Download Functions of the Cell Wall in the Interactions of Plant Cells: Analysis

Plant Cell Physiol. 39(4): 361-368 (1998)
JSPP © 1998
Mini Review
Functions of the Cell Wall in the Interactions of Plant Cells: Analysis Using
Carrot Cultured Cells
Shinobu Satoh'
University of Tsukuba, Institute of Biological Sciences, Tsukuba, Ibaraki, 305-8572 Japan
Interactions between cells and between tissues are important in the development and morphogenesis of higher
plants. Attempts to characterize the role of the cell wall in
such interactions have benefited from the use of carrot
(Daucus carota L.) cultured cells in vitro as a model system. The development of carrot cells in culture can be divided into three processes: the acquisition of embryogenic
competence; the development of the embryo; and the
maturation and dormancy of the embryo. Induction of
non-embryogenic callus is accompanied by weakened intercellular attachment, decreased levels of endogenous ABA
and a decrease in responsiveness to exogenous ABA. Cell
wall polysaccharides are known to be involved in various
developmental and morphogenetic events. In carrot cultured cells, possible roles in intercellular attachment have
been proposed for arabinan and xylose in the neutral sugar
regions of pectins, and various extracellular proteins have
been shown to be involved in somatic embryogenesis in
vitro. Some of these proteins are also present around and/
or in zygotic embryos, possibly being involved in the formation and functions of zygotic embryos and seeds. A 57-kDa
extracellular soluble glycoprotein that binds to insulin-like
peptides and an 18-kDa extracellular insoluble cystatin that
inhibits the proteinases of germinating seeds of carrot
might be involved in cellular signal transduction and intertissue interaction, respectively, in carrot seeds.
cell wall is important for various phenomena associated
with the development of higher plants because it can play
an essential role as a site of interactions with abiotic and biotic environments and in the communication between different cells, tissues and organs, as well as in the growth and
differentiation of individual cells. However, studies of the
functions of the plant cell wall have focused mainly on
matrix polysaccharides and their roles in the regulation of
cell growth. Indeed, the metabolism of hemicellulose during cell growth, under the control of plant hormones,
has been characterized in detail (Fry 1995, Nishitani 1995,
Sakurai 1991). By contrast, other aspects of the functions
of the cell wall remain poorly understood. Recently, the induction was reported of leaf primodia in apical meristems
of tomato by a cell wall protein, expansin, which increases
the extensibility of the cell wall (Fleming et al. 1997). Modulation of cell fate by the cell wall has also been observed
during embryogenesis of Fucus (Berger et al. 1994). These
results serve to emphasize the significance of the cell wall in
plant development. This review provides a summary of our
recent results, including recent progress in the field that is
related to the functions of the cell wall and the apoplast in
plant development, with a focus on possible cell-to-cell and
tissue-to-tissue interactions in plants. Many of the results
mentioned were obtained by use of carrot cells in culture as
a model system.
Key words: Carrot (Daucus carota) cultured cells — Cysteine proteinase inhibitor — Extracellular glycoprotein —
Pectin — Seed — Somatic embryo.
Higher plants are composed of numerous cells that are
organized to maintain and control the functions and development of the whole body. These cells, unlike those of
animals, have a rigid cell wall, a type of apoplastic space,
that encompasses the protoplast and is composed mainly of
polysaccharides, proteins and phenolic compounds. The
Abbreviations: EDGP, extracellular dermal glycoprotein;
EICC, extracellular insoluble cystatin of carrot; EIP18, 18-kDa extracellular insoluble protein; GP57, 57-kDa glycoprotein.
1
Recipient of the JSPP Young Investigator Award, 1997.
