Immunolocalization of calreticulin in protoplasts and somatic embryos of Daucus carota L. grown in suspension culture

ACTA BIOLOGICA CRACOVIENSIA Series Botanica A2J2: 87-92, 2000

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Marta Libik1*andLeslaw Przywara2**

* e-mail: libik@zfr.pan.krakow.pl

** e-mail: przywara@grodzki.phils.uj.edu.pl

'Department of Plant Physiology, Polish Academy of Sciences, ul. Slawkowska 17, 31-017 Cracow, Poland

department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52, 31-044 Cracow, Poland

Received February 7,2000; revision accepted March 13,2000

Ca2+ acts as a secondmessenger which controls a wide variety of cellularfunctions. Ca2+ homeostasisis accomplished bya complex ofmechanisms including pumps, ion channels and calcium buffers within storage compartments.

Calreticulin is the low-affinity, high-capacity Ca2+-binding protein of most eucaryotic cells. The distribution of calreticulin wasstudiedinisolatedprotoplasts and during differentstagesofsomatic embryogenesis incarrot (Daucus carota L. cv. StValery LungaRossa) suspension culture. Immunofluorescence stainingof protoplasts showed the presence of calreticulin inthecytoplasm. Nocalreticulinwas observed withinvacuoles.During somatic embryogenesis calreticulin was distributed mainlyin the protoderm of developing embryos in different stages.

Key words:Calreticulin, immunofluorescence, protoplast, somatic embryogenesis, Daucus carotaL.

INTRODUCTION

As in animal cells, Ca2+ regulates a variety of differ­

ent physiological processes in plants. Cytosolic free calcium [Ca2+]c is a convergence point for many dis­

parate signalling pathways (Trewavas and Malho, 1998). The source of Ca2+ during signaling in plant cells is not certain but it may occur through altered influx and efflux at the plasma membrane or through shuttling of internal Ca2+ between cytosol and internal compartments (Xing et al., 1994). Plant cells, like many other cells, maintain their cytoplas­

mic concentration between 10'7 and 10’6M, most like­

ly by active transport of Ca2+ out of the cell or into intracellular organelles such as vacuoles, endoplas­

mic reticulum (ER), chloroplasts and mitochondria.

Storage of Ca2+ within intracellular organelles is of great importance because it allows for rapid and

massive Ca2+ mobilization during signaling events, and provides the buffering capacity necessary to pre­

vent cytosolic Ca2+ overload. The two membrane sys­

tems (tonoplast and endoplasmic reticulum) are potentially the most important sites for control of in­

tracellular Ca2+ concentration in plants. Within the endoplasmic reticulum, sequestered, releasable Ca2+ does not occur as a free ion but rather is com­

plexed by Ca2+-binding proteins resident in the lumen of ER. Of these, calreticulin is generally con­

sidered to be the major contributor to the Ca2+ stor­

age capacity of the compartment, accounting for about half of the total Ca2+ stored (Meldolesi et al., 1996).

Calreticulin is a low-affinity, high-capacity Ca2t-binding protein that binds ~20 mols of Ca2+ per mole of protein and has an apparent molecular mass of 50-60 kDa in various species (Navazio et al.,

PL 1SSN 0001-5296 © Polish Academy of Sciences, Cracow 2000

1998a). Calreticulin has been purified from several different plant species: Spinacia oleracea (Menegaz- zi et al., 1993; Navazio et al., 1995), Hordeum vul- gare (Chen et al., 1994), Arabidopsis thaliana (Benedetti and Turner, 1995), Nicotiana tabacum (Denecke et al., 1995), Zea mays (Kwiatkowski et al., 1995; Napier et al., 1995), Pisum sativum (Hassan et al., 1995),Daucus carota (Libik et al., 1996; Libik, 1997), Liriodendron tulipifera (Navazio et al., 1998b), Ginkgo biloba (Nardi et al., 1998) and Eu- glena gracilis (Navazio et al., 1998a). In plants, calreticulin has been found mainly in the ER (Opas et al., 1996a) and nuclear envelope (Denecke et al., 1995; Napier et al., 1995) but it was also detected in the microtubule arrays of the phragmoplast and associated with the spindle apparatus of dividing cells (Denecke et al., 1995).

The ubiquitous distribution of calreticulin in every eucaryotic cell type (with the exception of yeast and erythrocytes), its intracellular localization in different compartments, and remarkable, phylo- genetically conserved sequence organized into dif­

ferent structural and functional domains has pro­

foundly modified the simple image of calreticulin from a classic Ca2+-buffering protein within the ER lumen (Fliegel et al., 1989) to a multifunctional factor playing a key role in various intracellular and intercellular events (Krause and Michalak, 1997).

