J. Plant Res. 111: 133-148, 1998 Journal of Plant Research (~) by The Botanical Society of Japan 1998

JPR Symposium

Dynamic Function and Regulation of Apoplast in the Plant Body

Naoki Sakurai

Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi Hiroshima, 739 Japan

Apoplast is the internal environment of plant. Our body energy. The direction of two flows is reverse. Usually, the posses the intemal environment that consists of blood, two routes are allotted to xylem vessel and sieve tube. lympha, and tissue fluid. Plant cells are also cultivated and A German plant scientist, E. ML~nch (1930) coined the term surrounded by a liquid medium in the apoplast. As well as apoplast. He termed the water path apoplast, and the other various important functions of the internal environment in part symplast. He noticed that not only xylem vessel but our body, apoplast function is also prerequisite for the plant also cell wall space is the water path and recognized them life. There are so far seven distinct functions of apoplast. as a single continuum of transportation system of water, but (1) Growth regulation with apoplastic enzymes by altering ignored the space for gas exchange. In terms of circulation cell-wall properties through degradation, synthesis, orienta- of mass flow in plant body described above, apoplast should tion and cross-linking of supra molecules of cell walls, such include the air space for gas exchange. Therefore, the as cellulose, non-cellulosic polysaccharides, proteins, and description that plant body consists of apoplast and symplast lignin; (2) Skeleton sustained by cellulose microfibrils, lignin is a simple and clear definition of plant body. One main and various types of structural proteins with distinctively high function of apoplast is the transportation of sugar with solar content of hydroxyproline, proline or glycine; (3) Skin to energy, though a burdensome problem whether or not sieve defend symplast from desiccation, pathogens' attack and tube is to be included in apoplast remains. harmful environmental factors, such as ozone and sulfur A French physiologist, C. Bernard (1813---1878), coined two dioxide; (4) Transportation route for not only well-known terms, internal secretion (1859) and internal environment molecules of water, inorganic ions, and sugar, but also plant (1865). He described that internal environment is a true hormones, oligosaccharides and proteins; (5) Homeostasis physiological environment inherent to individual organism, of the internal environment by controlling ionic balance, pH and every external influence can reach living cells only and water content; (6) Adhesion of cell to cell; (7) Gas through this internal field. This idea led to the concept, exchange space of leaf for photosynthesis. The present "homeostasis" raised by an American physiologist, W.B. article reviews the recent 'advances in studies of several Cannon (1871--,1945) in 1932. I would emphasize that apo- aspects of the dynamic function and regulation of apoplast. plast is the internal physiological environment of plant body. Table 1 summarizes the function of apoplast. There are Key. words~ Cell wall--Defense--Fruit ripening-- seven classified functions, though some of them are still Glucanase -- IAA -- Transportation speculative. Molecules that exist in apoplast and play a role in the specified function, are listed in the table as apoplastic molecules. Enzymes involved in the functions Stephen Hales (1677--.1761) tried to find a heart in plant are listed in the next column. Enzymes that are confirmed body. He might have been influenced by Harvey's famous or suggested to be localized to apoplast are marked with theory of blood circulation published in 1628. Hales' con- asterisks. The classification of Table 1 is analogous to that clusion was somehow strange; there were two hearts, one is of our body. We have bones (as endoskeleton), skin, and root and the other leaf. His conclusion, however, includes blood vessel (as transportation route). Our lymphatic system reality. Some plants generate root pressure. Transpiration is fighting against pathogens to recognize invading organ- of water through leaf stomata is definitely the motive force isms by an immune system. Our individual cells are for water movement from soil. Is there circulation system in attached each other by cell-cell adhesion protein such as plant ? Does plant circulate blood ? These might be the transmembrane proteins (cadherins, connexins, integrins, and next questions that Hales wanted to answer. selectins) and extracellular matrix (fibronectin). Our air Plant does not circulate liquid, but water passes through space for gas exchange is, of course, lung. The only plant body in one direction. Carbon dioxide that enters difference between animal and plant for the above classifi- through leaf stomata into internal air space is converted to cation, is the growth regulated by apoplast in plant. The sugar by the aid of photosynthesis. Produced sugar that plant cells extend or expand many folds after cell division, contains solar energy flows down to stem and root. In this while our body essentially grows (extends and expands) by sense, plant has a definitive route for flow of water and solar cell division, except for the fat cells which can expand after 134 N. Sakurai

