dynamic function and regulation of apoplast in the plant body

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

Naok i Sakura i

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

Apoplast is the internal environment of plant. Our body posses the intemal environment that consists of blood, lympha, and tissue fluid. Plant cells are also cultivated and surrounded by a liquid medium in the apoplast. As well as various important functions of the internal environment in our body, apoplast function is also prerequisite for the plant life. There are so far seven distinct functions of apoplast. (1) Growth regulation with apoplastic enzymes by altering cell-wall properties through degradation, synthesis, orientation and cross-linking of supra molecules of cell walls, such as cellulose, non-cellulosic polysaccharides, proteins, and lignin; (2) Skeleton sustained by cellulose microfibrils, lignin and various types of structural proteins with distinctively high content of hydroxyproline, proline or glycine; (3) Skin to defend symplast from desiccation, pathogens' attack and harmful environmental factors, such as ozone and sulfur dioxide; (4) Transportation route for not only well-known molecules of water, inorganic ions, and sugar, but also plant hormones, oligosaccharides and proteins; (5) Homeostasis of the internal environment by controlling ionic balance, pH and water content; (6) Adhesion of cell to cell; (7) Gas exchange space of leaf for photosynthesis. The present article reviews the recent 'advances in studies of several aspects of the dynamic function and regulation of apoplast.

Key. words~ Cell wall- -Defense--Fruit ripening-- Glucanase - - IAA - - Transportation

Stephen Hales (1677--.1761) tried to find a heart in plant body. He might have been influenced by Harvey's famous theory of blood circulation published in 1628. Hales' conclusion was somehow strange; there were two hearts, one is root and the other leaf. His conclusion, however, includes reality. Some plants generate root pressure. Transpiration of water through leaf stomata is definitely the motive force for water movement from soil. Is there circulation system in plant ? Does plant circulate blood ? These might be the next questions that Hales wanted to answer.

Plant does not circulate liquid, but water passes through plant body in one direction. Carbon dioxide that enters through leaf stomata into internal air space is converted to sugar by the aid of photosynthesis. Produced sugar that contains solar energy flows down to stem and root. In this sense, plant has a definitive route for flow of water and solar

energy. The direction of two flows is reverse. Usually, the two routes are allotted to xylem vessel and sieve tube.

A German plant scientist, E. ML~nch (1930) coined the term apoplast. He termed the water path apoplast, and the other �9 part symplast. He noticed that not only xylem vessel but also cell wall space is the water path and recognized them as a single continuum of transportation system of water, but ignored the space for gas exchange. In terms of circulation of mass flow in plant body described above, apoplast should include the air space for gas exchange. Therefore, the description that plant body consists of apoplast and symplast is a simple and clear definition of plant body. One main function of apoplast is the transportation of sugar with solar energy, though a burdensome problem whether or not sieve tube is to be included in apoplast remains.

A French physiologist, C. Bernard (1813---1878), coined two terms, internal secretion (1859) and internal environment (1865). He described that internal environment is a true physiological environment inherent to individual organism, and every external influence can reach living cells only through this internal field. This idea led to the concept, "homeostasis" raised by an American physiologist, W.B. Cannon (1871--,1945) in 1932. I would emphasize that apoplast is the internal physiological environment of plant body.

Table 1 summarizes the function of apoplast. There are seven classified functions, though some of them are still 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 are listed in the next column. Enzymes that are confirmed or suggested to be localized to apoplast are marked with asterisks. The classification of Table 1 is analogous to that of our body. We have bones (as endoskeleton), skin, and blood vessel (as transportation route). Our lymphatic system is fighting against pathogens to recognize invading organ- isms by an immune system. Our individual cells are attached each other by cell-cell adhesion protein such as transmembrane proteins (cadherins, connexins, integrins, and selectins) and extracellular matrix (fibronectin). Our air space for gas exchange is, of course, lung. The only difference between animal and plant for the above classification, is the growth regulated by apoplast in plant. The plant cells extend or expand many folds after cell division, while our body essentially grows (extends and expands) by cell division, except for the fat cells which can expand after

134 N. Sakura i

Table 1 Functions of apoplast and related molecules in plants

Function Apoplastic molecules Related enzymes Remarks

1. Growth regulation a) Degradative

b) Synthetic

c) Directional

d) Cessational

2. Skelton

3. Skin a) Dessication defence

b) Pathogen

c) Air pollutant Ozone Sulfur dioxide

4. Transportation route

5. Homeostasis of internal environment a) Ion balance b) pH c) Water content

6. Adhesion (cell to cell)

7. Air-space 8. Unknown

1, 3 : 1, 4-/~-Glucan 1, 3 : 1, 4-/%Glucanase* Xyloglucan 1, 4-~-Glucanase*

Endo-xyloglucan tTansferase* Callose 1, 3-,8-Glucanase* Expansin ? Pectin Polygalaoturonase Cellulose UDPG- pyrophosphorylase Pectin and callose Transferase (Golgi) Cellulose 1, 4-,8-Glucan synthase

Xyloglucan Diferulic acid (DFA) Cellulose Lignin, H202, Phenylpropane Glycine-rieh proteins Proline-rich proteins Extensin

