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Removal of the fibrillar network surrounding

Cichorium

somatic

embryos using cytoskeleton inhibitors: analysis of proteic

components

Audrey Chapman, Ste´phane Helleboid, Anne-Sophie Blervacq, Jacques Vasseur,

Jean-Louis Hilbert *

Laboratoire de Physiologie Cellulaire et Morphogene`se Ve´ge´tales,USTL/INRA,Uni6ersite´ des Sciences et Technologies de Lille,Baˆtiment SN2, F-59655Villeneu6e dAscq Cedex,France

Received 29 June 1999; received in revised form 13 August 1999; accepted 14 September 1999

Abstract

InCichorium‘474’, the embryo globular stage was characterised by a fibrillar network linking peripheral neighbouring cells. To test a putative connection between this fibrillar network and the cytoskeleton (CTK), we have used CTK disrupting agents (cold; colchicine; cytochalasine B) onCichoriumsomatic embryos. Scanning electron microscopy observations showed that these three treatments induced the disappearance of the fibrillar network and suggested that this network could take part of the CTK-plasma membrane (PM)-extracellular matrix (ECM) continuum. The treatment media containing the removal fibrillar network were used to analyse its proteic component by 2D-PAGE. Using a Sun SPARCstation computer, the comparison of the gels corresponding to the different treatments allowed us to detect a group of 25 common proteins recovered in the medium after each treatment and in somatic embryogenesis conditioned medium. During the short treatments applied on somatic embryos, a large amount of high molecular weight glycoproteins corresponding to arabinogalactan proteins (AGPs) have been quantified withb-D-glucosyl Yariv

reagent and identified with monoclonal antibodies raised against AGP epitopes (JIM13, JIM16, LM2). The implication of the fibrillar network and AGPs in the continuum CTK-PM-ECM are discussed in attempt to elucidate their possible function during Cichoriumsomatic embryogenesis. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Arabinogalactan proteins;Cichorium; Cytoskeleton; Extracellular matrix; Extracellular proteins; Somatic embryogenesis

www.elsevier.com/locate/plantsci

1. Introduction

Cells of the main organisms produce molecules secreted in the extracellular space to form a dy-namic structure, the extracellular matrix (ECM). In animal systems, the ECM has been shown to play an active role in developmental processes by the perception of environmental signals, such as cellular polarity, differentiation, cell division, cell death and cell migration [1]. While the

involve-ment of the cytoskeleton (CTK)-plasma mem-brane (PM)-ECM continuum as a mechanochemical transducer was well established in animals [2], compelling evidence has been ob-tained in favour of the presence of such continuum in cellular adhesion and communication in plants [3]. Recent views of the ECM, referred to as the cell wall [4], proposed a more dynamic role in plant growth and development [5]. Wyatt and Carpita [6] have described specific adhesions be-tween PM and ECM, or bebe-tween ECM and sub-strate molecules as conductor of mechanical signals from the environment. It has been sug-gested that the interaction between the CW and PM was important in plant developmental pro-cesses [7]. These adhesion sites between ECM and Abbre6iations:AGPs, Arabinogalactan proteins;bGlcY,b-D

-gluco-syl Yariv reagent; CTK, cytoskeleton; ECM, extracellular matrix; PM, plasma membrane; SEM, scanning electron microscopy.

* Corresponding author. Tel.: +33-3-20436678; fax: + 33-3-20337244.

E-mail address:hilbert@univ-lille1.fr (J.-L. Hilbert)

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PM have been linked to a specific stage of devel-opment or in reply to abiotic or biotic stress [8]. Specialized structures at the cell surface are in-volved in cell adhesion and guarantee communica-tion between the cell and the environment or between cells. Adhesion proteins related antigeni-cally to animal ECM, trans-membrane and CTK-connecting proteins like integrin [7], fibronectin [9] and vitronectin [10] have been found in plants. At animal cell surfaces and in the associated ECM, large proteoglycans mostly composed of gly-cosaminoglycan chains are linked covalently to protein cores and have diverse roles in chemical signalling and cell adhesion [11]. The location of arabinogalactan proteins (AGPs) at the cell sur-face is indicative of possible functional similarities with classes of animal proteoglycans, although biochemically distinct from the proteoglycans oc-curring in animal systems. AGPs may have roles in processes involving maintenance of cellular en-vironments, cell interactions and the developmen-tal cell morphology [12 – 14]. These AGPs may also be important in determining sexual develop-ment in plants because these glycoproteins are not found in the germ tissue of anther and ovule but reappeared in the embryo at heart stage [15].