Embryogenic competence of carrot cells—Plant cells
can divide, differentiate and regenerate whole plants even
after they have differentiated to specific types of cell in
mature plants. This ability, known as totipotency, was first
demonstrated by the discovery of the formation of somatic embryos of carrot (Daucus carota L.; Reinert 1958,
Steward et al. 1958). Somatic embryos are embryos derived
from somatic cells and they are capable of developing into
young plantlets via a series of morphological changes that
closely resemble those that occur during the development
of zygotic embryos. In both cases, embryos are characterized by globular, heart-shaped and torpedo-shaped profiles
in sequence. Somatic embryogenesis can be induced in various plants, but that in carrot has become one of the best-established model systems and has been used in attempts to
elucidate the physiological, biochemical and molecular biological mechanisms that underlie plant embryogenesis
(Kiyosue et al. 1993, Nomura and Komamine 1986, Sung et
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Functions of the cell wall
al. 1984, Zimmerman 1993).
In the case of carrot cells in culture, auxin plays an
essential role in the induction of somatic embryogenesis
(Fig. 1). When segments of carrot tissue are cultured on
auxin-containing medium, grainy yellowish callus, which is
called embryogenic callus, is formed on explants. This callus consists of small cells with dense cytoplasm that are called embryogenic cells. Formation of carrot somatic embryos can be readily induced by transfer of embryogenic
cells that have been cultured in medium supplemented with
auxin to auxin-free medium. Such embryogenesis can be
divided into two main processes: (i) the process whereby differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells but during
which their development into embryos is suppressed in the
presence of exogenous auxin; and (ii) the process whereby
the embryogenic cells display their embryogenic competence and develop into somatic embryos in the absence
of exogenous auxin.
When the selection of small clusters of cells from embryogenic callus culture is continued for several months in
the presence of auxin, embryogenic cells sometimes lose
their embryogenic competence and form small clusters of
cells in a process that is exclusively a result of multiple putative spontaneous mutations, which are a consequence of
somaclonal variations in embryogenic callus. Such clusters
are called non-embryogenic callus and they are unable to
form somatic embryos after transfer to auxin-free medium
(Steward et al. 1958, Satoh et al. 1986). The establishment
of lines of non-embryogenic callus has helped in the analysis of the embryogenic competence of carrot cells. The acquisition and loss of embryogenic competence are specific
to the cell culture system. These phenomena are not found
in intact plants but they provide a good system for investigations of the fundamental mechanisms of differentiation
of plant cells.
Development of somatic embryos and ABA—The system for culture of carrot cells in vitro allows us to monitor
developmental events in somatic embryos. In higher plants,
after fertilization, embryogenesis initially involves several
tiny cells that are located deep within maternal tissues, such
as flowers and young fruits. Therefore, the analysis of the
biological and biochemical events that occur in the development of zygotic embryos is rather difficult. Recent progress
in this field has been made as a result of genetic analysis of
various mutants of Arabidopsis with defects in zygotic embryogenesis (West and Harada 1993). However, investigations of the physiological events that occur in embryos and
developing seeds also require biochemical and molecular
biological analysis. Somatic embryos develop into young
plantlets via morphological changes that resemble those associated with the development of zygotic embryos. Moreover, common developmental programs of gene expression
are involved in both somatic and zygotic embryogenesis.
Thus, it is possible to identify some features of somatic embryogenesis and then to confirm and evaluate these same
features in developing zygotic embryos and seeds. In this
way, somatic embryogenesis provides a good system for
studies of zygotic events.
When carrot somatic embryos are cultured in hormone-free medium, embryos develop into seedlings and
plantlets because of the absence of a supply of ABA. By
contrast, zygotic embryos mature and become dormant under the control of ABA that is supplied mainly by maternal
tissues. In other words, ABA induces the synthesis of storage materials and of so-called late embryogenesis abundant
(LEA) proteins, as well as the acquisition of tolerance to
dehydration at the late stage of zygotic embryogenesis
(Thomas 1993). These phenomena, observed in late zygotic
embryos, can be also induced in somatic embryos by the addition of ABA to the culture medium (Iida et al. 1992, Zimmerman 1993). The endogenous level of ABA in embryogenic callus is high enough for the induction of expression
of some genes that are normally expressed in late zygotic
embryos, whereas the level of ABA in non-embryogenic callus is quite low (Kiyosue et al. 1992a). Moreover, non-embryogenic callus responds to exogenously applied ABA to
only a very limited extent (Kiyosue et al. 1992b).