Cutin Mucilage Thionins Pectin (plant) 1, 3 : 1, 6-,8-Glucan (pathogen) Chitin (pathogen) ATP ? Chitin-binding-protein Extensin ? Proline-rich protein (S-glyceproteins)

Ascerbic acid Ferulic acid, H202 H20 Inorganic ions Hormone (IAA, Cytokinin) Sugars Oligosaccharides Oligopeptides Suberin

P, CI, K, Ca, B etc H + Cell walls Mucilage Arabinogalactan Integlin-like protein CO2, 02 Lectin Extensine Glycine-rich proteins Proline-rich proteins Arabinogalaotan proteins

Endo-xyloglucan llansferase* Peroxidase (POX)* Cellulose synthase PAL*, CAD*, POX*, SOD

Lipid tTansfer protein* Transferase (Golgi)

Polygalacturonase * 1, 3-,8-Glucanasas* Chitinases* ATPase*

S- RNase*

Ascerbate peroxidase* Peroxidese *

IAAId oxidase* Invertase* Hydrolase ? Protease ?

Channel H+-ATPase

Transferase (Golgi)

Endo-1, 3 : 1, 4-,8-glucanase*

Poaceae

Dicots Fruit softening Dicots Pollen tube Dicots Fruit softening Gibbrellin Pollen Cortical microtubles Gibberellin Dicots

Secondary wall Secondary wall Vascular tissues

Epidermis Root

Extracellular Extracellular Extracellular

Extracellular Xylem exudate Xylem exudate

Vacuole Plasma membrane Cell wall pore Cactus

Spongy tissue

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 recent advances of some aspects of the function and regulation of apoplast listed in Table 1.

Growth Regulation

Growth regulation in the apoplast can be classified into four phases; a) stimulation of growth by degradation of non- cellulosic polysaccharides by auxin action, b) stimulation of growth by synthesis of cell wall polysaccharides in response to gibberellin, c) directional regulation of cellulose microfibrils by gibberellin action, and d) cessation of growth by forming cross-links among phenylpropanoids, cell wall proteins, non-cellulosic polysaccharides and cellulose.

Growth regulation by degradation of non-cel lu los ic polysaccharides by auxin action

This regulation involves degradative changes in cell wall architecture induced by auxin. In Poaceae, major non- cellulosic polysaccharides, 1, 3 : 1, 4-/%glucan, are degraded by the action of auxin, leading to the decrease in viscosity of wall matrix polysaccharides, and to cell wall loosening that causes a decrease in water potential of symplast (Sakurai 1991). There have been dozens of reports of wall-bound exo- and endo-glucanases that are speculated to cause the glucan degradation leading to elongation (Table 2). Cell- wall bound glucanases have been thought to be responsible for the auxin-induced degradation of 1, 3 : 1, 4-/%glucan, but the sufficient purification of responsible enzymes for an amino acid sequence analysis has not yet been carried out until recently. One of the glucanases, exo-1, 3:1, 4-/~- glucanase, was recently isolated and purified from the cell walls of dark grown barley seedlings by LiCI extraction (Kotake e ta / . 1997). The N-terminus amino acid sequence was almost identical to that of the /~-glucan exohydrolase (Exo II) enzyme found in germinated barley grains (Hrmova et al. 1996). Another isozyme (Exo I) reported in the germinated grains was not found in the barley cell walls (Kotake et al. 1997). The direct evidence to show the auxin stimulated the

gene(s) for the above glucanases is still lacking. Recently Inouhe and Nevins (1997) proposed that the non-enzymatic proteins regulate the activities of wall-bound glucanases in maize coleoptiles.

Two isozymes (El and Ell) of endo-1, 3 : 1, 4-~-glucanases were found in germinated barley grains (Woodward and Fincher 1982). Although these isozymes may not be bound to cell walls, they certainly function as a apoplastic enzyme on the seed germination to digest the endosperm cell walls, which facilitates the access of (z-amylase to endosperm starch granules (Fincher 1989). The gene for isoenzyme El was also transcribed in young leaves and roots and the expression was stimulated by iAA in young leaves but inhibited in young roots (Slakeski and Fincher 1992a), suggesting the involvement of El in the growth regulation. It, however, is surprising that the gene for El was not expressed in the coleoptiles (Slakeski and Fincher 1992b).

In most dicots, the target polysaccharides in auxin-induced elongation growth are xyloglucan. The degradation of xyloglucan induced by auxin action has been reported in many dicot plants (Labavitch 1981, Nishitani 1995). The details in this aspect of apoplastic function are reviewed in this series (Nishitani 1998).

Growth of pollen and pollen tube is somehow different from those of other plant tissues. Endo-~-l, 3-glucanase is necessary for normal development of microspore that is surrounded by callose on maturation (Tsuchiya et a/. 1995) and for pollen tube growth (Roggen and Stanley 1969). Cell walls of pollen tube also contains callose (1, 3-/%glucan) that is not ubiquitous in other plant tissues, except for cell plate and healing tissue at a wounded site (Li et al. 1997). Recently two abundant cell wall glycoproteins (66 and 69 kDa) have been reported to accumulate in pollen tubes of tobacco (Capkova et al. 1997). Cultivation in the continuous presence of an inhibitor of glycosylation of the proteins reduced callose deposition in the secondary cell wall and inhibited the pollen tube growth, suggesting the role of the glycoproteins in wall formation during the pollen tube growth.