In the present work, our goal was to assess the possible involvement of a fibrillar network, ob-served on peripheral cells of globular somatic em-bryos ([16], Chapman et al. submitted results), in the continuum ECM-PM-CTK. In this context, we used the Cichorium ‘474’ as a model of direct somatic embryogenesis induced in liquid system [17], which offers the opportunity to study ex-creted proteins during the acquisition of morpho-genetic competence and expression of the embryogenic program. Indeed, conditioned media of plant cultures contain a complex set of molecules, some of which probably derived from the cell wall and can be used to study the plant ECM.

To test the hypothesis of a connection between the fibrillar network and the continuum ECM-PM-CTK, we have investigated at first the effects of CTK disrupting agents (cold, colchicine, cy-tochalasine B) on Cichorium somatic embryos. In the second part, electrophoretic separation and computer analyses of the treatment media contain-ing the disorganised fibrillar network were used to analyse its proteic composition in attempt to eluci-date its possible function during somatic embryo-genesis in Cichorium.

2. Material and methods

2.1. Plant material and culture conditions

Plantlets of aCichoriumhybrid ‘474’ (Cichorium intybus L. var.sati6um xCichorium endi6iaL. var. latifolia) were propagated by direct somatic em-bryogenesis [18]. The plantlets grown from root embryos [17] were subcultured for 2 – 6 months on solid Heller medium [19] containing 15 mM su-crose, 2.25 mM inositol and vitamins according to Morel and Wetmore [20]. The growth conditions were 12 h light, 24°C/12 h dark, 20°C, photope-riod with a photon flux rate of 50 mmol m−2s−1

[18]. To induce somatic embryogenesis, roots of 2 month-old plantlets were cut and placed in half-strength Murashige and Skoog [21] medium, con-taining 10.1 mM KCl instead of KNO3, 1.7 mM

glutamine, microelements [19], 5×10−5 M

Fe-EDTA, vitamins [20], 45 mM sucrose, 10−7 M

1-naphtaleneacetic acid and 2.5×10−6 M

6-dimethylallylaminopurine. The pH was adjusted to 5.4 before autoclaving. The induction was per-formed in agitated liquid medium (25 ml) on an orbital shaker (160 rpm) at 35°C and in darkness. After 11 days of culture, somatic embryos were separated from roots and fractionated in size through inox filters (200 to 1000 mm).

2.2. Scanning electron microscopy (SEM)

Samples for SEM were taken after 11 days of Cichorium‘474’ root culture. The embryo material was fixed in 1% OsO4for 3 h at room temperature

[22], washed in distilled water and dehydrated through a graded series of ethanol. In the final step, ethanol was dried by amyl acetate and plant material was gradually dried to the critical point with liquid CO2 using the Emscope critical point

dryer CPD 750. The samples were mounted on metal blocks, sputtered with gold-palladium (Po-laron SEM coating system E S100), examined and photographed using a JEOL JSM-35CS scanning electron microscope.

2.3. Experimental treatments

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disrupting agents (cold, colchicine, cytochalasine B) immediately after 11 days of culture in stan-dard conditions. Somatic embryos from 11 day-old culture were placed in a 0.05 M Tris – HCl buffer (pH 7.2) for 4 h (Tris treatment) or for 8 h in the induction medium at 4°C (cold treatment). Another serie of somatic embryos were treated with 5 mM colchicine (Sigma, St Louis, MO) in the induction medium at 30°C for 7 h (colchicine treatment) or with 70mM cytochalasine B (Sigma) in the induction medium at 30°C for 8 h (cytocha-lasine treatment). As control, we used 11 day-old conditioned medium. After each treatment, a part of embryos were placed on solid Heller medium for several days to study the lethality of the treat-ment. Treatment media were stored for further proteic analyses.