Table 1 Functions of apoplast and related molecules in plants

Function Apoplastic molecules Related enzymes Remarks 1. Growth regulation a) Degradative 1, 3 : 1, 4-/~-Glucan 1, 3 : 1, 4-/%Glucanase* Poaceae Xyloglucan 1, 4-~-Glucanase* Dicots Fruit softening Endo-xyloglucan tTansferase* Dicots Callose 1, 3-,8-Glucanase* Pollen tube Expansin ? Dicots Pectin Polygalaoturonase Fruit softening b) Synthetic Cellulose UDPG- pyrophosphorylase Gibbrellin Pectin and callose Transferase (Golgi) Pollen c) Directional Cellulose 1, 4-,8-Glucan synthase Cortical microtubles Gibberellin d) Cessational Xyloglucan Endo-xyloglucan llansferase* Dicots Diferulic acid (DFA) Peroxidase (POX)* 2. Skelton Cellulose Cellulose synthase Secondary wall Lignin, H202, Phenylpropane PAL*, CAD*, POX*, SOD Secondary wall Glycine-rieh proteins Vascular tissues Proline-rich proteins Extensin 3. Skin a) Dessication defence Cutin Lipid tTansfer protein* Epidermis Mucilage Transferase (Golgi) Root b) Pathogen Thionins Pectin (plant) Polygalacturonase* Extracellular 1, 3 : 1, 6-,8-Glucan (pathogen) 1, 3-,8-Glucanasas* Extracellular Chitin (pathogen) Chitinases* Extracellular ATP ? ATPase* Chitin-binding-protein Extensin ? Proline-rich protein (S-glyceproteins) S- RNase* c) Air pollutant Ozone Ascerbic acid Ascerbate peroxidase* Sulfur dioxide Ferulic acid, H202 Peroxidese* 4. Transportation route H20 Inorganic ions Hormone (IAA, Cytokinin) IAAId oxidase* Sugars Invertase* Extracellular Oligosaccharides Hydrolase ? Xylem exudate Oligopeptides Protease ? Xylem exudate Suberin 5. Homeostasisof internalenvironment a) Ion balance P, CI, K, Ca, B etc Channel Vacuole b) pH H + H+-ATPase Plasma membrane c) Water content Cell walls Cell wall pore Mucilage Transferase (Golgi) Cactus 6. Adhesion (cell to cell) Arabinogalactan Integlin-like protein 7. Air-space CO2, 02 Endo-1, 3 : 1, 4-,8-glucanase* Spongy tissue 8. Unknown Lectin Extensine Glycine-rich proteins Proline-rich proteins Arabinogalaotan proteins PAL phenylalanin ammonialyase; CAD, cinnamyl alcohol dehydrogenase; POX, peroxidase; SOD, superoxide dismutase. Dynamic Function and Regulation of Apoplast ]3_~ cell division even at middle age. In this review, I disucss gene(s) for the above glucanases is still lacking. Recently recent advances of some aspects of the function and regu- Inouhe and Nevins (1997) proposed that the non-enzymatic lation of apoplast listed in Table 1. proteins regulate the activities of wall-bound glucanases in maize coleoptiles. Growth Regulation Two isozymes (El and Ell) of endo-1, 3 : 1, 4-~-glucanases were found in germinated barley grains (Woodward and Growth regulation in the apoplast can be classified into Fincher 1982). Although these isozymes may not be bound four phases; a) stimulation of growth by degradation of non- to cell walls, they certainly function as a apoplastic enzyme cellulosic polysaccharides by auxin action, b) stimulation of on the seed germination to digest the endosperm cell walls, growth by synthesis of cell wall polysaccharides in response which facilitates the access of (z-amylase to endosperm to gibberellin, c) directional regulation of cellulose microfi- starch granules (Fincher 1989). The gene for isoenzyme El brils by gibberellin action, and d) cessation of growth by was also transcribed in young leaves and roots and the forming cross-links among phenylpropanoids, cell wall pro- expression was stimulated by iAA in young leaves but inhib- teins, non-cellulosic polysaccharides and cellulose. ited in young roots (Slakeski and Fincher 1992a), suggesting the involvement of El in the growth regulation. It, however, Growth regulation by degradation of non-cellulosic polysac- is surprising that the gene for El was not expressed in the charides by auxin action coleoptiles (Slakeski and Fincher 1992b). This regulation involves degradative changes in cell wall In most dicots, the target polysaccharides in auxin-in- architecture induced by auxin. In Poaceae, major non- duced elongation growth are xyloglucan. The degradation cellulosic polysaccharides, 1, 3 : 1, 4-/%glucan, are degraded of xyloglucan induced by auxin action has been reported in by the action of auxin, leading to the decrease in viscosity of many dicot plants (Labavitch 1981, Nishitani 1995). The wall matrix polysaccharides, and to cell wall loosening that details in this aspect of apoplastic function are reviewed in causes a decrease in water potential of symplast (Sakurai this series (Nishitani 1998). 1991). There have been dozens of reports of wall-bound Growth of pollen and pollen tube is somehow different exo- and endo-glucanases that are speculated to cause the from those of other plant tissues. Endo-~-l, 3-glucanase is glucan degradation leading to elongation (Table 2). Cell- necessary for normal development of microspore that is wall bound glucanases have been thought to be responsible surrounded by callose on maturation (Tsuchiya et a/. 1995) for the auxin-induced degradation of 1, 3 : 1, 4-/%glucan, but and for pollen tube growth (Roggen and Stanley 1969). Cell the sufficient purification of responsible enzymes for an walls of pollen tube also contains callose (1, 3-/%glucan) that amino acid sequence analysis has not yet been carried out is not ubiquitous in other plant tissues, except for cell plate until recently. One of the glucanases, exo-1, 3:1, 4-/~- and healing tissue at a wounded site (Li et al. 1997). glucanase, was recently isolated and purified from the cell Recently two abundant cell wall glycoproteins (66 and 69 walls of dark grown barley seedlings by LiCI extraction kDa) have been reported to accumulate in pollen tubes of (Kotake eta/. 1997). The N-terminus amino acid sequence tobacco (Capkova et al. 1997). Cultivation in the continuous was almost identical to that of the /~-glucan exohydrolase presence of an inhibitor of glycosylation of the proteins (Exo II) enzyme found in germinated barley grains (Hrmova et reduced callose deposition in the secondary cell wall and al. 1996). Another isozyme (Exo I) reported in the germinated inhibited the pollen tube growth, suggesting the role of the grains was not found in the barley cell walls (Kotake et al. glycoproteins in wall formation during the pollen tube growth. 1997). The direct evidence to show the auxin stimulated the Expansin is the protein that is speculated to facilitate

Table 2. Apoplastic glycanases Glycanase Plant References Glucanase Endo-1, 3 : 1, 4-,8- corn Huber and Nevins 1980, 1981,1982, Hatfield and Nevins 1987, Inouhe and Nevins 1991 barley Woodward and Fincher 1982, Slakeski et al. 1990 Endo-1, 4-,8- avocado Hatfield and Nevins 1986 Exo-1, 3 : 1, 4-,8- pea Wu et al. 1996, Matsumoto et al. 1997 azuki Tabuchi et al. 1997 soybean Koyama et al. 1981 corn Huber and Nevins 1980, 1981, 1982, 1_,3- brador and Nevins 1989, Inouhe and Nevins 1991 Exo-1, 3-,8- barley Hrmova et al. 1996, Kotake et al. 1997 1,3-,8- pollen Tsuchiya et al. 1995 ,8-galactosidase Chick pea Dopico et al. 1989a ~ -galactosidase Chick pea Dopico et al. 1989b Endo-xyloglucan transferase azuki Nishitani 1992 136 N. Sakurai breaking of the hydrogen bonding between cellulose microfi- days in intact plants, while auxin-induced elongation lasts brils and other non-cellulosic polysaccharides, which is only several hours. Even in auxin-induced elongation extensively reviewed in this series (Shieh and Cosgrove 1998). growth, the addition of carbon source in the incubation Another degradative events related to plant growth is the medium extended the effect of auxin on the growth. It is fruit softening (Huber 1983, Fischer and Bennett 1991). likely that newly formed non-cellulosic polysaccharides Pectin degradation during fruit softening was most exten- maintains the extensibility or elasticity of the cell walls. sively studied. Polygalacturonase that hydrolyzes pectic Precisely controlled coordination of degradation and synthe- substances in the fruit cell walls is induced by ethylene that sis of cell wall polysaccharides regulated by hormones is accelerates fruit ripening. The facts, however, that trans- complex and still challenging. genic tomato fruit expressing only 1% of the polygalactur- onase of the normal level sustained appreciable pectin Directional regulation of cellulose microfibrils by gibberellin solubilization but did not soften (Smith et al. 1990) and the action transgenic ripening mutant augmented with sufficient The regulation of orientation of cellulose microfibrils is the polygalacturonase activity to solubilize polygalacturonans other mechanism by which GA stimulates elongation growth did not show fruit softening (Giovannoni et al. 1989), have de- (Shibaoka 1994), along with the increase in cell wall synthe- emphasized the role of pectin degrading enzyme in fruit sis. The orientation of the microfibrils is organized by softening. Xyloglucan degradation in tomato (Sakurai and cortical microtubules. Orientation of microtubules is gover- Nevins 1993), persimmon (Cutillas-lturralde et al. 1994), and ned by GA action. Though the mechanism by which GA avocado (O'Donoghue and Huber 1992, Sakurai and Nevins reoriented microfibrils is still obscure, the extensin in the cell 1997) and cellulose degradation in avocado (O'Donoghue et walls stabilized microtubules in BY-2 protoplasts, suggesting al. 1994) have been reported as the primary determinant of the association of cortical microtubules with cell walls the fruit softening. The precise determination of fruit texture through transmembrane proteins (Akashi et al. 1990). will be required to conclude the primary events of cell wall Whether GA acts on diverse sites to cause different bio- changes caused by a specific enzyme. Recently, a laser chemical and morphological events in parallel ways, or it Doppler method has been applied to the firmness measure- triggers one key reaction to cause the sequence of events ment of fruit (Muramatsu et al. 1997). This technique can leading to the cellulose re-orientation remains to be answer- remotely measure the physical properties of apoplast. ed.

