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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 361 362 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 363 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. 364 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- 365 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. 366 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. 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