2.4. Extracellular protein extraction

Treatment medium and 1, 5, 11-day old condi-tioned media from embryo cultures were passed through a Faltenfilter (MN 7131/4; Osi, Elan-court, France) and then through a Millipore 0.22 mm, dialysed during 72 h against distilled water, and lyophilised. Protein extraction was performed as described by Helleboid et al. [23]. In the final step, the pellet was resuspended in the UKS [24] lysis buffer. After centrifugation (30 000×g, 20 min, 22°C), the supernatants were stored at

−70°C until analysis. Proteins were assayed using DC Protein Assay Kit (Bio-Rad, Hercules, Calif., USA).

2.5. Gel electrophoresis

Mono-dimensional gel electrophoresis (SDS-PAGE) of extracellular proteins was realised as described by Laemmli [25] with a 10% or 12.5% acrylamide running gel and a 4.5% acrylamide stacking gel in a Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA).

Two-dimensional polyacrylamide gel elec-trophoresis (2D-PAGE) of about 150 – 180 mg ex-tracted protein was performed as described by Boyer et al. [26] except that ampholytes (ampholi-nes BioRad) were composed of 10% ampholytes pH 5 – 7 and 90% ampholytes pH 3 – 10 to a final concentration of 4%. Isoelectrofocusing (IEF) was run at room temperature at 1200 V for 17 h followed by 1500 V for 0.5 h. The IEF gels were

extruded, equilibrated and loaded on a uniform 12.5% acrylamide gel in second dimension separa-tion. Gels were run at 350 V in the electrophoresis buffer (50 mM Tris; 384 mM glycine; 0.1% (w/v) SDS) using a Bio Rad electrophoresis Multi-cell. Silver staining was performed according to Blum et al. [27] and the gels were dried on a BioRad model 543 slab-gel drier.

2.6. Computer analysis of 2D gel

The silver-stained gels were scanned with a Sharp JX330 scanner and analysed using the Bio Image software running on a Sun SPARCstation 20. For each treatment, changes of protein accu-mulation in the medium were identified from at least three gels corresponding to two different protein extractions and electrophoretic separa-tions. Using the comigrated standard markers (2D-SDS-PAGE standards, Bio Rad), the isoelec-tric point (pHi) and molecular weight (MW) of the proteins were calculated.

2.7. Glycoprotein and AGP staining on gel

After SDS-PAGE, glycoproteins were identified directly on the gel by periodic acid-Schiff (PAS) staining as described by Dubray and Bezard [28] except for final rinsing which were made in 0.5% sodium metabisulfite or by 30 mM b-D-glucosyl

Yariv reagent (bGlcY) in NaCl 1% for 3 h at room temperature and de-staining (50% v/v methanol, 7.5% v/v acetic acid in distilled water) for 12 h.

2.8. Quantification of AGPs by single radial gel diffusion

The presence of AGPs in treatment medium and in conditioned medium of somatic embryogenesis was tested by single radial diffusion [29]. All sam-ples were tested at least five times. Protein samsam-ples and arabic gum (Sigma Chemicals) were allowed to diffuse into a 1% (w/v) agarose gel containing 0.15 NaCl and 10 mg/l bGlcY for 16 h at room temperature. The bGlcY was synthesised from phloroglucinol and p-aminophenyl-D

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curve of arabic gum (0, 0.05, 0.1, 0.15, and 0.20 mg/ml).

2.9. Immunoblot assays of AGPs

Proteins separated by SDS-PAGE were trans-ferred onto a nitrocellulose membrane by electrob-lotting (100 V, 1 h) using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). For im-munoblot assays, the membranes were blocked overnight in TBS (25 mM Tris – HCl, pH 7.4; 0.5 M NaCl) containing 2% PVP prior to incubation with the primary antibodies (1:1000) raised against AGP epitopes: JIM13, JIM16 and LM2 [31,32]. Following three washes with TBST (25 mM Tris – HCl, pH 7.4; 0.5 M NaCl; 0.1% Triton X100), blots were incubated for 2 h with alkaline-phos-phatase-conjugated goat anti-rat antibodies (1:2000) (Jackson Immunoresearch Lab, USA) and washed as mentioned before. The alkaline-phosphatase signal was developed using 0.03% nitroblue tetrazolium and 0.015% 5-bromo-chloro-3-indoyl phosphate in a solution 10 mM NaCHCO3 and 1 mM MgCl2 at pH 9.8. The

prestained low range molecular weight marker proteins were phosphorylase B (142.9 kDa), serum albumin (97.2 kDa), ovalbumin (50 kDa), car-bonic anhydrase (35.1 kDa), trypsin inhibitor (29.7 kDa) and lysosyme (21.9 kDa) (Bio-Rad).