Stimulation of growth by synthesis of cell wall polysacchar- Cessation of growth by forming cross links among cell wall ides constituents Growth is also controlled by the synthesis of cell wall The final phase of growth regulation is cessation. Xylog- materials secreted from symplast (non-cellulosic polysac- lucan, the major non-cellulosic polysaccharides in dicots, is charides) and on the plasma membrane (cellulose). This believed to cross-link to cellulose molecules by hydrogen type of regulation was reported in GA-induced elongation of bonds (Hayashi 1989). Cleavage and reconnection of xylog- intact lettuce hypocotyls (Kawamura et al. 1976), of excised lucan molecules are mediated by endoxyloglucan transfer- oat stem segments (Montague and Ikuma 1975), and of ase (Nishitani 1995). The gene expression was confined at excised epicotyl segments of azuki bean (Nishitani and the tissue that had terminated elongation growth. This Masuda 1982). The GA-induced growth was more promi- topic is reviewed in this symposium (Nishitani 1998). nent in the presence of sucrose, suggesting that the GA Extensin was first coined as a structural protein necessary action requires sugar source. There seems two possible for the extension growth (Lamport 1970), but later, it rather mechanisms by which GA stimulates elongation growth of terminates the growth by cross-linking each other and stems. One is the elevation of UDP-sugar level to acceler- constructing a rigid network with cellulose microfibrils. ate the synthesis of cell wall polysaccharides (Montague and Ferulic acid (FA), a well-known precursor for lignin formation, Ikuma 1978). The other is the decrease in water potential of is bound to .some matrix polysaccharides and binds each symplast by accumulating sugar, partly because of an other to generate diferulic acid cross-linking that causes increase in wall-bound invertase activity by GA action rigid cell walls to cease elongation growth (Kamisaka et al. (Miyamoto and Kamisaka 1988a, b). Although the cell wall 1990). The reduced level of PAL and TAL activity by invertase is regarded as crucial to supply sink tissues with osmotic stress decreased the level of cell wall-bound FA carbohydrates via an apoplastic pathway (SchwebeI-Dugue and DFA, maintaining the elasticity of the cell walls (Wa- et al. 1994), the mechanism by which GA stimulates wall- kabayashi et al. 1997). This topic is dealt by Hoson (1998) in bound invertase remain unknown. Cytokinin induced cell this review series. wall-bound (extracellular) invertase but not the intracellular ones in suspension culture of Chenopodium rubrum (Ehne8 Skeleton and Toitsch 1997). Wall-bound invertase was also reported to regulate seed development (Cheng et al. 1996). Length of cellulose molecules differs in primary and sec- There is still not a concrete picture how the synthesis of ondary walls. The degree of polymerization (DP; number of cell wall participates in regulating elongation growth. GA- glucose unit per molecule) of woody cellulose is several induced elongation growth is usually observed for several thousands in primary walls and around 14,000 in secondary Dynamic Function and Regulation of Apoplast ~37 walls (Haigler 1985). Two distinctive populations of DPs of organized network-structure, re-shed the light on the role of cellulose molecules were found in primary walls of actively pectin in the skeletal structure of the cell walls. This aspect dividing suspension-cultured cells, one with DP of abut 500 of pectin function is reviewed in this series (Matoh 1988). and the other with DP of 2,000-3,000 (Blaschek et al. 1982). There are five classes of non-enzymatic cell-wall proteins Barley stems had averaged DP of 1,000 (Kokubo et al. 1991). (Showalter 1993); the extensin, the glycine-rich proteins It seems that higher DP is found in secondary walls of tissues (GRPs), the proline-rich proteins (PRPs), the solanaceous that sustain heavy part of plant, such as tree trunk, probably lectins, and the arabinogalactan proteins (AGPs). Though because higher DP is necessary for stronger association of the function of most of the proteins is still unknown, the one cellulose molecule with another by hydrogen bonds. extensin and the PRPs are likely involved in skeletal function, Cellulose exhibits very high Young's modulus, indicating high since their high content of tyrosine raises the possibility of strength to applied force. isodityrosine cross links among the extensin, PRPs and Curiously enough, there is little direct evidence that lignin GRPs. The predominant localization of GRP on vascular plays a role in reinforcing secondary walls of plant body, tissues infers the reinforcement of the cell walls and/or the though the covalent cross-links between lignin and polysac- reduction of friction of fluid transported. charides was proposed (liyama et al. 1994). A com- putational study demonstrated that coniferyl alcohol, one of Skin the lignin precursors, and its trimer could absorb on the surface of cellulose microfibrils (Houtman and Atalla 1995). Desiccation defense The author claimed that the structure of lignin is not amor- The fact that cuticle mutation of Sorghum bicolor with phous, as is often suggested, and the synthesis or the course altered epicuticular wax structure and less cuticle deposition of polymerization is modified by the polysaccharide compo- increased epidermal conductance to water vapor and sus- nents of the cell walls, such as cellulose. The compu- ceptibility to the fungal pathogen (Jenks et al. 1994), demon- tational analysis, however, does not still guarantee the role of strated the primary and indispensable function of cuticle in lignin in physical rigidity of cell wall architecture. Recently an aerial environment, i.e., the defense against desiccation Turner and Somerville (1997) identified the mutant of and pathogen (Post-Beitternmiller 1996). Cutin is a compo- Arabidopsis that produced less cellulose and exhibited less nent of plant cuticle, a continuous layer of surface waxes of stiffens of inflorescence stems, but contained the same epidermis of plant body except for roots, and prevents an un- amount of phenolics extracted from the cell walls as that of controlled evaporation of water. Though the constituents of wild type. The results suggest that lignin seems not to cutin are many known fatty acids, their insolubility and contribute to the stiffness of tissues. structural heterogeneity have hampered investigations of Lignin is usually found in the secondary cell walls of biosynthesis of cutin. Recent NMR technique applied to an vascular tissue, such as xylem and phloem. Inner wall intact cutin sample revealed that cutin is composed of surface of xylem vessel is lined with lignin. Hydrophobicity hydroxylated fatty acids and phenyl propanoids such as p- of lignin may reduce friction of water with vessel walls. coumaric acid (Stark et al. 1989). Cutin contains high Recent immunocytological study reveals that antibody aliphatic and less aromatic residue than suberine that is also against phenylalanine ammonia lyase (PAL) or cinnamyl hydrophobic substance controlling water path in apop~ast. alcohol dehydrogenase (CAD) is bound specifically to secon- Although the precise biosynthesis of cutin is still unknown, dary walls of tracheary elements derived from Zinnia meso- there are several genes involved in the wax formation of phyll cells (Nakashima et al. 1997). Activity of PAL bound epidermis in Arabidopsis, CER1 (Aarts et al. 1995), CER2 (Xia tightly to cell walls increased on the differentiation of tra- et al. 1996), and CER3 (Jannoufa et al. 1996), and in maize, cheary elements. If PAL and CAD are present in apoplast, Glossy2 (Tacke et al. 1995). The some gene products seem one may anticipate in apoplastic space the existence of the to be involved in the fatty acid biosynthesis and chain enzyme precursors, such as phenylalanine, p-coumaryl elongation pathway (Post-Beitternmiller 1996). aldehyde, coniferyl aldehydes, sinapyl aldehyde, and enzyme Beside the gene analyses of cutin biosynthesis, un- products, such as cinnamic acid, p-coumaric alcohol, expected factors have been recently focused on cutin coniferyl alcohol, and sinapyl alcohol. Although it is prob- biosynthesis. Lipid-transfer proteins (LTPs) that were first able that the aldehyde molecules can readily move across found in plants twenty years ago and had been thought to be plasma membrane, the existence of these aldehyde mole- involved in movement of lipids, such as membrane cules in the apoplast has not yet been demonstrated. biogenesis, have been recently identified as the key proteins Another important agent for lignin formation in apoplast is for cutin formation (Hendrik et al. 1994, cf. Kader 1996). hydrogen peroxide. The existence and generation mecha- LTPs are basic, ca. 9-kDa proteins and predominantly local- nism of H202 in apoplast has been widely accepted (Ogawa ized to the apoplast. Isolation of LTP genes, in fact, et al. 1997). revealed the presence of a signal peptide, indicating that Pectin was thought to be localized on middle lamella, and LTPs could enter the secretary pathway. LTPs can transfer not to be involved in the rigidity of the plant cell walls. The phosphatidylcholine, phosphatidylinositol to membranes. fact, however, that boron and calcium were excluively found Although the precise mechanism of transfer of lipid to cutin in the cell walls, and both ions perticipates in the joint of high by LTPs is still unknown, the specific expression of LTP gene molecular weight of pectin polymers to make a highly- to epidermal cells in maize (Sossouztzov et al. 1991), carrot 138 N. Sakurai

