).

The calreticulin was associated preferentially with plasmodesmata and pit ®elds even in plasmolysed cells

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 481±488

484 FrantisÏek BalusÏka et al. with contracted protoplasts (Figure 2e). As revealed by double immunolocalization, calreticulin co-localized tightly with callose at plasmodesmata and pit ®elds of plasmolysed cells (Figure 2f,g). In sharp contrast to the calreticulin ¯uorescence, the HDEL (Figure 2i) and BiP (Figure 2j) signal localized exclusively to contracting protoplasts in plasmolysed cells. Controls Importantly, use of appropriate pre-immune serum for the calreticulin antibody did not result in any labelling (Figure 2c). Moreover, as already mentioned above, we used a monoclonal antibody speci®c against HDEL proteins in order to con®rm the speci®c enrichment of calreticulin within plasmodesmata-associated ER domains. The BiP signal, similarly like HDEL ¯uorescence, showed contrasting behaviour and remained within contracting protoplasts. As a further positive control, we used the same procedure to immunolocalize other antigens in maize roots which resulted in contrasting labelling patterns (data not shown, but see BalusÏka et al., 1997 for actin, Mews et al., 1997 for cyclins, SÏamaj et al., 1998 for arabinogalactan protein epitopes, and Jahn et al., 1998 for plasma membrane H+-ATPase). Immunogold localization of calreticulin within plasmodesmata In order to specify more precisely the subcellular localization of the maize calreticulin, we employed the immunogold electron microscopy technique. Con®rming our light microscopy observations, plasmodesmata were labelled in most cells of the root tip, irrespective of whether they occurred singly or clustered into pit ®elds (Figure 3a±c). In addition, elements of cortical ER were also often labelled with the calreticulin antibody (not shown). Sections treated with the pre-immune serum lacked gold labelling of any subcellular structures (Figure 3d). Discussion In cells of the maize root tip, we have found preferential localization of ER-resident calreticulin within cortical ER elements associated with plasmodesmata. This feature also persists in plasmolysing cells where most of the cytoplasm retracts from the cell periphery and is organized in the form of contracted protoplasts. Immunolocalization using a monoclonal anti-HDEL antibody (Napier et al., 1992) revealed general cytoplasmic ¯uorescence, thus demonstrating the distribution of proteins containing this ER-targeting sequence throughout the endomembrane system. While calreticulin also has an HDEL sequence, its preferential distribution within a subset of ER domains

indicates that there must be a mechanism that selectively enriches calreticulin within cell periphery-associated domains of the plant ER (reviewed by Staehelin, 1997). Recently, calreticulin from Nicotiana plumbaginifolia was localized to protoplast surfaces under the control of auxin action (Borisjuk et al., 1998). The cell periphery association of calreticulin was also reported for animal cells where it acts either as a cell surface receptor with lectin properties (White et al., 1995) or as a cell adhesion factor putatively due to its binding to integrin (Coppolino et al., 1995; Coppolino et al., 1997; Dedhar, 1994; Rojiani et al., 1991). Calreticulin localizes preferentially to cortical ER associated with plasmodesmata The ER represents an extremely complex and pleiomorphic organelle of plant cells, rearranging continuously throughout the cytoplasm (e.g. Lichtscheidl and Weiss, 1988; Quader, 1990). Whereas the `internal ER' is highly dynamic and tends to form tubular cisternae, the cortical ER consists of large lamellar sheets which tightly underlie the plasma membrane and change their form and arrangements only slowly (reviewed by Lichtscheidl and Hepler, 1996). Of prime signi®cance in this respect is our original observation that calreticulin is speci®cally enriched at cellular peripheries in association with the cortical ER of plasmodesmata. This particular ®nding supports numerous earlier suggestions that plant-speci®c cortical ER plays a pivotal role in various responses of plant cells to their environment (reviewed by Hepler et al., 1990). ER elements closely underlying the plasma membrane represent the most critical domain with respect to immediate calcium events induced by external stimuli (Hepler et al., 1990; Lichtscheidl and Hepler, 1996). Although another ER-based calcium-binding protein, calsequesterin, was found to be enriched in specialized ER domains of animal cells (Villa et al., 1991), this feature was not reported for calreticulin until now (for review see Meldolesi and Pozzan, 1998). Therefore, our data are original with respect to the speci®c localization of calreticulin to specialized ER domains in eukaryotic cells. In higher plants, plasmodesmata harbour a distinct subcompartment of the cortical ER domain (Staehelin, 1997), providing the structural basis for membraneous continuity between neighbouring cells (e.g. Lucas et al., 1993; McLean et al., 1997). Until now, however, only few proteins have been identi®ed within these specialized cellto-cell channels, and none of them has been fully sequenced and unequivocally identi®ed (reviewed by Kragler et al., 1998). Our study identi®es calreticulin not only as the ®rst sequenced plant protein localized to plasmodesmata of higher plant cells but also as the ®rst ER-resident protein found within plasmodesmata. Other proteins localized to plasmodesmata include the moveã Blackwell Science Ltd, The Plant Journal, (1999), 19, 481±488