3. Results

3.1. Effects of CTK inhibitors on the fibrillar network surrounding Cichorium somatic embryos

After 11 days in agitated culture medium, Ci -chorium somatic embryos emerged from cortical parenchyma cells of roots (Fig. 1A). Lack of syn-chronisation allowed observation of globular em-bryos of various sizes among the cortical root cells. The peripheral cells of the embryos were linked by a fibrillar network, which tended to cover the entire embryo surface (Fig. 1B) prior to the protoderm differentiation. This extracellular structure, designated as fibrillar network, was con-sidered as a part of the ECM. The network was formed of smooth fibres of different length. Con-trary to the embryo cells, cortical parenchyma cells of roots had a smooth surface (data not shown). To test the hypothesis of a connection

between the fibrillar network and the continuum CTK-PM-ECM, we have used classical treatments known to destabilise the CTK: cold [33], col-chicine with tubulin as target [34,35] and cytocha-lasine B with actin as target [36]. An 8 h cold treatment in the induction medium removed the fibrillar network and let embryo cells undamaged (Fig. 1C). The convex peripheral cells of the em-bryos have a smooth surface and the intercellular junctions were broken. The effect of colchicine for 7 h at 30°C showed the beginning of the network removal (Fig. 1D). The surface of embryo periph-eral cells was collapsed. A deposit covered each cell and the intercellular junction. An 8 h cytocha-lasine B treatment in the induction medium re-moved also the fibrillar network (Fig. 1E). The somatic embryo exhibited the same external mor-phology, but some globular structures (1 mm in diameter) appeared at the cell surface. These re-sults favour the idea that the fibrillar network surrounding Cichorium somatic embryos seemed to be linked to the CTK and consequently partici-pated in the continuum CTK-PM-ECM. Any of these treatments were lethal for the embryos, which developed into plantlets after the fibrillar network regeneration. Moreover, a 4 h Tris treat-ment removed also the fibrillar network (Fig. 1F) and gave a spongy aspect to the superficial embryo cells. Because Tris – HCl buffer was commonly used in protein extraction, this result was consis-tent with a proteic composition of this structure in accordance with the preliminary observations of Dubois et al. [37] which revealed a destruction of the fibrillar network using protease.

3.2. Analysis of proteins released following treatments with CTK inhibitors

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the larger amounts were recorded by using Tris and cytochalasine B treatments. These protein lev-els were higher comparing with protein accumula-tion in medium from 11-day-old embryogenic root culture (mg/g FW), except for cold treatment. These results demonstrate the correlation between the rapid accumulation of proteins in treatment media (greater than protein amount of 11-days embryogenic culture medium) with the disruption of the previously described fibrillar network and so suggest its proteic composition. To analyse the

proteins released in the different treatment media, they were separated by two-dimensional-PAGE and the silver-stained gels were analysed using a Sun SPARCstation computer. For each condition, a synthetic gel was obtained from three gels corre-sponding to three different protein extraction and electrophoretic separations (Fig. 2). In all extrac-tions, the proteins detected by 2D-gel elec-trophoresis were separated in the pHi range from 3 to 8 and molecular weight (MW) from 10 to 100 kDa. The profile of extracellular proteins varied in

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Table 1

Protein assays in 11-day-old conditioned medium of Cicho-rium somatic embryogenesis and somatic embryo treatment medium by BioRad DC protein assay Kita

mg/25 ml of

Protein amount mg/g of explant, fresh weight (FW)

Cytochalasine (8 h) 320.598.6 53.491.5

aThe displayed values correspond to the average of three

assay replicates.