Intercellular attachment in plant morphogenesis—In
multicellular organisms, such as higher animals and plants,
intercellular attachment is an important factor in the control of morphogenesis. Unlike the functions of animal
cells, the functions of plant cells are restricted spatially by a
rigid cell wall, and epigenic cell-to-cell attachment after cell
division is not usually observed. However, exceptions include the fusion of carpels in the formation of the pistil
(Vanderschoot et al. 1995). Both slippage and separation
of cells occur commonly in actively growing regions of
plants and during the formation of spongy parenchyma tissues. The separation of cells is also observed in the abscission of organs and the ripening of fruits (Osborne and
Jackson 1989).
During long-term culture of carrot embryogenic callus
in suspension, the callus gradually begins to form small
clusters of cells and loses embryogenic competence, becoming non-embryogenic callus. In culture systems derived
from various other plants, such as tobacco and rice, loss by
callus of morphogenic competence (the capacity for embryogenesis and formation of adventitious buds) is generally
observed, with an accompanying reduction in the size of
cell clusters as a result of the loosening of intercellular attachments. It seems likely, therefore, that close intercellular attachments are essential for morphogenetic processes
such as embryogenesis and organogenesis. To examine this
issue, we compared cell wall polysaccharides between carrot embryogenic and non-embryogenic callus in an attempt
to characterize mechanisms of intercellular attachment that
are involved in morphogenesis in higher plants.
Functions of the cell wall
Carrot embryogenic callus forms large clusters of
cells, while non-embryogenic callus forms small clusters
(Satoh et al. 1986). Microscopic observations revealed
differences in the features of intercellular contacts between
the two types of callus: intercellular contacts in embryogenic callus were tighter than those in non-embryogenic callus
(Kikuchi et al. 1995). Differences between embryogenic
callus and non-embryogenic callus were also found in the
polysaccharides of the pectic fraction when pectic, hemicellulosic and cellulosic fractions were prepared from
cell walls (Kikuchi et al. 1995). The level of neutral sugars
was higher than that of acidic sugars in embryogenic callus,
and the converse was true in non-embryogenic callus.
Gas-chromatographic analysis of neutral sugars in pectic
fractions revealed that embryogenic callus was rich in
arabinose while non-embryogenic callus was rich in galactose. An analysis of the pectins from several cultures, in
which the sizes of clusters exhibited some variation, showed that the molar ratio of arabinose to galactose and the
level of xylose were correlated with the average size of cell
clusters (Kikuchi et al. 1996a, b). These results suggest that
the neutral-sugar region that contains arabinose and xylose
might play an important role in the determination of intercellular attachment, namely, in the determination of the
size of cell clusters.
The separation of cells was observed upon the addition of colchicine to the culture medium of soybean cells,
with the release into the medium of 18-kDa galacturonan,
which could, by itself, induce cell separation (Hayasi and
Yoshida 1988). It was proposed that the pectic substance
might exert its effect by stimulating the production of ethylene as a soluble, biologically active factor.
Calcium bridges of pectin—Pectins are composed of
an acidic main chain and neutral side chains (Talmadge et
al. 1973). The main chain is composed of linearly linked galacturonic acid residues, with rhamnose residues scattered
along the polygalacturonic acid chains. The side chains are
mainly composed of arabinose and galactose, and they are
linked to the scattered rhamnose residues in the main
chain. The functions of pectin are thought to involve formation of 'calcium bridges' via an ionic bond between
a Ca 2+ ion and carboxyl groups, and such bridges are
thought to be responsible for the intermolecular linkage of
pectins. The cementing activity of pectin has been proposed to be related directly to the number of calcium bridges
and the level of polygalacturonic acids.