(Sterk et al. 1991), Arabidopsis (Thoma et al. 1994), and barley These results indicated that 02- generation in the cell wall (Gausing 1994), but not in the roots of various plants (see ref. fractions seems to be catalyzed by cell wall-bound perox- cited in Kader 1996), strongly suggested the role of LTPs in ydase and that the plant cell walls alone are able to respond cutin formation. The movement, however, of cutin to the to the elicitor non-specifically and to the suppressor in a plant surface is still the mysterious aspect of cuticular wax species-specific manner. Such kind of specificity was also formation. found in the cell wall-bound ATPase of pea. The Mrs are Root mucilage, mainly composed of polysaccharides, different from those of plasma membrane. Its activity was secreted from symplast to the apoplast has been proposed specifically reduced by the suppressor of pea pathogen and not only to accumulate water from soil (Oades 1978) but also not inhibited by neomycin that inhibits the activity of plasma to from a primary site for colonization of the root by microbial membrane ATPase (Shiraishi et al. 1997), suggesting that the symbiosis and pathogens (Bacic et al. 1986, Hinch and putative receptors for the suppressor tightly bind to cell wall- Clarke 1980). The role of root mucilage in protecting from bound ATPase or ATPase is the receptor itself (Kiba et al. desiccation has been recently challenged (McCully and 1996). This kind of response of cell walls not via symplast Boyer 1997). In some cacti, mucilage in the stems play a seems to be a knee jerk in plants. role as a capacitor for apoplastic water (Nobel et al. 1992). Self-incompatibility is not a pathogenic defense of apo- Root mucilage represents an important apoplastic pool for AI plastic function, but the self-recognition system not to in wheat (Archambault et al. 1996). produce zygotes after self-pollination (Newbigin eta/. 1993, Nasrallah and Nasrallah 1993). The self-incompatibility in Pathogen Brassica is controlled by the S-locus that contains at least Apoplast of some plants contains a small toxic proteins two genes, SLG (S locus glycoprotein) and SRK (S locus against pathogens. The existence of thionins, a group of receptor kinase). The S-glycoprotein has been demonstrat- low-molecular-weight polypeptides (ca. 5 kDa) with toxic ed to have RNase activity (McClure et al. 1989). The effects on bacteria, fungi, yeast and animal and plant cells, antibody, generated using synthetic peptides corresponding was suggested in 1885 as a substance lethal to brewer's to the SLG gene, labeled the intercellular matrix in the stigma yeast in wheat flower (Bohlmann and Ape11991). All thionins and transmitting tissue of the style, through which the pollen known so far conserves 6 or 8 cysteine residues, disulfide tube grows, and the cell walls in the epidermis of the bridges of which renders thionins a high heat stability. placenta (Anderson et al. 1989), demonstrating that the S- Though the thionins found in endosperm of cereals may glycoprotein is secreted to the apoplast. Although the function as storage proteins, a new group of thionins found involvement of these two types of genes and proteins in self- in barley leaves may play an important role during the incompatibility are clearly demonstrated, the direct mecha- defense against pathogens (Bohlmann et al. 1988, Ebrahim- nism by which the pollen growth is inhibited is still unknown. Nesbat et al. 1989). The new thionins could be detected in the cell wall and the central vacuoles (Reimann-Philipp et al Air pollutant 1989a), especially in the outer cell walls of the epidermis Ozone is the phytotoxic air pollutant in industrialized (Reimann-Philipp et al. 1989b). The results strongly suggest countries. The phytotxicity of ozone is due to its high that these thionins are part of a resistance mechanism of oxidant capacity and to its generation of toxic superoxide barley plants against pathogens. anion, hydroxyl radicals and hydrogen peroxide. Ascorbate The oligosaccharide signals derived from plant cell walls peroxidase was found in the of pumpkin (Ranieri et al. 1996), or pathogen walls are well known in a defense strategy of in Vigna angularis (Takahama and Oniki 1994), in Sedum plant against pathogens (Darvill and Albersheim 1984, Lamb album (Castillo and Greppin 1986) and in bean (Peters et al. and Dixon 1990, Lamb et al. 1989, Yoshikawa et al. 1993). 1988). The activity of apoplastic ascorbate peroxidase, and Microbial polygalacturonase or pectic lyase digests levels of antioxidant and phenols in the apoplast increased in polygalacturonans of plant cell walls on infection, and the response to ozone fumigation to some plants, suggesting oligogalacturonide fragments elicits the phytoalexin to attack that ozone stimulates the antioxidant systems in the apoplast the pathogen (Jin and West 1984). On infection of path- and that ascorbate peroxidase activity, ascorbic acid levels ogens, plant chitinase or glucanase secreted to cell walls or and phenols are an important system for defense against air into the vacuoles, generates chitosan oligomers or 3, 6-/~- pollutant. The fact that chloroplast ascorbate peroxidase is glucan oligomers from the pathogen cell walls that, in turn, not involved in protection against ozone (-I-orsethaugen et al. elicit phytoalexin (Simmons 1994, Bol et al. 1990). It is 1997), suggests the role of apoplast in detoxification of interesting that vacuolar chitinase or glucanase are acidic, ozone. Apoplast peroxidase is also involved in sulfur diox- while extracellular ones are basic. ide detoxification (Pfanz and Oppmann 1991). It has been recently found that the "isolated" cell walls of pea and cowpea plants is able to generate superoxide in Transportation Route for Hormones and Proteins response to fungal signal molecules (Kiba et al. 1997). A suppressor synthesized by a pea pathogen, which suppres- It is well known that ions and water are transported ses the normal response of pea to the pathogen, inhibited through xylem vessel and assimlilated sugars through sieve the activity of superoxiside generation in the "isolated" pea tube. The sugars do not entirely move through cytoplasm cell walls, but not in the cowpea cell walls (Kiba et al. 1997). via plasmodesmata from the mesophyll cells to the phloem, Dynamic Function and Regulation of Apoplast 139 but via apoplast (Giaquinta 1983, Wilson and Lucas 1986). Table 3. Endogenousconcentration of IAA in apoplast and Sorbitol, a major photoassimilate translocated in the phloem symplast of etiolated squash hypocotylsa of woody Rosaceae, is also predominantly loaded to the Age Part IAA concentration (x 10-8M) phloem via apoplast (Moing et al. 1997). Water and solute morement in the apoplast were recently reviewed (Canny Apoplast Symplast 1995). Besides the function of apoplast of tranportation day 2 upper 40.5 5.4 route for ions and photoassimilates, apoplast also serves the lower 4.2 2.7 route for other substances, which has not drawn much day 3 upper 9.7 3.9 attention. lower 1.8 1.7

