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
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
By SEM, we have observed the removal of the fibrillar network as a result of the different treat-
ments cold; colchicine; cytochalasine B; Tris – HCl. The treatment medium containing the
removed fibrillar network offered the opportunity to analyse and determine its components. As de-
scribed by Woessner [38] in animal and Reuzeau and Pont Lezica [3] in plant systems, ECMs were
intricate networks of glycoproteins and carbohy- drates secreted by the cell. For these reasons, our
first experiments consisted to detect proteins in the treatment media. Protein assays Table 1 revealed
an accumulation of proteins in each treatment and
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 mgg 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
Fig. 1. A – B SEM of Cichorium ‘474’ root-induced embryos after 11 d in induction medium. A Somatic embryos se emerge from cortical cells cc after 11 d of root culture in liquid medium; bar, 100 mm. B The fibrillar network nt covers peripheral
cells of the embryo ec; bar, 5 mm. C – E SEM of Cichorium ‘474’ embryos after CTK inhibitor treatments. C 8 h cold treatment 4°C in induction medium. Embryo cells ec are undamaged; bar, 5 mm. D 7 h colchicine 5 mM treatment 30°C
in induction medium. A deposit d covers each embryo cells ec; bar, 5 mm. E 8 h cytochalasine B treatment 70 mM; 30°C in induction medium. Globular structures gs are observed at the surface of embryo cells ec; bar, 5 mm. F 4 h Tris – HCl
treatment 0.05 M; pH 7.2; 30°C. Embryo cells ec have a spongy aspect; bar, 5 mm.
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 Kit
a
mg25 ml of Protein amount
mgg of explant, fresh weight FW
medium 288.9 9 7.7
28.2 9 0.07 11-day-old embryo-
genic root culture medium
360.0 9 10.5 Tris 4 h
60.1 9 1.8 149.8 9 6.7
Cold 8 h 24.2 9 1.2
37.5 9 1.2 225.2 9 7.0
Colchicine 7 h Cytochalasine 8 h
53.4 9 1.5 320.5 9 8.6
a
The 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 ap- plied on somatic embryos obtained after 11 days
of root culture. The synthetic gels recorded 255, 50, 92, and 289 proteins, respectively for Tris – HCl
Fig. 2A, cold Fig. 2B, colchicine Fig. 2C and cytochalasine B Fig. 2D treatments. Using Tris –
HCl, 255 proteins were detected in the medium after treatment. Considering its common use as
protein extraction buffer, it might explain the high number of proteins released in the treatment
medium. In contrast, a low number of protein 50 were detected after cold treatment and the gel was
deprived of non-specific coloration. We can note that cytochalasine B is a toxic molecule with a
high molecular weight 479.6 kDa which pre- vented the migration of high molecular weight
proteins 1,2,3,4,5,6. It was detected on gels be- tween the pHi of 4.5 to 5.4 Fig. 2D. The com-
parison of the gels corresponding to all the treatments allowed us to detect a group of 25
common proteins recovered in the medium after each treatment. These 25 proteins were numbered
from 1 to 25 on the proteic cards Fig. 2. The pHi and the molecular weight of the 25 proteins were
determined Table 2. Moreover, the conditioned medium of 11 days Cichorium root culture Fig.
2E included the 25 common proteins found in the treatment media. They represented the major
proteins of the proteic pattern. None of these proteins were detected in the 11 day-old condi-
tioned medium of a of a non-embryogenic line Fig. 2F and in the medium of this line treated
with Tris – HCl, cold, colchicine and cytochalasine B treatment media data not shown. These results
suggested that the 25 proteins could be compo- nents of the fibrillar network removed by Tris –
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
Fig. 2. A – D Computer gel images, obtained after spot quantification of 2D-PAGE silver stained polyacrylamide gels of excreted proteins during different treatments of Cichorium somatic embryos. A 4 h Tris – HCl treatment 0.05 M; pH 7.2; 30°C. B 8 h
cold 4°C treatment in induction medium. C 7 h colchicine treatment 5 mM; 30°C in induction medium. D 8 h cytochalasine B treatment 70 mM; 30°C in induction medium. E – F 11 day-old conditioned medium. E Embryogenic Cichorium. F
Non-embryogenic line. Molecular mass markers are soybean trypsin inhibitor 21.5 kDa, bovine carbonic anhydrase 31 kDa, rabbit muscle GAPDH 36 kDa, bovine muscle actin 43 kDa and bovine serum albumin 66.2 kDa.
Table 2 Common proteins removed from Cichorium somatic embryos
after different treatments: Tris–HCl, cold, colchicine, and cytochalasine B
a
pHi Spot number
MW 1
63.0 4.7
62.9 2
4.8 4.9
63.0 3
63.4 4
5.0 5.3
5 62.9
5.4 62.8
6 55.7
7 5.5
60.2 8
7.3 5.4
44.0 9
43.7 10
5.6 5.8
11 47.5
6.0 48.2
12 49.1
13 6.9
7.6 14
48.9 6.8
36.2 15
35.4 16
5.5 5.2
17 37.8
5.0 33.3
18 35.4
19 5.5
20 4.4
31.0 4.5
31.0 21
4.4 22
29.3 4.5
29.3 23
24 23.5
4.6 4.7
23.4 25
a
Numbers 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
