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 a Cichorium hybrid ‘474’ Cichorium
intybus L. var. sati6um x Cichorium endi6ia L. 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°C12 h dark, 20°C, photope-
riod with a photon flux rate of 50 mmol m
− 2
s
− 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 KNO
3
, 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 OsO
4
for 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 CO
2
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 To test the putative nature of the fibrillar net-
work and its implication in the continuum ECM- PM-CTK,
Cichorium somatic
embryos were
exposed to Tris – HCl buffer and various CTK
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 70 mM 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 71314; 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 wv
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
2
D 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 vv methanol, 7.5 vv 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 samples
and arabic gum Sigma Chemicals were allowed to diffuse into a 1 wv agarose gel containing
0.15 NaCl and 10 mgl bGlcY for 16 h at room temperature. The bGlcY was synthesised from
phloroglucinol and p-aminophenyl-
D
-glycopyra- noside precursors Sigma Chemicals, St. Louis,
MO, USA according to the method of Yariv et al. [30]. The amount of AGPs in the samples was
determinated by comparison with the standard
curve of arabic gum 0, 0.05, 0.1, 0.15, and 0.20 mgml.
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
NaCHCO
3
and 1 mM MgCl
2
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