When the carboxyl groups of polygalacturonic acid
are methylesterified, the cementing activity decreases (Bonner 1950). Changes in the degree of methylesterification of
pectin during the cell growth have been reported. The pectic polysaccharides present in the walls of young or actively
growing plant cells were found to be highly methylesterified
whereas walls of mature cells contain strongly acidic pectins (Asamizu et al. 1984, Goldberg et al. 1986, Yamaoka
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and Chiba 1983). In carrot cultured cells, highly acidic pectin and highly methylesterified pectin were reported to be
present in the middle lamella and in primary cell walls, respectively (Liners and van Cutsem 1992). However, calcium bridges are not sufficient to explain the mechanism of intercellular attachment because the cementing of cells to one
another is determined not only by the rigidity of pectin but
also by the interactions between pectin molecules and other
components, such as hemicellulose and/or cellulose. Good
candidates for bridges between pectin and hemicellulose
might be the neutral-sugar side chains of pectin, but the
functions of such neutral sugars are not well understood.
Neutral sugar chains of pectin—We purified pectins
from carrot callus by chromatography on gel-filtration and
ion-exchange columns and analyzed the structure of the
neutral-sugar regions (Kikuchi et al. 1996a). Linkage analysis showed that the neutral-sugar chains of non-embryogenic callus and embryogenic callus had galactan-rich neutralsugar chains whose galactan moieties were 6-linked and
linear, as well as arabinan-rich neutral-sugar chains whose
arabinan moieties were 5-linked and branched from the 3
position, respectively. Possible structural models of the
neutral-sugar chains of callus pectins are shown in Fig. 2.
Each chain has three regions, namely, basal arabinan, middle galactan and terminal arabinan regions. The basal
arabinan region is a 5-linked linear chain. The middle galactan region is a 6-linked linear chain with limited branching
from the 3 and 4 positions. The terminal arabinan region is
3- and 5-linked and branched. The differences between embryogenic and non-embryogenic callus appear to be located
mainly in the middle galactan and terminal arabinan
regions. The middle galactan region is larger and slightly
more branched in non-embryogenic callus than in embryogenic callus, and the terminal arabinan region is larger and
slightly more branched in embryogenic callus than in nonembryogenic callus. These results suggest the significance
of the terminal arabinan of pectin molecules in the intercellular attachments of carrot cultured cells.
Variations in pectic polysaccharides were also noted in
a study of two lines of morphologically different cultured
cells derived from Catharanthus roseus and a difference in
arabinose content between the two types of cell was demonstrated (Suzuki et al. 1990). A high level of arabinose and a
low level of galactose were also found in the apical region
of pea root (Tanimoto 1988). These results support the hypothesis that arabinose might play a significant role in morphogenesis.
The pectin purified from carrot callus contained terminally linked xylose at a level above 15%, even though
xylose is usually an uncommon component of pectins.
Terminally linked xylose might be linked directly to positions 2 and 3 of galacturonic acid chains (Kikuchi et al.
1996b). The level of terminally linked xylose in embryogenic callus was twice that in non-embryogenic callus.
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Functions of the cell wall
Embryogenic callus
Plant
Somatic embryo
Seedling
+Auxin
Acquisition of
embryogenic
competence
High level of
endogenous ABA
+Auxin
Development
of embryo
Loss of
competence
Non-embryogenic callus
^
Germination
Low level of
endogenous ABA
+ABA
Maturation
Dormancy
Late embryo
Weak intercellular attachment
Low level of endogenous ABA
Low responsiveness to ABA
Fig. 1 System for culture of carrot cells. Because embryogenic callus already has some characteristics of the embryo and its endogenous level of ABA is quite high in the presence of auxin, similar expression of genes is sometimes observed in embryogenic callus and in
late embryos supplied with ABA. Non-embryogenic callus derived from embryogenic callus has decreased ability to produce and to respond to ABA, as well as reduced mutual attachment of cells.