Expansin is the protein that is speculated to facilitate

Table 2. Apoplastic glycanases

Glycanase Plant References Glucanase

Endo-1, 3 : 1, 4-,8-

Endo-1, 4-,8- Exo-1, 3 : 1, 4-,8-

corn

barley

avocado pea azuki

Huber and Nevins 1980, 1981,1982, Hatfield and Nevins 1987, Inouhe and Nevins 1991 Woodward and Fincher 1982, Slakeski et al. 1990 Hatfield and Nevins 1986 Wu et al. 1996, Matsumoto et al. 1997 Tabuchi et al. 1997

Exo-1, 3-,8- 1,3-,8-

,8-galactosidase ~ -galactosidase Endo-xyloglucan

transferase

soybean corn

barley pollen Chick pea Chick pea

azuki

Koyama et al. 1981 Huber and Nevins 1980, 1981, 1982, 1_,3- brador and Nevins 1989, Inouhe and Nevins 1991 Hrmova et al. 1996, Kotake et al. 1997 Tsuchiya et al. 1995 Dopico et al. 1989a Dopico et al. 1989b

Nishitani 1992

136 N. Sakurai

breaking of the hydrogen bonding between cellulose microfibrils and other non-cellulosic polysaccharides, which is extensively reviewed in this series (Shieh and Cosgrove 1998).

Another degradative events related to plant growth is the fruit softening (Huber 1983, Fischer and Bennett 1991). Pectin degradation during fruit softening was most extensively studied. Polygalacturonase that hydrolyzes pectic substances in the fruit cell walls is induced by ethylene that accelerates fruit ripening. The facts, however, that transgenic tomato fruit expressing only 1% of the polygalacturonase of the normal level sustained appreciable pectin solubilization but did not soften (Smith et al. 1990) and the transgenic ripening mutant augmented with sufficient polygalacturonase activity to solubilize polygalacturonans did not show fruit softening (Giovannoni et al. 1989), have de- emphasized the role of pectin degrading enzyme in fruit softening. Xyloglucan degradation in tomato (Sakurai and Nevins 1993), persimmon (Cutillas-lturralde et al. 1994), and avocado (O'Donoghue and Huber 1992, Sakurai and Nevins 1997) and cellulose degradation in avocado (O'Donoghue et al. 1994) have been reported as the primary determinant of the fruit softening. The precise determination of fruit texture will be required to conclude the primary events of cell wall changes caused by a specific enzyme. Recently, a laser Doppler method has been applied to the firmness measurement of fruit (Muramatsu et al. 1997). This technique can remotely measure the physical properties of apoplast.

days in intact plants, while auxin-induced elongation lasts only several hours. Even in auxin-induced elongation growth, the addition of carbon source in the incubation medium extended the effect of auxin on the growth. It is likely that newly formed non-cellulosic polysaccharides maintains the extensibility or elasticity of the cell walls. Precisely controlled coordination of degradation and synthesis of cell wall polysaccharides regulated by hormones is complex and still challenging.

Directional regulation of cellulose microfibrils by gibberellin action

The regulation of orientation of cellulose microfibrils is the other mechanism by which GA stimulates elongation growth (Shibaoka 1994), along with the increase in cell wall synthesis. The orientation of the microfibrils is organized by cortical microtubules. Orientation of microtubules is gover- ned by GA action. Though the mechanism by which GA reoriented microfibrils is still obscure, the extensin in the cell walls stabilized microtubules in BY-2 protoplasts, suggesting the association of cortical microtubules with cell walls through transmembrane proteins (Akashi et al. 1990). Whether GA acts on diverse sites to cause different bio- chemical and morphological events in parallel ways, or it triggers one key reaction to cause the sequence of events leading to the cellulose re-orientation remains to be answered.

Stimulation of growth by synthesis of cell wall polysaccharides

Growth is also controlled by the synthesis of cell wall materials secreted from symplast (non-cellulosic polysaccharides) and on the plasma membrane (cellulose). This type of regulation was reported in GA-induced elongation of intact lettuce hypocotyls (Kawamura et al. 1976), of excised oat stem segments (Montague and Ikuma 1975), and of excised epicotyl segments of azuki bean (Nishitani and Masuda 1982). The GA-induced growth was more promi- nent in the presence of sucrose, suggesting that the GA action requires sugar source. There seems two possible mechanisms by which GA stimulates elongation growth of stems. One is the elevation of UDP-sugar level to acceler- ate the synthesis of cell wall polysaccharides (Montague and Ikuma 1978). The other is the decrease in water potential of symplast by accumulating sugar, partly because of an increase in wall-bound invertase activity by GA action (Miyamoto and Kamisaka 1988a, b). Although the cell wall invertase is regarded as crucial to supply sink tissues with carbohydrates via an apoplastic pathway (SchwebeI-Dugue et al. 1994), the mechanism by which GA stimulates wall- bound invertase remain unknown. Cytokinin induced cell wall-bound (extracellular) invertase but not the intracellular ones in suspension culture of Chenopodium rubrum (Ehne8 and Toitsch 1997). Wall-bound invertase was also reported to regulate seed development (Cheng et al. 1996).