HCl buffer and the three treatments destabilising the CTK. The cold treatment seemed to be an appropriated experimental condition to study the fibrillar network proteins because the proteic pat-tern obtained after the treatment was most similar to the pattern of the 25 common proteins and because no damage for somatic embryo morphol-ogy was observed using this treatment compared to others. However, the 11-day old conditioned medium more abundantly obtained and containing also the 25 proteins was another possible candi-date for further proteic analyses.

3.3. Accumulation of AGPs in the medium in response to CTK inhibitors

To initiate the characterisation of the released proteins in the treatment medium, we tested the extracellular protein glycosylation, since animal ECM is composed of proteoglycans. The 11 day-old medium proteins were separated by SDS-PAGE and submitted to periodic acid Schiff (PAS) staining. As shown on Fig. 3A, high molec-ular weight glycoproteins (\150 kDa) were strongly detected. These proteins could correspond to AGPs, which are known as high molecular weight secreted components of the plant cell sur-face. Moreover, AGPs are otherwise known to be detected as a smear bands in SDS-PAGE and difficultly fixed in gels following electrophoresis using 2D PAGE standard procedures [39]. To detect the putative AGPs in the treatment medium, the bGlcY, a synthetic phenyl glycoside that interact specifically with AGPs, has been used. Analysis of conditioned medium and treat-ment medium proteins (Tris, cold, colchicine, cy-tochalasine B) by SDS-PAGE followed by probing

with bGlcY revealed its reactivity with a smear of

material with apparent molecular weight of 100 – 200 kDa (Fig. 3B) as observed with PAS. These diffused bands were characteristic of AGPs. These analyses showed that high molecular weight glyco-proteins, not detected in the precedent 2D-PAGE and highly accumulated in the treatment medium, seemed to correspond to AGPs.

To identify AGPs, total proteins extracted from treatment media and 11-day-old conditioned medium were submitted to immunoblots using monoclonal antibodies (JIM13, JIM16, LM2). As shown on immunoblots with AGPs monoclonal antibodies for cold treatment medium (Fig. 3C), relation with the specificity of the treatments

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the AGPs detected occurred as smeared bands with size greater than 110 kDa. LM2 reacted most abundantly with the proteins of the cold treatment medium than JIM13 and JIM16 antibodies. Simi-lar result was obtained for the three other

treat-ments (Tris, colchicine, cytochalasine B) (data not shown). First characterisation showed that AGPs recognised by LM2 antibodies were predominant. To quantify AGPs, treatment and conditioned media were collected and proteins were subjected

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Table 2

Common proteins removed fromCichoriumsomatic embryos after different treatments: Tris–HCl, cold, colchicine, and cytochalasine Ba

aNumbers refer to protein spots shown in Fig. 2; their

molecular masses (MW) and pHi were indicated after estima-tion by references to standards (BioRad SDS PAGE).

The correlation between the removal of the fibril-lar network and high accumulation of AGPs in the treatment medium suggest that one possible com-ponent of the fibrillar network could be AGPs.

4. Discussion

In Cichoriumsomatic embryogenesis, the globu-lar stage is characterised by a fibrilglobu-lar network at the embryo cell surface [16] prior to the protoderm differentiation. SEM and TEM observations al-lowed us to describe as a three dimensional net-work linking together peripheral embryonic cells. This network was then define as an intermediate stage of growth during somatic embryogenesis process where a balance between extensibility and cohesion is necessary (Chapman et al. submitted results). Preliminary experiments showed that en-zymes such as protease and pronase E as well as lipid solvents and pectinase greatly altered the embryo cell surface and induced the removal of this fibrillar network [16]. Such structure has been observed on somatic embryos of Drosera rotundi-folia,Zea mays[40] andPinus nigra [41]. However, only a few plant species have been shown to possess strands surrounding embryo cell surface,

to the single radial gel diffusion according to Van Holst and Clarke [29] (Table 3). Comparing with the standard curve of arabic gum, we have deter-minated the amounts of AGPs in the samples. They were detected at high level in the four treat-ment media, particularly for the cytochalasine treatment (190 mg AGPs/mg total proteins). Dur-ing somatic embryogenesis process, AGPs were detected at low level on the 1-day-old conditioned medium (5.26 mg AGPs/mg total proteins) and increased at an up thirty-eight fold higher level to day 11 of the embryogenic culture (190mg AGPs/