Non-embryogenic callus
E1CC(EIP18)
EDGP(GP57)
Embryogenic callus
Fig. 3 Illustration of the localization of two extracellular proteins in a mature carrot seed. The two proteins, namely, the soluble glycoprotein EDGP (extracellular dermal glycoprotein) and
the insoluble protein EICC (extracellular insoluble cystatin of carrot), were originally detected in the culture medium of suspensioncultured carrot callus and are probably synthesized by the zygotic
embryo at the late stage of embryogenesis. They accumulate in the
interspace between the embryo and the endosperm, in particular
around the radicle (EDGP) and in the cell wall of the embryo and
in the space facing the inner side of the endosperm (EICC) in the
seed. EDGP is capable of binding to insulin-like peptides of
animals and plants and might possibly be involved in cellular
signal transduction in seeds. EICC inhibits a proteinase from germinating seeds of carrot and might function to protect the embryo
from the proteinases produced for the digestion of the endosperm. E and ES indicate embryo and endosperm, respectively.
Functions of the cell wall
A similar xylose-linked polygalacturonic acid (xylogalacturonan) has also been found in apple pectin (Schols et al.
1995).
The terminal xylose of xylogalacturonan and the
arabinose of neutral-sugar blocks might be involved in intercellular attachments and in development of the plant
through their binding to the hemicellulosic polysaccharides
of cell walls via some as yet unidentified factors.
Intermolecular linkages of pectins—To characterize
possible linkages between pectin neutral sugars and hemicellulose, we examined the levels of phenolic acids in embryogenie and non-embryogenic callus, since diferiilic acid linkages formed by peroxidase in cell walls are known to be
important in the intermolecular linkages of the noncellulosic polysaccharides and in cell growth in monocotyledonous plants (Fry 1986). In culturs of rice cells, amino
acids added to the medium induced formation of a suspension of finely dispersed cells while, at the same time, levels
of ferulic and diferulic acid decreased. Thus, it is possible
that feruloyl and diferuloyl esters between matrix polysaccharides might be involved in the aggregation of cultured
rice cells (Kato et al. 1994). However, our results exclude
the possibility that phenolic acids might be the main functional elements in the intermolecular linkages in carrot cell
walls because the total amount of phenolic acids was the
same in both embryogenic and non-embryogenic callus and
the level of phenolic acids was extremely low (unpublished
data). The possible presence of covalent crosslinks between
xyloglucans and rhamnogalacturonans via arabinan residues was reported recently in cotton cell walls (Fu and
Mort 1997). Confirmation of such linkages should lead to
further progress in the elucidation of mechanisms of intercellular attachment and their impotance in plant development.
Somatic embryogenesis and extracellular proteins—
Zygotic embryogenesis, including maturation and dormancy, progresses with interactions between maternal tissues
and the endosperm. By contrast, somatic embryogenesis occurs in vitro and is independent of maternal and other nonembryonic tissues. Thus, culture of somatic embryos can
provide clues to help us determine whether or not physiological events in seeds are derived from properties of the
embryo itself.
The extracellular surface of each plant cell is composed of the cell wall and the middle lamela, both of which
consist mainly of polysaccharides and proteins. The proteins can be classified as structural proteins (e.g., extensin,
hydroxyproline-rich glycoprotein, glycine-rich protein, pro-
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line-rich protein), enzymes and other soluble proteins (arabinogalactan protein, lectin) (Cassab and Varner 1988,
Keller 1993, Showalter 1993). In cells and somatic embryos
in suspension culture in liquid medium, the proteins involved in the physiological functions of the cell wall are
known sometimes to be released into the medium (Cordewener et al. 1991, Kreuger and Van Hoist 1993, Satoh et
al. 1986, Van Engelen et al. 1995). Therefore, we can analyze the materials that function in cell walls, without contamination by cytoplasmic substances, if we collect and analyze the culture medium of cells and somatic embryos.