There is still not a concrete picture how the synthesis of cell wall participates in regulating elongation growth. GA- induced elongation growth is usually observed for several

Cessation of growth by forming cross links among cell wall constituents

The final phase of growth regulation is cessation. Xylog- lucan, the major non-cellulosic polysaccharides in dicots, is believed to cross-link to cellulose molecules by hydrogen bonds (Hayashi 1989). Cleavage and reconnection of xyloglucan molecules are mediated by endoxyloglucan transferase (Nishitani 1995). The gene expression was confined at the tissue that had terminated elongation growth. This topic is reviewed in this symposium (Nishitani 1998).

Extensin was first coined as a structural protein necessary for the extension growth (Lamport 1970), but later, it rather terminates the growth by cross-linking each other and constructing a rigid network with cellulose microfibrils. Ferulic acid (FA), a well-known precursor for lignin formation, is bound to .some matrix polysaccharides and binds each other to generate diferulic acid cross-linking that causes rigid cell walls to cease elongation growth (Kamisaka et al. 1990). The reduced level of PAL and TAL activity by osmotic stress decreased the level of cell wall-bound FA and DFA, maintaining the elasticity of the cell walls (Wa- kabayashi et al. 1997). This topic is dealt by Hoson (1998) in this review series.

Skeleton

Length of cellulose molecules differs in primary and secondary walls. The degree of polymerization (DP; number of glucose unit per molecule) of woody cellulose is 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 cellulose molecules were found in primary walls of actively dividing suspension-cultured cells, one with DP of abut 500 and the other with DP of 2,000-3,000 (Blaschek et al. 1982). Barley stems had averaged DP of 1,000 (Kokubo et al. 1991). It seems that higher DP is found in secondary walls of tissues that sustain heavy part of plant, such as tree trunk, probably because higher DP is necessary for stronger association of one cellulose molecule with another by hydrogen bonds. Cellulose exhibits very high Young's modulus, indicating high strength to applied force.

Curiously enough, there is little direct evidence that lignin plays a role in reinforcing secondary walls of plant body, though the covalent cross-links between lignin and polysaccharides was proposed (liyama et al. 1994). A com- putational study demonstrated that coniferyl alcohol, one of the lignin precursors, and its trimer could absorb on the surface of cellulose microfibrils (Houtman and Atalla 1995). The author claimed that the structure of lignin is not amor- phous, as is often suggested, and the synthesis or the course of polymerization is modified by the polysaccharide components of the cell walls, such as cellulose. The compu- tational analysis, however, does not still guarantee the role of lignin in physical rigidity of cell wall architecture. Recently Turner and Somerville (1997) identified the mutant of Arabidopsis that produced less cellulose and exhibited less stiffens of inflorescence stems, but contained the same amount of phenolics extracted from the cell walls as that of wild type. The results suggest that lignin seems not to contribute to the stiffness of tissues.

Lignin is usually found in the secondary cell walls of vascular tissue, such as xylem and phloem. Inner wall surface of xylem vessel is lined with lignin. Hydrophobicity of lignin may reduce friction of water with vessel walls. Recent immunocytological study reveals that antibody against phenylalanine ammonia lyase (PAL) or cinnamyl alcohol dehydrogenase (CAD) is bound specifically to secondary walls of tracheary elements derived from Zinnia mesophyll cells (Nakashima et al. 1997). Activity of PAL bound tightly to cell walls increased on the differentiation of tracheary elements. If PAL and CAD are present in apoplast, one may anticipate in apoplastic space the existence of the enzyme precursors, such as phenylalanine, p-coumaryl aldehyde, coniferyl aldehydes, sinapyl aldehyde, and enzyme products, such as cinnamic acid, p-coumaric alcohol, coniferyl alcohol, and sinapyl alcohol. Although it is probable that the aldehyde molecules can readily move across plasma membrane, the existence of these aldehyde molecules in the apoplast has not yet been demonstrated.

Another important agent for lignin formation in apoplast is hydrogen peroxide. The existence and generation mechanism of H202 in apoplast has been widely accepted (Ogawa et al. 1997).

Pectin was thought to be localized on middle lamella, and not to be involved in the rigidity of the plant cell walls. The fact, however, that boron and calcium were excluively found in the cell walls, and both ions perticipates in the joint of high molecular weight of pectin polymers to make a highly-

organized network-structure, re-shed the light on the role of pectin in the skeletal structure of the cell walls. This aspect of pectin function is reviewed in this series (Matoh 1988).

There are five classes of non-enzymatic cell-wall proteins (Showalter 1993); the extensin, the glycine-rich proteins (GRPs), the proline-rich proteins (PRPs), the solanaceous lectins, and the arabinogalactan proteins (AGPs). Though the function of most of the proteins is still unknown, the extensin and the PRPs are likely involved in skeletal function, since their high content of tyrosine raises the possibility of isodityrosine cross links among the extensin, PRPs and GRPs. The predominant localization of GRP on vascular tissues infers the reinforcement of the cell walls and/or the reduction of friction of fluid transported.