Auxin a, Tsurusaki et al. (1997a). Recently, auxin (indole-3-acetic acid, IAA) has been found to be synthesized in apoplast (Tsurusaki et al. 1997b). by exogenous IAA was observed from the level of 10.7 M that Furthermore, the endogenous level of IAA was higher in the is higher than the actual endogenous IAA concentration apoplast than in the symplast (-I'surusaki eta/. 1997a). even in the actively growing tissue. Is the endogenous IAA I had been thinking of one mystery of endogenous level of really effective in controlling the stem growth? To clarify this IAA in plants. It was the fact that the optimum concentra- discrepancy, we separately determined the IAA levels of tion of IAA exogenously applied to plant stem tissues in apoplast and symplast of etiolated squash hypocotyls by a elongation growth (10-s M) (Nissen 1985) was 1,000times GC-SIM technique with I~_,6-1AA as an internal standard higher than the actual endogenous level (10 8 M) in stem (Table 3). Apoplastic IAA concentration of upper part of the tissues (Akiyama et al. 1983). A typical dose response curve stems was 8 times higher than symplastic concentration on of IAA in elongation growth of excised stem segments is day 2. On day 3, the apoplastic concentration of the upper shown in Fig. 1. The stimulation of stem elongation induced part decreased to 1X10 -7 M but it is still effective in inducing elongation. The symplastic concentrations, however, on day 3 in the upper and lower parts never exceeded 0.6x10 -7 M that are not effective in elongation. The concentration, 4• z M, found in the apoplast on day 2 in the upper part SY AP level level Optimum seems to be appropriate to account for the vital role of endogenous IAA in controlling intact growth of stems. Small increment or decrement of IAA concentration ranging from 10-7 to 10-6 M is sufficient to effectively regulate the 0 growth rate. The growth rate of etiolated squash hypocotyls reached maximum on day2 and decreased after day3 (Sakurai eta/. 1987a). The upper part of the hypocotyl consisted of younger and shorter cells than the lower part, and actively grews on day 2 (Sakurai et al. 1987b). There- Cm fore it is highly probable that the apoplast IAA regulates the stem growth but not the symplast one. 0 How does apoplastic IAA affect the elongation growth of cells ? Venis et al. (1990) have reported that an imperme- able IAA analogue that retains auxin activity, caused elonga- tion growth and changes in membrane potential, even it did not enter into the cells (Venis et al. 1990). Auxin-binding protein (ABP1) has been postulated to mediate auxin action 10-9 10-8 10-7 10~ 10-5 on cell elongation process (Macdonald 1997). This protein IAA concentration (M) contains the endoplasmic reticulum (ER) retention signal (KDEL). The electron microscopic immuno-cytochemistry, Fig. 1. Typical dose response curve of cell elongation of plant however, identified the localization of ABP1 not only on ER cell to exogenous IAA. Plant cells of excised segments elongates in response to exogenously applied IAA. The but also on cell walls (Jones and Herman 1993). The minimum IAA concentration that elicits cell elongation is positive labeling of Golgi apparatus also suggests that the usually around 10 7 M. The optimum concentration is ABP1 is transported to the cell walls via the secretory system. around 10-5 M. The concentration range from 10-7 to Furthermore, the auxin receptor on plasma membrane that is 10 5 M is effective in regulatingelongation response, since involved in auxin-induced elongation growth may face the relationbetween the responseand the concentration (in outside (Lobbler and Kl&mbt 1985). These results strongly log scale) is linear. The endogenous IAA levelof symplast was below 5• 8 M and that of apoplast was 4• -7 M suggest that auxin exerts its effect on the elongation growth inactively growing regions of etiolated squash hypocotyls. from outside of the cells. This theory also favors to explain The apoplastic IAA concentration, but not symplasticone, the response of tissues to auxin that is moved basipetally effectively regulates elongation growth. with polarity. The next question was where the apoplast 140 N. Sakurai