Some proteins found in the culture medium are known
to be involved in the development of somatic embryos.
Namely, a 53- to 57-kDa grycoprotein released from proembryogenic masses of Citrus cultured cells was reported to
suppress somatic embryogenesis in vitro of Citrus (Gavish
et al. 1992). Furthermore, a 38-kDa cationic peroxidase
and a 32-kDa acidic endochitinase, purified from the culture medium of carrot cells, reversed the inhibition of carrot somatic embryogenesis by tunicamycin, an inhibitor of
glycosylation (Cordewener et al. 1991), and the arrest of
somatic embryogenesis in a temperature-sensitive mutant
of carrot (De Jong et al. 1992), respectively. In another
study, addition of arabinogalactan proteins obtained from
the culture medium and from carrot seeds modified the embryogenic potential of cultured carrot cells (Kreuger and
Van Hoist 1993). While these observations suggest the involvement of extracellular glycoproteins in somatic embryogenesis in vitro, the roles of the various extracellular proteins in the formation and functions of zygotic embryos
and seeds in intact plants remain obscure.
By contrast to the above results, we found that some
of the extracellular proteins detected in the medium of carrot cultured cells could also be found around and/or in
zygotic embryos in carrot seeds (Satoh and Fujii 1988,
Satoh et al. 1995). Therefore, we have focused on the functions in carrot seeds of such proteins, namely, EDGP (a
soluble extracellular dermal glycoprotein, previously designated GP57) and EICC (an insoluble extracellular cystatin
of carrot, previously designated EIP18).
A soluble glycoprotein that binds insulin-like peptides
from cultured cells and in seeds of carrot—When we analyzed the soluble glycoproteins that had been released from
carrot cells into the culture medium by SDS-PAGE, we
found that the presence of two glycoproteins, with molecular masses of 65 kDa and 57 kDa (GP57), respectively, was
related to the formation of somatic embryos (Satoh et al.
1986). The 65-kDa glycoprotein was released specifically
Fig. 2 A schematic illustration showing possible structures of pectins and the intermolecular associations of pectin molecules in embryogenic and non-embryogenic callus of carrot. The pectin of embryogenic callus contains more xylogalacturonan, as well as a larger terminal arabinan region and a smaller middle galactan region in its neutral-sugar chain, than the pectin of non-embryogenic callus. The
pectin molecules are linked to each other by calcium bridges (Ca) and might be linked to hemicellulose molecules via putative interactions (question marks) that involve the terminal arabinan regions of neutral-sugar chains and the xylose of the xylogalacturonan in pectin molecules. GA, R, X, G and A indicate galacturonic acid, rhamnose, xylose, galactan and arabinan, respectively.
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Functions of the cell wall
from embryogenic callus that had been cultured in medium
without 2,4-D, in which callus can form somatic embryos.
GP57 was released from the same embryogenic callus,
when the callus was cultured in medium with 2,4-D, in
which no somatic embryos were formed. Non-embryogenic callus released only GP57 in the presence and in the
absence of 2,4-D. In an immunohistochemical study with a
monoclonal antibody raised against purified GP57 that was
specific to the peptide portion of the glycoprotein, GP57
was found in the space between the zygotic embryo and the
endosperm of dry seeds and it was especially concentrated
around the radicle (Fig. 3). Moreover, the level of GP57 decreased during germination (Satoh and Fujii 1988). GP57 is
probably synthesized and released from the zygotic embryo
at the later stages of embryogenesis in the developing seed.
It is likely that GP57 is synthesized by zygotic embryos and
callus but not by somatic embryos because of a difference
in the level of endogenous ABA and responsiveness to
ABA.
GP57 was also found in the dermal tissues of vegetative organs, in the endodermis and epidermis of young
roots, in the periderm of mature taproots and in the epidermis of petioles and young leaves, and expression of the
gene for GP57 was induced by wounding in the mature
taproot (Satoh et al. 1992). The stronger expression of the
gene in non-embryogenic callus might have been related to
wounding. Therefore, the name of the glycoprotein, GP57,
was changed to EDGP (extracellular dermal glycoprotein).