Skin

Desiccation defense The fact that cuticle mutation of Sorghum bicolor with

altered epicuticular wax structure and less cuticle deposition increased epidermal conductance to water vapor and susceptibility to the fungal pathogen (Jenks et al. 1994), demonstrated the primary and indispensable function of cuticle in an aerial environment, i.e., the defense against desiccation and pathogen (Post-Beitternmiller 1996). Cutin is a component of plant cuticle, a continuous layer of surface waxes of epidermis of plant body except for roots, and prevents an un- controlled evaporation of water. Though the constituents of cutin are many known fatty acids, their insolubility and structural heterogeneity have hampered investigations of biosynthesis of cutin. Recent NMR technique applied to an intact cutin sample revealed that cutin is composed of hydroxylated fatty acids and phenyl propanoids such as p- coumaric acid (Stark et al. 1989). Cutin contains high aliphatic and less aromatic residue than suberine that is also hydrophobic substance controlling water path in apop~ast. Although the precise biosynthesis of cutin is still unknown, there are several genes involved in the wax formation of epidermis in Arabidopsis, CER1 (Aarts et al. 1995), CER2 (Xia et al. 1996), and CER3 (Jannoufa et al. 1996), and in maize, Glossy2 (Tacke et al. 1995). The some gene products seem to be involved in the fatty acid biosynthesis and chain elongation pathway (Post-Beitternmiller 1996).

Beside the gene analyses of cutin biosynthesis, un- expected factors have been recently focused on cutin biosynthesis. Lipid-transfer proteins (LTPs) that were first found in plants twenty years ago and had been thought to be involved in movement of lipids, such as membrane biogenesis, have been recently identified as the key proteins for cutin formation (Hendrik et al. 1994, cf. Kader 1996). LTPs are basic, ca. 9-kDa proteins and predominantly localized to the apoplast. Isolation of LTP genes, in fact, revealed the presence of a signal peptide, indicating that LTPs could enter the secretary pathway. LTPs can transfer phosphatidylcholine, phosphatidylinositol to membranes. Although the precise mechanism of transfer of lipid to cutin by LTPs is still unknown, the specific expression of LTP gene to epidermal cells in maize (Sossouztzov et al. 1991), carrot

138 N. Sakurai

(Sterk et al. 1991), Arabidopsis (Thoma et al. 1994), and barley (Gausing 1994), but not in the roots of various plants (see ref. cited in Kader 1996), strongly suggested the role of LTPs in cutin formation. The movement, however, of cutin to the plant surface is still the mysterious aspect of cuticular wax formation.

Root mucilage, mainly composed of polysaccharides, secreted from symplast to the apoplast has been proposed not only to accumulate water from soil (Oades 1978) but also to from a primary site for colonization of the root by microbial symbiosis and pathogens (Bacic et al. 1986, Hinch and Clarke 1980). The role of root mucilage in protecting from desiccation has been recently challenged (McCully and Boyer 1997). In some cacti, mucilage in the stems play a role as a capacitor for apoplastic water (Nobel et al. 1992). Root mucilage represents an important apoplastic pool for AI in wheat (Archambault et al. 1996).

Pathogen Apoplast of some plants contains a small toxic proteins

against pathogens. The existence of thionins, a group of low-molecular-weight polypeptides (ca. 5 kDa) with toxic effects on bacteria, fungi, yeast and animal and plant cells, was suggested in 1885 as a substance lethal to brewer's yeast in wheat flower (Bohlmann and Ape11991). All thionins known so far conserves 6 or 8 cysteine residues, disulfide bridges of which renders thionins a high heat stability. Though the thionins found in endosperm of cereals may function as storage proteins, a new group of thionins found in barley leaves may play an important role during the defense against pathogens (Bohlmann et al. 1988, Ebrahim- Nesbat et al. 1989). The new thionins could be detected in the cell wall and the central vacuoles (Reimann-Philipp et al 1989a), especially in the outer cell walls of the epidermis (Reimann-Philipp et al. 1989b). The results strongly suggest that these thionins are part of a resistance mechanism of barley plants against pathogens.

The oligosaccharide signals derived from plant cell walls or pathogen walls are well known in a defense strategy of plant against pathogens (Darvill and Albersheim 1984, Lamb and Dixon 1990, Lamb et al. 1989, Yoshikawa et al. 1993). Microbial polygalacturonase or pectic lyase digests polygalacturonans of plant cell walls on infection, and the oligogalacturonide fragments elicits the phytoalexin to attack the pathogen (Jin and West 1984). On infection of pathogens, plant chitinase or glucanase secreted to cell walls or into the vacuoles, generates chitosan oligomers or 3, 6-/~- glucan oligomers from the pathogen cell walls that, in turn, elicit phytoalexin (Simmons 1994, Bol et al. 1990). It is interesting that vacuolar chitinase or glucanase are acidic, while extracellular ones are basic.

It has been recently found that the "isolated" cell walls of pea and cowpea plants is able to generate superoxide in response to fungal signal molecules (Kiba et al. 1997). A suppressor synthesized by a pea pathogen, which suppresses the normal response of pea to the pathogen, inhibited the activity of superoxiside generation in the "isolated" pea cell walls, but not in the cowpea cell walls (Kiba et al. 1997).