Maize calreticulin localizes to plasmodesmata in root apex 485

Figure 3. Immunogold EM microscopy. Speci®c labelling of individual plasmodesmata (arrowheads in (a±c) and clustered plasmodesmata in pit ®elds (arrows in (a,c)) is shown for the longitudinal (black asterisks in (a), (c)) and transverse (stars in (a) and (b)) walls of the inner cortex cells. (d) Pre-immune serum does not label any subcellular compartment (transverse wall indicated by star) of inner cortex cells. Bar = 150 nm for (a) and (b); bar = 100 nm for (c) and (d). Images are taken at a ®nal magni®cation of 3 46 000 (a,b) and 3 56 000 (c,d).

ment protein encoded by viral genomes (e.g. Ding et al., 1992), the pathogenesis-related protein of maize (Murillo et al., 1997), the protein kinase-like protein originally thought to be putative plant connexin (Mushegian and Koonin, 1993), and myosin-like proteins (Blackman and Overall, 1998). Actin and unconventional plant myosin VIII are two further candidates for plasmodesmata proteins (e.g. Ding et al., 1996; Reichelt et al., 1996; White et al., 1994). Calreticulin within plasmodesmata could be expected to participate in their gating via its calcium-buffering activities. The reason for this hypothesis is that unconventional myosin VIII has the potential to be regulated by calcium (Knight and Kendrick-Jones, 1993) and calreticulin within plasmodesmata could gate their permeability via modulation of actual calcium levels. In accordance with this hypothesis, increased cytoplasmic calcium was reported to inhibit transport across plasmodesmata (Erwee and Goodwin, 1983; Tucker, 1988). Moreover, Tucker and Boss (1996) suggest that plasmodesmata are designed to allow propagation of calcium waves across plant tissues. Recent progress in plasmodesmata research has clearly revealed that these unique cytoplasmic channels seem to be actively gated via actomyosin-based forces (Ding et al., 1996; reviewed by Overall and Blackman, 1996 and McLean et al., 1997). It might well be that the structural conformation of plasmodesmata is actively maintained via actomyosin-based forces (for a hypothetical model see Overall and Blackman, 1996), as it seems to be the case also of nuclear pore complexes (e.g. Berrios et al., 1991; Rakowska ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 481±488

et al., 1998). For instance, depletion of calcium in the nuclear envelope altered the conformation of nuclear pore complexes and inhibited nuclear transport (Perez-Terzic et al., 1996). Moreover, ATP depletion exerts dramatic effects on nuclear pore conformation, inhibiting the translocation step (Rakowska et al., 1998). On the other hand, it is known that azide treatment and anaerobic stress both increase the size exclusion limit of plasmodesmata (Cleland et al., 1994; Tucker, 1993). Thus, although both nuclear pore complexes and plasmodesmata seem to require ATP, and putatively actomyosin-based forces, for maintaining their structural conformation and functional organization, there is a fundamental difference between these two types of channels. Calreticulin remains enriched at callosic pit ®elds even in plasmolysed cells that have contracted their protoplasts In order to probe further the cell periphery-associated localization of calreticulin, we have exposed growing maize roots to mannitol treatment leading to plasmolysis. The latter process is known to be accompanied by stimulation of callose synthesis at plasmodesmata and pit ®elds (e.g. Eschrich, 1957; Oparka et al., 1994) as well as by re-arrangements of the cortical ER elements (Oparka et al., 1994). Here we report that in plasmolysed maize root cells, in which most of the cytoplasm has retreated from the cell walls in the form of contracting protoplasts, calreticulin remains associated almost exclusively with callosic pit ®elds at the cell periphery. This ®nding strongly suggests that calreticulin-enriched cortical ER domains