The amino acid sequence of EDGP contains a short motif
that is present at the active site of aspartyl proteases, such
as pepsin, but no proteolytic activity was detected in preparations of EDGP. These results suggest the possibility that
EDGP might be involved in the response of plants to biotic
and/or abiotic stress.
A homolog of EDGP has been found in soybean,
namely, 7S basic globulin. This protein is released when
soybean seeds are soaked in hot water for several hours
(Hirano et al. 1992). This basic globulin is capable of binding bovine insulin and insulin-like growth factors from
animals, and carrot EDGP has the same property (Komatsu and Hirano 1991). Furthermore, the basic globulin was
reported by Watanabe et al. (1994) to have protein kinase
activity. A 4-kDa peptide, designated leginsulin, that can
bind to the basic globulin and compete with insulin for
binding to the basic globulin was isolated from young
taproots of germinating soybean seeds. Leginsulin had a
stimulatory effect on the phosphorylation activity of basic
globulin. From these results, it was proposed that the basic
globulin-leginsulin system might be involved in cellular
signal-transduction in plant seeds (Watanabe et al. 1994).
The physiological functions of EDGP (GP57) and soybean 7S basic globulin are still unclear. Further efforts are
needed to reveal the true functions of these novel extracellular proteins in plant seeds.
Insoluble cystatin in the culture medium and in seeds
of carrot—We identified another unusual extracellular protein (an 18-kDa extracellular insoluble protein; EIP18) in
the culture medium of carrot cells. This insoluble protein
contained no glycan moieties (Satoh et al. 1995). EIP18
was found in amorphous particles that were suspended in
the culture medium of callus and it was not solubilized by
treatment of these particles with EDTA, with Triton X-100
plus NaCl or with LiCl. However, it was partially solubilized by treatment with NaSCN and was entirely solubilized
by treatment with urea. An immunohistochemical study
revealed that in the carrot plant, this extracellular insoluble
protein is present only in the seeds, being located both in
the cell wall of the embryo and at the inner edge of the
endosperm that faces the interspace between the embryo
and the endosperm (Fig. 3).
The cloning and sequencing of a cDNA for EIP18
revealed that EIP18 has a signal sequence and that the protein is homologous to inhibitors of cysteine proteinases,
namely, phytocystatins (family-4 cystatins; Barrett 1987).
Therefore, we changed the name of EIP18 to EICC (extracellular insoluble cystatin of carrot; Ojima et al. 1997). Although some plant cystatins are known to have a signal sequence, there is no information as to whether they are
secreted from cells or are accumulated in protein bodies.
EICC is the only plant cystatin whose secretion from cells
has been confirmed to date.
The most significant characteristic of EICC is its insolubility. EICC is the first cystatin that has been found in an
insoluble form under non-denaturing conditions. The insolubility of EICC is not considered to be due directly to ionic
and hydrophobic bonds but to be due, at least in part, to
hydrogen bonds. We found no characteristics that might be
expected to cause the insolubility of the protein in the primary sequence of EICC. No glycine-rich, proline-rich or
hydroxyproline-rich domains, which have often been
found in the insoluble proteins of plant cell walls (Keller
1993, Showalter 1993), nor any hydrophobic region other
than the signal sequence, were found in EICC. Because the
EICC expressed in yeast cells was secreted from the cells
and deposited in an insoluble form in the yeast cell wall,
the insolubility appears to be due to the structure of the
EICC polypeptide itself (Ojima et al. 1997).
Physiological functions of the extracellular insoluble
cystatin—A suspension of EICC purified from the culture
medium of carrot callus had an inhibitory effect on two cysteine proteinases, namely, papain and chymopapain, but
not on bromelain. This specificity is identical to that of
some other cystatins (Barrett 1987), but EICC also weakly
inhibited the activity of trypsin, a serine proteinase. Moreover, EICC also had an inhibitory effect on a crude preparation of proteinase(s) from germinating carrot seeds (Ojima
et al. 1997). EICC was required at high levels for the inhibition of proteinases (molar ratio of EICC to papain, 100 :
Functions of the cell wall
1), probably as a consequence of the insolubility of EICC.