IAA came from, secreted from symplast or synthesized in the root with cytokinin transportation in xylem sap is also found apoplast ? in grafting experiment with wild type and mutant of pea, the Kuraishi (1974) reported that a dwarf mutant of barley, latter causes a substantial decrease in the concentration of strain "uzu", grew slower than the corresponding isogenic zeatin riboside in the xylem sap (Beveridge et al. 1997). The normal strain, because the mutant produced less IAA. We wild type sections normalize the cytokinin concentraion in demonstrated that the cell wall fraction of strain uzu the sap of mutant roots, whereas mutant sections cause wild produced less IAA from indole-3 acetaldehyde than that of type roots to reduce the concentraion in the sap, suggesting normal strain, but there was no difference of the activity of the role of the shoot in the regulaiton of cytokinin export from IAA synthesis in cytoplasmic fraction between these two the root. strains, indicating that the less IAA in the dwarf strain results Cytokinin is also found in phloem sap of Xanthium strumar- from the less activity of IAA synthesis in the cell walls, and ium (Phillips and Cleland 1972) and Lupinus albus (Taylor et the main site of IAA synthesis in barley is apoplast (Tsurusaki al. 1990). The results do not directly show that plant leaf is et al. 1997b). Interestingly, the optimum pH (7.0) and Km the site of cytokinin production, since pink-pigmented value (5/~M) of the apoplastic enzyme activity to convert facultatively methylotrophic bacteria make up more than indoleacetaldehyde to IAA were different from those of the 90% of the bacteria on plant leaves, and they produced symplastic activity (pH 6.0 and 31/zM), suggesting that the zeatin and zeatin riboside (Holland 1997). apoplast enzyme is different from the symplast one, and the apoplastic enzyme activity is not a contaminant of cytoplas- Other hormones mic one. Root senses the soil drying and increases the level of ABA The fact that IAA is synthesized in apoplast leads to an in xylem sap (Liang et al. 1997). This increase may be due interesting hypothesis. It is commonly or unconsciously to the less capability of degradation of ABA in root tissues. accepted that the plant perceives the changes in environ- Leaf abscission process is also reported to be initiated by the mental signals with a high sensitivity because of its immobil- increase in ABA level in xylem sap, which requires the ity, and adapts their metabolic activity to the changes. If previous accumulation of ABA in roots (Gomez-Cadenas et the apoplast is able to synthesize IAA, all the cell do not have al. 1996). to respond to the external signals. When one susceptible 1-Aminocyclopropane-l-carboxylic acid, a precursor of cell responds to the important but weak physical signal and ethylene, is also delivered from root to shoot through xylem starts to synthesize IAA in the apoplast, the synthesized vessel and the level increased 3 folds in response to flood apoplastic IAA also influences neighboring cells as well as stress in tomato, though the level of ABA and nitrate in xylem the cell itself through auxin-receptor bound on plasma vessel did not (Else et al. 1995). Polyamines identified by membrane. This type of signal perception and transduction HPLC were also found in xylem exudates of several plants is known as an autocrine signaling in animal. A cell (Fridman et al. 1986). The major component was putrescine secretes signaling molecules that can bind back to its own and more putrescene was found in older than in younger receptors. The autocrine signaling is most effective when sunflower plants. neighboring cells should be stimulated simultaneously. It is Recently, the existence of gibberellin in xylem sap of tea probable that apoplast is used as a field of transduction of tree has been reported (Oyama et al. 1997). Therefore, autocrine signaling of hormones, although the involvement of virtually almost all the plant hormones are transported the apoplastic activity of IAA synthesis in the polar transport through xylem vessel. of auxin remains to be answered. Proteins Cytokinins The fact that xylem sap contains proteins stems from the Cytokinin is believed to be synthesized in root tips and original work done by Wilson in 1923 (Wilson 1923). He translocated into shoot through xylem vessel (Torrey 1976). found the activities of catalase, peroxidase, and reductases It has been recently claimed that cytokinins are produced by in guttation fluid (essentially xylem sap). Recent studies on the microbial symbionts of plants, not by plants themselves xylem sap proteins confirmed that the acidic proteins includ- (Holland 1997). Nevertheless the xylem vessel is still the ing peroxydase with pl 4.6 are present in xylem sap (Biles et route of cytokin transportation, since the many root-as- al. 1989), partly because the xylem cell walls may be nega- sociated bacteria are demonstrated cytokinin producers, t- tively charged (Biles and Abeles 1991). Stem exudate of Zeatin riboside and dihydrozeatin riboside are the major watermelon was shown to contain proteins that inhibited the components of transported cytokinin found in xylem sap growth of Fusarium oxysporum, the causal agent for Fusar- (Heindl eta/. 1982, Jameson et all 1987, Soejima, et al. 1992, ium wilt of watermelon (Biles et al. 1990). Jones 1973), or conjugated zeatin in rice (Soejima et al. 1992). There were five main protein bands appeared on SDS- The fluxes increased during late flowering and early pod PAGE collected from xylem sap of squash (Satoh et al. 1992). formation in soybean, and the removal of pod of soybean, Two of them (40 and 75 kDa) were highly mannosilated, and which delayed the leaf senescence, increased the level of increased for about 24 hr after cutting. Another polype- cytokinins in xylem sap (Nooden eta/. 1990), suggesting that ptides (32 kDa) appeared soon after cutting, disappeared and pod or pod formation depressed cytokinin production in or then reappeared again 48-64 hr after cutting. The level of translocation from the root. The cross-talk of shoot and the last two polypeptides (14 and 19 kDa) were present Dynamic Function and Regulation of Apoplast 141