486 FrantisÏek BalusÏka et al. might be implicated in a highly elusive adhesion of the plasma membrane to extracellular matrices of higher plants. In contrast to the behaviour of calreticulin during plasmolysis, labelling with the anti-HDEL and anti-BiP antibodies remains restricted within contracting protoplasts, indicating that other ER-resident proteins show contrasting behaviour during plasmolysis. Intriguingly, calsequesterin-enriched ER domains of chicken Purkinje neurons are also uniquely depleted of BiP (Villa et al., 1991). It is possible that calreticulin and other ER-resident proteins relocate within ER networks of plasmolysing plant cells during their protoplast contraction but this attractive possibility requires further investigations. Nevertheless, such a ®nding would be consistent with observations that a large 60 kDa ER lumenal protein is capable of rapid diffusion throughout the ER-nuclear envelope compartment in animal cells (Subramanian and Meyer, 1997). In conclusion, we have identi®ed ER-resident maize calreticulin as a component of cortical ER elements associated speci®cally with plasmodesmata. In addition to, or as part of, its roles in calcium storage, signalling and as a glycoprotein-folding chaperone, calreticulin might turn out to be important for the structure and/or function of plasmodesmata.

equipped with epi¯uorescence and standard exciter and barrier ®lters. Photographs were taken on Kodak T-Max 400 ®lms.

Western blot analysis To prepare the soluble protein fraction, maize roots were homogenized (10 min in a mortar) in an ice-cold solution of 250 mM Tris±HCl (pH 8.0), 25 mM EDTA, 330 mM sucrose, 5 mM DTT, 5 mM ascorbic acid and 1 mM phenyl-methylsulphonyl ¯uoride, followed by ®ltration through one layer of nylon mesh. All subsequent steps were carried out at 4°C. The post-mitochondrial supernatant (15 min, 10 000 g) was centrifuged for 50 min at 18 000 g. The resulting supernatant was designated as the soluble protein fraction. Then, samples were re-suspended in 200 ml of a solution containing 10 mM MES/bis-tris-phosphate (BTP) pH 7.5, 5 mM EDTA and 20% glycerol. SDS±PAGE was carried out using 15% (for calreticulin) or 10% (for HDEL peptide) acrylamide gels. Samples were adjusted to 1 mg ml±1 of protein heated to 80°C for 10 min, centrifuged and the supernatant loaded on gels (15 ml of sample per lane). After protein separation, the gels were stained with Coomassie blue or their content was transferred to nitrocellulose membrane using a transblot cell (Bio-Rad, MuÈnchen, Germany). The membrane blots were blocked in TBS containing 4% (w/v) BSA for 30 min, washed with TTBS (TBS buffer containing 0.05% Tween-20), and incubated with polyclonal calreticulin antibody diluted 1:10 000 or monoclonal HDEL antibody diluted 1:1000 in TBS containing 1% BSA. Following extensive washing in TTBS, blotted proteins were visualized using alkaline phosphatase-conjugated anti-rabbit and anti-mouse IgGs (both obtained from Sigma) diluted 1:100 000 in TBS containing 1% BSA.