Some of the EICC might be in an active form but associated with insoluble EICC on the surface of the insoluble
EICC.
Proteinase inhibitors have a variety of physiological
functions. In the seeds of many plants, large amounts of inhibitors specific for serine proteinases, such as trypsin, are
known to exist in protein bodies, and they are considered
to take a part in defense mechanisms by inhibiting the
digestive enzymes of insects. By contrast, cysteine proteinase inhibitors, such as oryzacystatins in rice are considered to be involved in the control of endogenous proteinases for the digestion of the endosperm and/or in the
protection of seeds from damage by blight and harmful insects (Kondo et al. 1989 a, b, Liang et al. 1991, Michaud et
al. 1993).
In carrot seeds, it is possible that EICC located in and
around the embryo protects the embryo from proteolysis by the cysteine proteinases that are synthesized for digestion of the endosperm during seed germination. EICC
might not penetrate the endosperm to inhibit the digestion
of endosperm because of its insolubility.
The expression of EICC was detected in maturing seed
and it was markedly promoted by the application of ABA
to somatic embryos in cell culture, as also observed in the
case of the expression of some late embryogenesis (LEA)
proteins. EICC might be synthesized in embryos, with promotion of its expression by endogenous ABA, and it might
accumulate in the cell walls of the embryo and in the space
around embryo during the formation of the seed.
We are now analyzing the cysteine proteinases that appear during the germination of carrot seeds. Our results to
date suggest a possible role for the novel extracellular insoluble cystatin in the control of endogenous proteolysis in
seeds and they should contribute to the further characterization of interactions of cells and tissues in seeds of higher
plants.
Concluding remarks—The first part of this review summarized the characteristics and the advantages of carrot
cells in culture for studies of plant development. Then the
significance of cell wall polysaccharides in the development
and morphogenesis of higher plants was considered, with a
focus on the functions of pectins in relationships between
cells. The possibility was then discussed that the arabinoseand xylose-containing neutral-sugar chains of pectin might
be responsible for the pectin-hemicellulose linkages and
for the intercellular attachment of cells in carrot callus. If
similar changes in the neutral-sugar chains of pectins could
be demonstrated in developmental processes in intact
plants, we would have a greater understanding of the roles
of intercellular attachment in morphogenesis and development in higher plants. Further analysis of mutant cells and
of plants with defects in intercellular attachment and morphogenesis should also help to confirm the significance of
367
pectin-hemicellulose linkages in both intercellular attachment and morphogenesis.
Evidence was presented that various extracellular proteins are involved in somatic embryogenesis in vitro. Some
of these proteins are also present in zygotic embryos and
seeds, possibly being involved in tissue-to-tissue interactions for the formation and functions of zygotic embryos
and seeds in intact plants. EDGP (GP57) and EICC
(EIP18) might be involved in cellular signal transduction
and in interactions between the embryo and endosperm, respectively. Further molecular biological research is now required for elucidation of the actual functions of these proteins in plants.
Future studies of the functions of the macromolecules,
such as polysaccharides and proteins, in an apoplastic
region, the cell wall, should enhance our understanding of
the molecular mechanisms of morphogenesis and the development of higher plants, in particular insofar as these
mechanisms relate to the interactions among cells.
The author expresses his sincere appreciation to Emeritus Professors T. Fujii and H. Harada and to Professors H. Kamada and
S. Sakai of the University of Tsukuba for their valuable help and
encouragement during all work in his laboratory. The author is
also grateful to all of his collaborators for their cooperation
throughout his research. Parts of this work were supported by a
Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
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(Received December 4, 1997; Accepted March 5, 1998)