Table 4. ApoplastpHs in plant tissues

Plant pH Method

Vicia faba 4.5~ Microelec~Tode Zea mays* 4.9___0.03b Confocalmicroscopy with NERF Vicia faba 5.2~5.9c Fluorescence Taxus baccata 5.25___0.25a 6-Glucoxy-7-hydroxyeoumarin Ginkgo bibloba 5.50+__0.15d ibid Rumex lunaria 5.60___0.20d ibid Nymphaea alba 5.85_0.25d ibid Beta vulgaris 6.00_0.25d ibid Prunus armeniaca** 6.0--,3.4e pH electrode Primula palinuri 6.15___ 0.15 e 6-GIucoxy- 7-hydroxycoumarin Hordeum distichum 6.3___0.3 f C,en'~ifugation Populus nigra 6.38___0.19d 6-Glucoxy-7-hydroxycoumarin Spinacia oleracea 6.40___0.30 d ibid Gossypium hirsutum 6.7~7.5g Pressurechamber Helianthus annuus 6.77~7.42h Centrifugation 6.22___0.12~ 5-Carboxyfluoresceinand FITC dextran Prunus persica** 7.0--,4.2e pH electrode

a, Aloni et al. (1988); b, Tayloret al. (1996); c, Muhling et al. (1995); d, Pfanz and Dietz (1987); e, Ugaldeet al. (1988), f, Tetlow and Farrar(1993); g, Hartung et aL (1988), h, Dannel et al. (1995), i, Hoffmann et aL (1992). *, root;, *% mesocarp; all the other tissues are leaves or needle. constitutively. suggesting the active efflux of H + from symplast to apoplast Besides proteins in xylem sap, fructose, myo-inositol and in acively growing tissues. There are many factors to oligosaccharides composed of mainly of galactose, change apopalst pH. Fusicoccin accelerates the acidifica- arabinose and glucose are the major components of tran- tion, while gibberellic acid inhibited it. The high dosage of sported sugar in xylem sap in squash (Satoh et al. 1992) application of nitrate to the sunflower plant increased the apoplast pH in the laves, but not the pH of xylem sap, Homeostasis of Internal Environment suggesting that cotransport of nitrogen with proton across the plasma membrane consumed apoplastic protons (Dannel Our blood pressure, temperature, pH, and ion and sugar et al. 1995). The dosage of ammonium form of nitrogen, concentration are precisely regulated. Hydrostatic pressure however, decreased the apoplast pH in the sunflower leaves of plant body is equivalent to our blood pressure. The (Hoffmann et al. 1992). On the contrary, pH change in regulation of apoplastic pH and ion concentration are also apoplast affects the hormonal balance in the plant. The likely (Grignon and Sentenac 1991). There is, so far, almost change in pH on dehydration enhanced the release of ABA no information of active regulation of temperature of plant from mesophyll cell of cotton leaves into the apoplastic fluid body, though salicylic acid is known to raise temperature of (Hartung et al. 1988). male reproductive tissues (Raskin 1992) pH condition of apoplast is essentially determined by the Plant apoplast responds to environmental stresses in activity of plasma membrane-bound H+-ATPase and mem- different ways (Dietz 1997). pH and redox control in the brane transport of solutes, but pectic substances in the cell apoplast serves a mechanism to respond to the environmen- walls also affects the ion concentrations and pH in apoplast. tal signals, such as gravity (Taylor eta/. 1996). Cation exchange capacity of the pectic substances serves a pH in the apoplast has been measured in many plant reservoir function of ions, especially K + and Ca ++ (Grignon systems by pH electrode with a combination of centrifugation and Sentenac 1991). About 50% of the fixed anionic method for collecting apoplast fluid or impermeable fluores- charges in the cell walls of Horse bean and Yellow lupine cent dyes (Table 4). The measured pH range of apoplast is were protonated at pH 5.0, although pure galacturonic acid from 4.5 to 7. Fruit tissue showed an extremely low pH on and non-esterified pectin showed a pK of 3.2 (Sentenac and maturation, because of leakage of organic acid. Most of Grignon 1981). Therefore pK of the cell walls is bout 5.0. the data of apoplast pH falls on 5.5 to 6.5. Aloni eta/. (1988) Since the apoplastic pH, at least in a limited tissue or cells, showed that the initial pH of mesophyll tissue of soybean can be immediately changed in response to light, water was estimated to be 4.5, and the tissue acidified the unbuf- deficit, IAA, and nitrogen dose, pH in the free space of fered solution placed on the tissue from pH 6.0 to 5.1, apoplast is dynamically regulated (Grignon and Sentenac ]42 N. Sakurai