Experimental procedures Plant material, experimental treatment and immuno¯uorescence microscopy Maize grains (Zea mays L., cv. Alarik) obtained from Force Limagrain (Darmstadt, Germany) were grown in moistened rolls of ®lter paper for 4 days in darkness at 20°C. To induce plasmolysis of root cells, roots were submerged into 700 mM mannitol for 2 h. Fixation was performed with 3.7% formaldehyde in stabilizing buffer (50 mM PIPES, 5 mM MgSO4 and 5 mM EGTA, pH 6.9) for 1 h. The composition of stabilizing buffer was optimized for the cytoskeleton but proved to be suitable for a wide range of diverse antigens including calreticulin (Mews et al., 1997; Jahn et al., 1998; SÏamaj et al., 1998; BalusÏka, unpublished data). Embedding, sectioning and immunolabelling were performed as described previously (BalusÏka et al., 1997). All antibodies used in the present study were diluted in PBS supplemented with 1% BSA and applied on sections for 60 min at room temperature. Polyclonal anti-calreticulin antibody (Napier et al., 1995) and its pre-immune serum were diluted 1:20, monoclonal anti-HDEL antibody (Napier et al., 1992) was diluted 1:10, and polyclonal anti-BiP antibody (e.g. Figure 10 in Pueyo et al., 1995) was diluted 1:100. Callose was visualized with monoclonal antibody raised against (1®3)-b-glucan (Biosupplies, Parkville Victoria, Australia) diluted 1:200. Subsequently, the sections were stained with FITC-conjugated anti-rabbit (calreticulin, BiP) and TRITC-conjugated anti-mouse (HDEL peptide) IgGs raised in goat (Sigma Chemical Co, St Louis, Missouri, USA) diluted 1:100 in PBS containing 1% BSA for 60 min at room temprature. Fluorescence was examined with an Axiovert 405M inverted microscope (Zeiss, Oberkochen, Germany)

Immunogold electron microscopy Sample preparation. Apical 2 mm long segments of maize root

tips were excised and ®xed in 3.7% formaldehyde in stabilizing buffer (50 mM PIPES, 5 mM MgSO4 and 5 mM EGTA, pH 6.9) for 1.5 h. After washing four times with stabilizing buffer and three times with PBS, the root segments were dehydrated in graded ethanol/PBS series and embedded in LR White resin (Hard Grade, BioCell, Cardiff, UK) which was allowed to polymerize for 5 days at 36°C in an aluminium oven in order to preserve the tissue antigenicity. A Reichert ultramicrotome OM U3 (Reichert, Vienna, Austria) was used to obtain ultra-thin sections which were collected on formvar-coated Ni grids.

Immunolabelling. Residual aldehydes on sections were blocked

with 0.05 M glycine in PBS and non-speci®c binding of proteins was avoided by applying 5% BSA and 5% normal goat serum for 30 min. Subsequently, grids were washed with washing mixture: 1% BSA and 0.1% gelatin ®sh in PBS for 5 min, then incubated with the polyclonal calreticulin antibody or with pre-immune serum, both diluted 1:20, for 1.5 h. The sections were washed ®ve times with washing medium and incubated with the second antibody (goat anti-rabbit IgGs conjugated to 20 nm gold particles, BioCell, Cardiff, UK) for 1.5 h. Further extensive washing with washing medium and PBS (four times for 10 min) was followed by post-®xation with 3% glutaraldehyde for 15 min. Subsequently, washing of sections with PBS and staining with uranyl acetate and lead citrate were performed. Labelled sections were examined with a Zeiss EM 10A (Zeiss, Oberkochen, Germany) at 60 kV.

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 481±488

Maize calreticulin localizes to plasmodesmata in root apex 487 Acknowledgements We thank Maarten Chrispeels (University of California, La Jolla, USA) for providing us with the anti-BiP antibody and Monika Polsokiewicz for skilful technical assistance. Financial support to AGRAVIS by the Deutsche Agentur fuÈr Raumfahrtangelegenheiten (DARA, Bonn, Germany) and the Ministerium fuÈr Wissenschaft und Forschung (DuÈsseldorf, Germany) is gratefully acknowledged. F.B. is partially supported by the Grant Agency VEGA (project no. 3009). This work was also supported by a research fellowship to J.SÏ. from the Alexander von Humboldt Foundation (Bonn, Germany).

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