Plant Science 150 2000 103 – 114
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, USTLINRA, Uni6ersite´ des Sciences et Technologies de Lille, Baˆtiment SN
2
, F-
59655
Villeneu6e d
’
Ascq Cedex, France Received 29 June 1999; received in revised form 13 August 1999; accepted 14 September 1999
Abstract
In Cichorium ‘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 on Cichorium somatic 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 with b-
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 Cichorium somatic embryogenesis. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Arabinogalactan proteins; Cichorium; Cytoskeleton; Extracellular matrix; Extracellular proteins; Somatic embryogenesis www.elsevier.comlocateplantsci
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 between 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
:
hilbertuniv-lille1.fr J.-L. Hilbert 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 8 5 - 5
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