1991). be very narrow, 98--.100% in apoplast space. When the The precise mechanism of homeostasis for phosphate ion relative humidity is 98%, negative water potential of the cell in the apoplast has been recently found (Mimura 1995). wall surface reaches --2.8 MPa, corresponding to 1.25 M of Since the available phosphate is present at very low concen- osmotic potential that exceeds the potential of most plant tration in the soil, plants always suffer phosphate deficient. cells. It is well known that absicic acid closes stomata, but When plant can uptake phosphate from the soil, it stores in it seems that this regulation does not work daily. Lu et al. the vacuoles of cells, especially in the old tissues. The (1997) proposed a mechanism where sucrose in the guard- stored phosphate is translocated to young tissues for cell cell wall is a physiological signal that regulates stomatal division and elongation growth on phosphate starvation. aperture. They showed that mesophyll-derived sucrose in Phosphate concentration in the cytoplasm is regulated in a guard-cell walls was sufficient to close stomatal opening by narrow range by an effective phosphate homeostasis in ca. 3/z m. Increase in the level of sucrose in the apoplast is which the phosphate in the vacuoles acts as buffer (Mimura caused either by high transpiration or high efflux of sucrose et al. 1990). The analysis of transportation of phosphate from mesophyll. The possible involvement of osmotic from an old to young barley leaf revealed that the phosphate potential and solutes of apoplast in the regulation of cell concentration of apoplast was maintained at 1 mM (Mimura expansion has been also pointed out (Grignon and Sentenac 1995). If the apoplastic phosphate concentration exceeds 1991, Canny 1995). this level, the vacuoles seems to uptake the extra phosphate Expression of one of the endo-1, 3 : 1, 4-/~-glucanase in the apoplast to maintain the apoplast concentration at 1 gene (Ell, see 1.a) is restricted to the aleurone layer of mM. The reason for the homeostatic regulation of phos- germinated barley grains. The gene for El, however, is phate in the apoplast remains to be answered. transcribed at relatively high levels in young leaves as well as in the scutellum and aleurone layer (Slakeski et a/. 1990). Adhesion (cell to cell) The El expression pattern implies that the isozyme El is involved in the separation of mesophyll cells and creation of Adhesion of cell to cell is indispensable for constructing a air space in leaf. ff each mes0phyU cell was completely multicellular tissue, but little is known about the responsible separated, there would be no translocation of photoas- substance. Classically, pectin is said to be localized on similates to phloem. The mechanism of the cell wall degra- middle lamella. When fruit softening was believed to be dation with remained junction between two cells at plas- caused by pectin degradation, the existence of pectin in the modesm, is intriguing. middle lamella seemed to be reasonable, since the pectic degradation might lead to separation of cells. Unequivocal Unknown evidence, however, for the pectin localization is still lacking. Quick-freeze, deep-etch electron microscopic observation Extensin, PRPs, GRPs, and AGPs are well known cell wall revealed that the treatment of carrot parenchyma tissues proteins, and various speculative proposals for their functions with polygalacturonase lost granular substances in the are described, but the biological significance is still contro- middle lamella, but there still remained a meshwork structure versial, though 3, 4-dehydroproline, the inhibitor of proryl (Tamura and Senda 1992), suggesting that the middle lamella hydroxylase, inhibits cell-wall assembly and cell division in does not consist solely of pectin. Heterogeneous composi- tobacco protoplast, suggesting that these hydroxyproline- tion of many cell types in the plant tissues made it difficult to rich proteins are essential for cell growth and development study the cell adhesion. Using suspension-cultured cells, (Cooper et al. 1994). Kikuchi et al. (1996) disclosed that the size of cell cluster of Several plant lectins induced the production of pisatin, a carrot cells was strongly and positively correlated with the phytoalexin of pea (Toyoda et al. 1995). Chitin-binding ratio of arabinose to galactose of neutral sugar chain of lectins are ubiquitous in plants, and most of the lectins are pectic fraction. The result suggests that not the calcium vacuolar proteins. Chitin-binding lectins, however, of bridge between acidic rhamnogalacturonans but the bran- Solanaceae. species, such as potato, thorn apple and ched arabinogalactan side-chain participates in the adhe- tomato, differ from all other chitin-binding lectins in that they sion of cells. are rich in arabinose, and contains the cystein/glycine-rich In animal cells, many types of transmembrane proteins and domain and the hydroxyproline/serine-rich domain (Raikhel extracellular matrix have been found to mediate cell-cell eta/. 1993). The second domain is extensively glycosylated adhesion. So far, there is only one report about the detec- and exists as a polyproline helix, a similar secondary struc- tion of such a protein, analogous to integrin, in root tips of ture to extensin or hydroxyproline-rich proteins. Chitin- Arabidopsis and Chara by immunofluorescence microscopy binding lectin is thought to have antifungal, and insect (Katembe eta/. 1997). The authors suggested the involve- antinutrient activity. ment of the integrin-like proteins in gravity perception. Ascorbate oxidase is secreted from symplast to apoplast in pumpkin (Esaka 1993) and bound to the cell walls of Vigna Air-space angularis (Takahama and Oniki 1994). Analysis of the promoter by transient expression assay in the pumpkin fruit Humiditiy control in the air space of apoplast is essential tissues suggested the existence of a cis-acting region for the water economy. The range of humidity control must responsible for IAA regulation (Kisu et al. 1997). Although Dynamic Function and Regulation of Apoplast ]43 the actual function of apoplastic ascorbate oxidase is still Orpin, T., Dedman, H., Tregear, G., Fernley, R. and unknown, ascorbate free radicals generated by ascorbate Clarke, A.E. 1989. Sequence variability of three alleles oxidase from ascorbic acid, enhanced the root growth of of the self incompatibility gene of Nicotiana a/ata. onion (Hidalgo et al. 1991) by increase in the uptake of Plant Cell 1: 483-491. nutrients into the vacuoles (Gonzales-Reyes et al. 1994). Archambault, D.J., Zhang, G. and Taylor, G.J. 1996. Accu- The association of apoplastic ascorbate oxidase with auxin- mulation of AI in root mucilage of an AI-resistant and an induced elongation was also reported in Vigna angularis AI-sensitive cultivar of wheat. Plant Physiol. 112: 1471-1478. (Takahama and Oniki 1994). Bacic, A., Moody, S. and Clarke, A. 1986. Structural analy- sis of secreted root slime from maize. Plant Physiol. Concluding Remarks 80: 771-777. Beveridge, C.A., Muffet, I.C., Kerhoas, L, Sotta, B., Miginiac, This past decade has witnessed many advances and new E. and Rameau, C. 1997. The shoot controls zeatin directions in apoplast research. Recently Robertson et al. riboside export from pea roots. Evidence from the (1997) extracted cell wall-bound proteins from Arabidopsis, branching mutant rms4. Plant J. 11: 339-345. carrot, French bean, tomato and tobacco, and 233 proteins Biles, C.L and Abeles, F.B. 1991. Xylem sap proteins. were selected on SDS-PAGE for protein sequencing, 146 Plant Physiol. 96: 597-601. proteins gave N-terminal data. They found that a signifi- Biles, C.L., Martyn, R.D. and Eilson, H.D. 1989. Isozymes cant proportion of wall proteins (74%) has not been previous- and general proteins from various watermelon cultivars ly described. These data represent a future protein and tissue types. HortScience 24: 810-812. resource and function for apoplast studies. BUes, C.L., Martyn, R.D. and Netzer, D. 1990. In vitro inhibi- The volume of apoplast was estimated at about 5--,10% tion activity of xylem exudates from cucurbits towards (Cosgrove and Cleland 1983, Sakurai and Kuraishi 1988) in Fusarium oxysporum microconidia. Phytoparasitica 1: stem tissues and 5---40% in leaves (Kramer 1983). If the 41-49. volume of vacuoles is estimated as 80% in the tissue, the Blaschek, W., Koehler, H., Selmer, U. and Franz, G. 1982. apoplast volume (10%) is compatible to the cytoplasmic Molecular weight distribution of cellulose in primary cell volume (10%). Apoplast is not a small space in the plant walls. Investigations with regenerating protoplasts, sus- body. A space surrounded by a membrane within a cell is pension cultured cells and mesophyll of tobacco. Planta 154: 550-555. toporogically outside. Therefore, the vacuole space is Bohlmann, H. and Apel, K. 1991. Thionins. Annu. Rev. 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