At present, four principal cellular functions are suggested to be affected by calreticulin (Borisjuk et al. 1998). Two of them - participation in Ca2+storage and signalling, as well as chaperone activity - are consistent with calreticulin localization within the ER lumen, whereas the calreticulin-dependent modulation of gene expression and its contribution to regulation events of cell adhesiveness require extra-ER sites (Opas et al., 1996b).

In the present study we report intracellular localization of calreticulin in carrot protoplasts and the distribution of this protein during somatic em­

bryogenesis in different stages of embryo develop­

ment in this species. On the basis of the results we discuss the hypothetical function of calreticulin in somatic embryogenesis.

MATERIALS AND METHODS

The carrot {Daucus carota L.) cultivar St Valery Lunga Rossa was used. Seeds were sterilized in 70%

ethanol for 3 min and in commercial bleach (ACE) for 30 min followed by washing in sterile distilled water. The seeds were germinated in petri dishes on

B5 medium (Gamborget al., 1968) in the dark. Cell suspension culture was obtained from callus derived from hypocotyl explants of 7-day-old seed­

lings. The hypocotyls were placed on B5 medium containing 0.5 mg I1 2,4-D and 0.25 mg I'1 BAP, and the callus produced was transferred to liquid B5 me­

dium with the same concentration of growth regula­

tors.

Somatic embryogenesis was induced by trans­

ferring callus tissue to B5 medium but without growth regulators. After initiation of embryogen­

esis the liquid cultures were maintained in Erlen- meyer flasks on a rotatory shaker. The method of transfer and cell density were as described by De Vries et al. (1988).

Immunofluorescence staining of isolated proto­

plasts and somatic embryos was performed using a modified procedure described by Opas et al. (1996a).

To isolate the protoplasts, on day 4 of suspension culture the cells were transferred to enzymatic sol­

ution (pH 4.8) containing 2% cellulase, 1% mece- rozyme R-10, 0.4 M mannitol, and 0.05 M sodium citrate. The cells were incubated in this solution for 16 h at 37°C in the dark. Isolated protoplasts were fixed in 3.5% paraformaldehyde for 10 min, washed in phosphate buffer (PBS; pH 7.0) and then trans­

ferred to slides covered with organosilane. The slides were incubated in RST-X-100 solution (pH 6.9) containing 100 mM Pipes, 1 mM EGTA and 4%

polyethylene glycol (PEG 8000) for 3 min. Then the material was incubated with primary polyclonal antibody against spinach calreticulin (diluted 1:200 in PBS) for 30 min at 24°C. After incubation the material was rinsed with PBS and incubated with secondary antibody (conjugated anti-rabbit IgG FITC diluted 1:50 in PBS) for 30 min at 24°C. After a final wash with PBS the slides were mounted in glycerin with Vinol 205S to prevent photobleaching.

All antibodies were centrifuged after dilution. Pri­

mary antibody was omitted to check the specificity of labelling.

Somatic embryos during subsequent stages of development were fixed in 3.5% paraformaldehyde for 2 h and washed in PBS (pH 7.0). The material was dehydrated with ethanol (10 min each at 30%, 50%, 70% and 90%, then 3 x 10 min at 100%) and embedded in polyethylene glycol (PEG 1500). Semi­

thin (5 pm) PEG sections were made with a Microm HM 340 E microtome (Adamas Instrumenten BV).

The sections were transferred to slides covered with organosilane. Immunolocalization of calreticulin in somatic embryos was performed as described pre­

viously for protoplasts. Slides were examined with a

CalreticulininDaucus carota 89

Fig. la-d. Carrotcell protoplast suspension inGamborg’sB5 medium supplementedwith 2,4-DandBAP.(a)Intactproto­

plasts. x 200, (b-d) Immunolocalization ofcalreticulin; immu­

nofluorescence generated by anti-spinachcalreticulin anti­

body followed by anti-rabbit antibody conjugated with FITC, (b-c)Protoplasts showing diffuse labelling in the cytoplasm, (d) Control material; first antibodywas omitted;protoplasts donot show immunofluorescence. Fig. lb x 390, Fig. lc, d x 760.

Nikon Labophot fluorescence microscope. Micro­

photos were recorded on Kodak film.

RESULTS

IMMUNOLOCALIZATION OF CALRETICULIN IN PROTOPLASTS

Cells of Daucus carota L. on day 4 of culture were less vacuolized and therefore most useful for inves­

tigation of calreticulin localization in subcellular structures. Immunofluorescence staining of isolated protoplasts (Fig. la) showed the presence of calre­

ticulin in the cytoplasm (Fig. lb,c) - there were bright light green granularities in all area of cyto­

plasm. The presence of calreticulin was expected to be determined in ER, but unfortunately a clear net­

work of endoplasmic reticulum was not observed. At the light microscope level of resolution it was not possible to discern whether or not the bright granu­

larities represent separate vesicles. Nevertheless, it was clear that the vacuoles were entirely calre- ticulin-negative. The control material confirmed the specificity of the reaction. Protoplasts incubated with secondary antibody only did not exhibit any flu­

orescence (Fig. Id).