These results indicated that 02- generation in the cell wall fractions seems to be catalyzed by cell wall-bound peroxydase and that the plant cell walls alone are able to respond to the elicitor non-specifically and to the suppressor in a species-specific manner. Such kind of specificity was also found in the cell wall-bound ATPase of pea. The Mrs are different from those of plasma membrane. Its activity was specifically reduced by the suppressor of pea pathogen and not inhibited by neomycin that inhibits the activity of plasma membrane ATPase (Shiraishi et al. 1997), suggesting that the putative receptors for the suppressor tightly bind to cell wall- bound ATPase or ATPase is the receptor itself (Kiba et al. 1996). This kind of response of cell walls not via symplast seems to be a knee jerk in plants.

Self-incompatibility is not a pathogenic defense of apoplastic function, but the self-recognition system not to produce zygotes after self-pollination (Newbigin eta/ . 1993, Nasrallah and Nasrallah 1993). The self-incompatibility in Brassica is controlled by the S-locus that contains at least two genes, SLG (S locus glycoprotein) and SRK (S locus receptor kinase). The S-glycoprotein has been demonstrated to have RNase activity (McClure et al. 1989). The antibody, generated using synthetic peptides corresponding to the SLG gene, labeled the intercellular matrix in the stigma and transmitting tissue of the style, through which the pollen tube grows, and the cell walls in the epidermis of the placenta (Anderson et al. 1989), demonstrating that the S- glycoprotein is secreted to the apoplast. Although the involvement of these two types of genes and proteins in self- incompatibility are clearly demonstrated, the direct mechanism by which the pollen growth is inhibited is still unknown.

Air pollutant Ozone is the phytotoxic air pollutant in industrialized

countries. The phytotxicity of ozone is due to its high oxidant capacity and to its generation of toxic superoxide anion, hydroxyl radicals and hydrogen peroxide. Ascorbate peroxidase was found in the of pumpkin (Ranieri et al. 1996), in Vigna angularis (Takahama and Oniki 1994), in Sedum album (Castillo and Greppin 1986) and in bean (Peters et al. 1988). The activity of apoplastic ascorbate peroxidase, and levels of antioxidant and phenols in the apoplast increased in response to ozone fumigation to some plants, suggesting that ozone stimulates the antioxidant systems in the apoplast and that ascorbate peroxidase activity, ascorbic acid levels and phenols are an important system for defense against air pollutant. The fact that chloroplast ascorbate peroxidase is not involved in protection against ozone (-I-orsethaugen et al. 1997), suggests the role of apoplast in detoxification of ozone. Apoplast peroxidase is also involved in sulfur dioxide detoxification (Pfanz and Oppmann 1991).

Transportation Route for Hormones and Proteins

It is well known that ions and water are transported through xylem vessel and assimlilated sugars through sieve tube. The sugars do not entirely move through cytoplasm 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). Sorbitol, a major photoassimilate translocated in the phloem of woody Rosaceae, is also predominantly loaded to the phloem via apoplast (Moing et al. 1997). Water and solute morement in the apoplast were recently reviewed (Canny 1995). Besides the function of apoplast of tranportation route for ions and photoassimilates, apoplast also serves the route for other substances, which has not drawn much attention.

Auxin Recently, auxin (indole-3-acetic acid, IAA) has been found

to be synthesized in apoplast (Tsurusaki et al. 1997b). Furthermore, the endogenous level of IAA was higher in the apoplast than in the symplast (-I'surusaki eta/. 1997a).

I had been thinking of one mystery of endogenous level of IAA in plants. It was the fact that the optimum concentration of IAA exogenously applied to plant stem tissues in elongation growth (10 -s M) (Nissen 1985) was 1,000times higher than the actual endogenous level (10 8 M) in stem tissues (Akiyama et al. 1983). A typical dose response curve of IAA in elongation growth of excised stem segments is shown in Fig. 1. The stimulation of stem elongation induced

0

�9

Cm

0

SY AP level level Optimum

10 -9 10 -8 10 -7 10 ~ 10 -5

IAA concentration (M) Fig. 1. Typical dose response curve of cell elongation of plant

cell to exogenous IAA. Plant cells of excised segments elongates in response to exogenously applied IAA. The minimum IAA concentration that elicits cell elongation is usually around 10 7 M. The optimum concentration is around 10 -5 M. The concentration range from 10 -7 to 10 5 M is effective in regulating elongation response, since the relation between the response and the concentration (in log scale) is linear. The endogenous IAA level of symplast was below 5• 8 M and that of apoplast was 4• -7 M inactively growing regions of etiolated squash hypocotyls. The apoplastic IAA concentration, but not symplastic one, effectively regulates elongation growth.

Table 3. Endogenous concentration of IAA in apoplast and symplast of etiolated squash hypocotyls a

Age Part IAA concentration (x 10-8M)

Apoplast Symplast

day 2 upper 40.5 5.4 lower 4.2 2.7

day 3 upper 9.7 3.9 lower 1.8 1.7

a, Tsurusaki et al. (1997a).

by exogenous IAA was observed from the level of 10 .7 M that is higher than the actual endogenous IAA concentration even in the actively growing tissue. Is the endogenous IAA really effective in controlling the stem growth? To clarify this discrepancy, we separately determined the IAA levels of apoplast and symplast of etiolated squash hypocotyls by a GC-SIM technique with I~_,6-1AA as an internal standard (Table 3). Apoplastic IAA concentration of upper part of the stems was 8 times higher than symplastic concentration on day 2. On day 3, the apoplastic concentration of the upper 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 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 growth rate. The growth rate of etiolated squash hypocotyls reached maximum on day2 and decreased after day3 (Sakurai e t a / . 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- fore it is highly probable that the apoplast IAA regulates the stem growth but not the symplast one.