mg total proteins). In a non-embryogenic cultivar, proteins from treatment media (Tris – HCl, cold, colchicine, cytochalasine B) were also submitted to Yariv test. In this case, AGPs remained at a low level for all the treatment media (less to 5 mg AGPs/mg total proteins). These results demon-strated that AGPs were up to 12- to 35-fold higher accumulated in the treatment media, 5- and 11-day old conditioned medium compared to similar treated non-embryogenic cultivar roots or 1-day old embryogenic hybrid root conditioned medium.

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Table 3

Quantification of AGPs in somatic embryo treatment medium and in conditioned medium ofCichoriumsomatic embryogenesis by the method of the single radial gel diffusion according to Van Holst and Clarke [29]a

Total proteins (mg)/well (halo diameter)2(mm)2

Extracellular Total AGPs (mg)/well mg AGPs/mg total proteins

proteins

25

Tris (4 h) 12 0.95 79.17

Cold (8 h) 15 25 0.95 63.33

25

Colchicine (7 h) 12 0.95 79.17

25 0.95

5 190

Cytochalasine (8 h) 38

J1 16 0.2 5.26

25 0.95 47.5

J5 20

25 0.95 190

5 J11

aExtracellular proteins were collected from 1-day- (J1), 5-days- (J5), 11-days- (J11) after induction of somatic embryogenesis.

The concentration of the sample was adjusted to fall within the standard curve. Values outside the standard curve were not accurate. The values are the mean values of five separate protein extractions.

certainly because early stages in somatic embryo-genesis have not been often described. Moreover, we have observed a surprising similarity with the animal ECM structure as mentioned by Reuzeau and Pont-Lezica [3] in comparing a fresh unfixed onion cell wall [42] and a frog otolithic membrane [43].

Recent identifications of plant surface molecules involved in cell-adhesion strongly suggested that a continuum, ECM-PM-CTK, might be a common structure present in eukaryotic cells. In such a way, further investigations were undertaken to test the putative involvement of the fibrillar network in the plant continuum. To check this hypothesis, we have used CTK disrupting agents (cold, colchicine, cytochalasine B) on Cichorium somatic embryos and observed by SEM its effects on the fibrillar network at the embryo cell surface. It is estab-lished that the selected treatments have different affinity with the CTK, but several experiments were needed to give more insights to support the continuum hypothesis. First, cold was assumed to depolymerise the CTK (microtubules) with elicita-tion by cytosolic calcium in Nicotiana [44]. Next, more specific drugs like colchicine and cytocha-lasine B, with respectively tubulin and actin as target, were also tested with the intention of im-proving the CTK involvement. Besides, Weede-burg and Seagull [45] have shown that colchicine allowed alteration of the cellulose microfibrils and inhibited CTK organisation. These results sug-gested a relation between the CTK and the CW. Similarly, in our model, the proposal of a connec-tion between the fibrillar network and the CTK was suggested by the removal of the fibrillar net-work following all the treatments. However, such

network could not be directly linked to the CTK but connected via membrane ligands and/or CTK-associated proteins as described by Wyatt and Carpita [6]. This original approach constituted a basis for a biochemical characterisation of the disorganised structure, which was consequently re-covered in the treatment medium

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proper-ties, but confirmed the proteic nature of the fibrillar network previously shown by Dubois et al. [16] with protease.