IMMUNOLOCALIZATION OF CALRETICULIN IN SOMATIC EMBRYOS

The distribution of calreticulin during somatic em­

bryogenesis was observed at four stages of embryo development: globular, late globular, heart- and tor­

pedo-shaped. Immunofluorescence staining results revealed that calreticulin was present mainly in the protoderm of developing embryos from globular to heart-shaped stage. It was clearly visible in the form of bright yellow fluorescence (Fig. 2a-c). In torpedo­

shaped embryos, fluorescence seemed to be dis­

tributed uniformly in all tissues (Fig. 2d). Compa­

rison of the labelled embryos with the control ma­

terial confirmed the specificity of immunofluores­

cence staining. Somatic embryos labelled with secondary antibody only did not display fluorescence and they were faintly green (Fig. 2e,f). The faint green light observed in the control material was probably a result of overlapping of the autofluores­

cence of the cellulose walls of somatic embryos and the yellow FITC fluorescence produced in the em­

bryos labelled with both antibodies. Addition of these two fluorescences might shift the light wave­

length toward yellow.

Fig. 2a-d. The presenceand localization of'calreticulin visualizedby immunocytochemical methods in carrot somatic embryos at different stages of development cultured in Gamborg's B5 medium. Immunofluorescence generated by anti-spinach calreticulin antibody followed by anti-rabbit antibody conjugated with FITC. In somatic embryos atglobular stage (a), transition stage (b) and heart-shapedstage(c>, calreticulin is localized mainly in the protoderm. Intorpedo-shaped stage (d), calreticulin is presentinalltissues. Fig. 2e—f. Control material;first antibody was omitted; somatic embryos donotshow fluorescence. Fig. 2a, d x 70; Fig.2b,c,e, f x 200.

DISCUSSION

In this report we described the intracellular locali­

zation in protoplasts and the distribution of one of the calcium-binding proteins, calreticulin, in somatic embryos of Daucus carota L. Immunodetection of this protein in isolated protoplasts using immuno­

fluorescence has been performed in only a few plant and animal species (Xing et al., 1994; Ioshii et al., 1995; Opas et al., 1996a). In animal cells it has been pos-sible to observe the localization of calreticulin in the endoplasmic reticulum, in the form of a clear ER network image after immunofluorescence staining.

In contrast, in plant cells the presence of this protein in ER has never been obvious, because of difficulties in distinguishing individual subcellular structures.

Plant cells are often highly vacuolated, and the cy­

toplasm containing particular organelles is limited to a narrow area close to the cell wall. In our experi­

ment we were able to state unequivocally that calre­

ticulin is present in the cytoplasm of the isolated protoplast. Moreover, we showed that this protein is absent in the vacuoles. These findings are in agree­

ment with an earlier study on the immunolocaliza­

tion of calreticulin in plant cells (Opas et al., 1996 a).

If calreticulin is a multifunctional protein it might be supposed to be present in the different subcellular structures, depending on the role it is to play. In animal cells, calreticulin was observed in the nucleus, plasma membrane and Golgi vesicles, in addition to ER (Meldolesi et al., 1996). The occur­

rence of calreticulin in the Golgi apparatus and in plasma membranes was demonstrated in Nicotiana plumbaginifolia protoplasts (Borisjuk at al., 1998). In light of this information, it would be useful to check, by means of electron microscopy, whether calreticulin also occurs in other organelles carrot cells.

It is well known that Ca2+ plays an important role in plant growth and development, and somatic embryogenesis is a model system for investigation

Calreticulin in Daucuscarota 91

of these processes. Our results from immunolocaliz­

ation of calreticulin in somatic embryos of Daucus carota L. are very similar to data on the distribution of calcium ions during somatic embryogenesis in this species (Timmers et al., 1996). The highest concen­

tration of Ca2+ was found in the protoderm of em­

bryos. That is in agreement with the generally ac­

cepted assumption that the main function of calre­

ticulin is to bind and store calcium in the cell. The highest concentrations of calmodulin and other pro­

teins (e. g., lipid transfer protein EP2 and glycopro­

teins) are also in the protoderm of somatic embryos (Lo Schiavo et al., 1990; Sterck et al., 1991; Timmers et al., 1995). The high concentration of many pro­

teins in the protoderm and the distribution of cal­

cium confirm the very important role of this tissue in somatic embryogenesis (De Jong et al., 1992). The function of this tissue is based on synthesis of sev­

eral enzymatic proteins necessary for correct devel­

opment of somatic embryos, and their secretion into the medium (De Vries and Hendriks, 1995).

ACKNOWLEDGEMENTS

We wish to thank Professor Paola Mariani of the University of Padua, Italy, who kindly provided us with polyclonal antibody against spinach calre­

ticulin and FITC-conjugated secondary anti-rabbit antibody.

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