How does apoplastic IAA affect the elongation growth of cells ? Venis et al. (1990) have reported that an impermeable IAA analogue that retains auxin activity, caused elongation 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 on cell elongation process (Macdonald 1997). This protein contains the endoplasmic reticulum (ER) retention signal (KDEL). The electron microscopic immuno-cytochemistry, however, identified the localization of ABP1 not only on ER but also on cell walls (Jones and Herman 1993). The positive labeling of Golgi apparatus also suggests that the ABP1 is transported to the cell walls via the secretory system. Furthermore, the auxin receptor on plasma membrane that is involved in auxin-induced elongation growth may face outside (Lobbler and Kl&mbt 1985). These results strongly suggest that auxin exerts its effect on the elongation growth from outside of the cells. This theory also favors to explain the response of tissues to auxin that is moved basipetally with polarity. The next question was where the apoplast

140 N. Sakurai

IAA came from, secreted from symplast or synthesized in the apoplast ?

Kuraishi (1974) reported that a dwarf mutant of barley, strain "uzu", grew slower than the corresponding isogenic normal strain, because the mutant produced less IAA. We demonstrated that the cell wall fraction of strain uzu produced less IAA from indole-3 acetaldehyde than that of normal strain, but there was no difference of the activity of IAA synthesis in cytoplasmic fraction between these two strains, indicating that the less IAA in the dwarf strain results from the less activity of IAA synthesis in the cell walls, and the main site of IAA synthesis in barley is apoplast (Tsurusaki et al. 1997b). Interestingly, the optimum pH (7.0) and Km value (5/~M) of the apoplastic enzyme activity to convert indoleacetaldehyde to IAA were different from those of the symplastic activity (pH 6.0 and 31/zM), suggesting that the apoplast enzyme is different from the symplast one, and the apoplastic enzyme activity is not a contaminant of cytoplasmic one.

The fact that IAA is synthesized in apoplast leads to an interesting hypothesis. It is commonly or unconsciously accepted that the plant perceives the changes in environmental signals with a high sensitivity because of its immobil- ity, and adapts their metabolic activity to the changes. If the apoplast is able to synthesize IAA, all the cell do not have to respond to the external signals. When one susceptible cell responds to the important but weak physical signal and starts to synthesize IAA in the apoplast, the synthesized apoplastic IAA also influences neighboring cells as well as the cell itself through auxin-receptor bound on plasma membrane. This type of signal perception and transduction is known as an autocrine signaling in animal. A cell secretes signaling molecules that can bind back to its own receptors. The autocrine signaling is most effective when neighboring cells should be stimulated simultaneously. It is probable that apoplast is used as a field of transduction of autocrine signaling of hormones, although the involvement of the apoplastic activity of IAA synthesis in the polar transport of auxin remains to be answered.

Cytokinins Cytokinin is believed to be synthesized in root tips and

translocated into shoot through xylem vessel (Torrey 1976). It has been recently claimed that cytokinins are produced by the microbial symbionts of plants, not by plants themselves (Holland 1997). Nevertheless the xylem vessel is still the route of cytokin transportation, since the many root-associated bacteria are demonstrated cytokinin producers, t- Zeatin riboside and dihydrozeatin riboside are the major components of transported cytokinin found in xylem sap (Heindl eta/. 1982, Jameson et all 1987, Soejima, et al. 1992, Jones 1973), or conjugated zeatin in rice (Soejima et al. 1992). The fluxes increased during late flowering and early pod formation in soybean, and the removal of pod of soybean, which delayed the leaf senescence, increased the level of cytokinins in xylem sap (Nooden eta/ . 1990), suggesting that pod or pod formation depressed cytokinin production in or translocation from the root. The cross-talk of shoot and

root with cytokinin transportation in xylem sap is also found in grafting experiment with wild type and mutant of pea, the latter causes a substantial decrease in the concentration of zeatin riboside in the xylem sap (Beveridge et al. 1997). The wild type sections normalize the cytokinin concentraion in the sap of mutant roots, whereas mutant sections cause wild type roots to reduce the concentraion in the sap, suggesting the role of the shoot in the regulaiton of cytokinin export from the root.

Cytokinin is also found in phloem sap of Xanthium strumarium (Phillips and Cleland 1972) and Lupinus albus (Taylor et al. 1990). The results do not directly show that plant leaf is the site of cytokinin production, since pink-pigmented facultatively methylotrophic bacteria make up more than 90% of the bacteria on plant leaves, and they produced zeatin and zeatin riboside (Holland 1997).

Other hormones Root senses the soil drying and increases the level of ABA

in xylem sap (Liang et al. 1997). This increase may be due to the less capability of degradation of ABA in root tissues. Leaf abscission process is also reported to be initiated by the increase in ABA level in xylem sap, which requires the previous accumulation of ABA in roots (Gomez-Cadenas et al. 1996).