After protein extraction, we have selected the proteins shared by all treatment media, considering that some of them were originated from the disor-ganised fibrillar network. The Sun SPARCstation comparison of the protein pattern corresponding to the proteins recovered in the four treatment media has revealed a common pattern of 25 proteins correlated with the removal of the fibrillar network. The 25 proteins detected after the four treatments were also present without any treatment in 11 day-old conditioned medium of embryogenic Ci -chorium, in which they represented the major accu-mulated proteins. At this time, it was previously established that most of the embryos have reached the size \200mm and exhibited a protoderm [47]. Thus, embryos with a protoderm never showed a fibrillar network and its proteic components were consequently recovered in the medium. The correla-tion between the fibrillar network disappearance and the protoderm differentiation observed by Dubois et al. [16] has been confirmed by the ultrastructural study of the outer CW differentia-tion during Cichorium somatic embryogenesis (Chapman et al. submitted results). Therefore, this could explain the presence of high amount of proteins implicated in the network dynamic at 11 days of culture, which coincide with the protoderm formation of the older embryos. In this case, we suggest that the network shown around cells of Cichoriumembryos represent a part of an organised extracellular complex that may promote the co-or-dination of early developmental stages of embryos. This fibrillar network can be a point of compari-son with animal cells where surface glycoproteins play a role in adhesion, recognition and are linked to the CTK. The animal ECM is characterised by glycosamino-glycans [48]. In Cichorium, specific glycoproteins withN-acetyl-galactosamine binding sites have been previously revealed with FITC-la-belled Dolichos biflorus agglutininon young globu-lar embryos [16]. In our study, a high accumulation

of bGlcY-reactive AGPs of similar size to that

recognised either by JIM13, JIM16 or LM2 was observed duringCichoriumsomatic embryogenesis and after the different treatment destabilising the CTK. Although the biological function(s) of AGPs remains uncertain, there are evidences for their involvement in cell proliferation [12,49], cell

expan-sion [50,51] and in regulation of somatic embryo development [13,14,52,53]. Recently, studies showed that AGPs are accumulated as soluble molecules in the medium of suspension-cultured plant cells [52,54,55]. It has been observed that some of these promote, while others inhibits, somatic embryogenesis [13,52,53]. Moreover, Smallwood et al. [32] have shown that the same AGP can react

withbGlcY and several monoclonal antibodies like

the LM2, JIM8, JIM13 and JIM15. Only the extremely variable carbohydrate composition of AGPs could give information on the nature of the putative different AGP(s) implicated in Cichorium somatic embryogenesis. In this model, we have shown that CTK destabilisation induces AGPs accumulation at high level in the treatment medium and modifies the surface of the embryos peripheral cells. This was in accordance with Serpe and Noth-nagel [12], who have shown that the perturbation of AGPs by bGlcY alters the alignment of micro-tubules in Rosa cell suspensions. The bGlcY can also destabilise the normal intercalation of new cell wall subunits, while exocytosis of secretory vesicles still occurs [56]. This AGPs-bGlcY complexes should be responsible for the lack proper cell wall assembly. These results support our precedent hy-pothesis that a relation could exist between AGPs and the CTK.

The current knowledge seems to predict that the molecules involved in plant and animal ECM could have common feature. Therefore, understanding the structure and role of plant cell surface in the perception and transduction of environmental sig-nals, and comparing the plant and animal ECM-PM-CTK continuum, will be a tremendous advancement. In this way, further microsequencing experiments of the 25 common proteins will be investigate to determine the nature of the proteins in relation with the fibrillar network. Moreover, AGP studies was currently underway to further precise their participation as fibrillar network com-ponents in the continuum and to elucidate its possible function during somatic embryo development.

Acknowledgements

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Many thanks to Dr J.P. Knox (Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds, UK) for generous gift of JIM13, JIM16 and LM2 antibodies. This research was supported by a ‘‘Contrat Plan-Etat-Re´gion’’ to the Laboratoire de Physiologie Cellulaire et Morpho-gene`se Ve´ge´tales and by a Doctoral Fellowship from the Ministe`re de l’Education Nationale, de l’Enseignement supe´rieur et de la Recherche to Audrey Chapman.

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Gambar

Fig. 1. (A–B) SEM of Cichoriumtreatment (4°C) in induction medium. Embryo cells (ec) are undamaged; bar, 5in induction medium
Table 1
Fig. 2. (A–D) Computer gel images, obtained after spot quantification of 2D-PAGE silver stained polyacrylamide gels of excretedproteins during different treatments ofNon-embryogenic line
Table 2Common proteins removed fromafter different treatments: Tris–HCl, cold, colchicine, andcytochalasine B Cichorium somatic embryosa
+2

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