1-Aminocyclopropane-l-carboxylic acid, a precursor of ethylene, is also delivered from root to shoot through xylem vessel and the level increased 3 folds in response to flood stress in tomato, though the level of ABA and nitrate in xylem vessel did not (Else et al. 1995). Polyamines identified by HPLC were also found in xylem exudates of several plants (Fridman et al. 1986). The major component was putrescine and more putrescene was found in older than in younger sunflower plants.

Recently, the existence of gibberellin in xylem sap of tea tree has been reported (Oyama et al. 1997). Therefore, virtually almost all the plant hormones are transported through xylem vessel.

Proteins The fact that xylem sap contains proteins stems from the

original work done by Wilson in 1923 (Wilson 1923). He found the activities of catalase, peroxidase, and reductases in guttation fluid (essentially xylem sap). Recent studies on xylem sap proteins confirmed that the acidic proteins includ- ing peroxydase with pl 4.6 are present in xylem sap (Biles et al. 1989), partly because the xylem cell walls may be nega- tively charged (Biles and Abeles 1991). Stem exudate of watermelon was shown to contain proteins that inhibited the growth of Fusarium oxysporum, the causal agent for Fusar- ium wilt of watermelon (Biles et al. 1990).

There were five main protein bands appeared on SDS- PAGE collected from xylem sap of squash (Satoh et al. 1992). Two of them (40 and 75 kDa) were highly mannosilated, and increased for about 24 hr after cutting. Another polypeptides (32 kDa) appeared soon after cutting, disappeared and then reappeared again 48-64 hr after cutting. The level of the last two polypeptides (14 and 19 kDa) were present

Dynamic Function and Regulation of Apoplast

Table 4. Apoplast pHs in plant tissues

Plant pH Method

Vicia faba 4.5 ~ Microelec~Tode Zea mays* 4.9___0.03 b Confocal microscopy with NERF Vicia faba 5.2~5.9 c Fluorescence Taxus baccata 5.25___0.25 a 6-Glucoxy-7- hydroxyeoumarin Ginkgo bibloba 5.50+__0.15 d ibid Rumex lunaria 5.60___0.20 d ibid Nymphaea alba 5.85_0.25 d ibid Beta vulgaris 6.00_0.25 d ibid Prunus armeniaca** 6.0--,3.4 e pH electrode Primula palinuri 6.15 ___ 0.15 e 6-GI ucoxy- 7-hydroxycoumarin Hordeum distichum 6.3___ 0.3 f C, en'~ifugation Populus nigra 6.38___0.19 d 6-Glucoxy-7- hydroxycoumarin Spinacia oleracea 6.40___ 0.30 d ibid Gossypium hirsutum 6.7~7.5g Pressure chamber Helianthus annuus 6.77~7.42 h Centrifugation

6.22___0.12 ~ 5-Carboxyfluorescein and FITC dextran

Prunus persica** 7.0--,4.2 e pH electrode

a, Aloni et al. (1988); b, Taylor et al. (1996); c, Muhling et al. (1995); d, Pfanz and Dietz (1987); e, Ugalde et 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.

141

constitutively. Besides proteins in xylem sap, fructose, myo- inos i t o l and

oligosaccharides composed of mainly of galactose, arabinose and glucose are the major components of transported sugar in xylem sap in squash (Satoh et al. 1992)

Homeostasis of Internal Environment

Our blood pressure, temperature, pH, and ion and sugar concentration are precisely regulated. Hydrostatic pressure of plant body is equivalent to our blood pressure. The regulation of apoplastic pH and ion concentration are also likely (Grignon and Sentenac 1991). There is, so far, almost no information of active regulation of temperature of plant body, though salicylic acid is known to raise temperature of male reproductive tissues (Raskin 1992)

Plant apoplast responds to environmental stresses in different ways (Dietz 1997). pH and redox control in the apoplast serves a mechanism to respond to the environmental signals, such as gravity (Taylor e t a / . 1996).

pH in the apoplast has been measured in many plant systems by pH electrode with a combination of centrifugation method for collecting apoplast fluid or impermeable fluores- cent dyes (Table 4). The measured pH range of apoplast is from 4.5 to 7. Fruit tissue showed an extremely low pH on maturation, because of leakage of organic acid. Most of the data of apoplast pH falls on 5.5 to 6.5. Aloni e ta / . (1988) showed that the initial pH of mesophyll tissue of soybean was estimated to be 4.5, and the tissue acidified the unbuf- fered solution placed on the tissue from pH 6.0 to 5.1,

suggesting the active efflux of H + from symplast to apoplast in acively growing tissues. There are many factors to change apopalst pH. Fusicoccin accelerates the acidifica- tion, while gibberellic acid inhibited it. The high dosage of application of nitrate to the sunflower plant increased the apoplast pH in the laves, but not the pH of xylem sap, suggesting that cotransport of nitrogen with proton across the plasma membrane consumed apoplastic protons (Dannel et al. 1995). The dosage of ammonium form of nitrogen, however, decreased the apoplast pH in the sunflower leaves (Hoffmann et al. 1992). On the contrary, pH change in apoplast affects the hormonal balance in the plant. The change in pH on dehydration enhanced the release of ABA from mesophyll cell of cotton leaves into the apoplastic fluid (Hartung et al. 1988).

pH condition of apoplast is essentially determined by the activity of plasma membrane-bound H+-ATPase and membrane transport of solutes, but pectic substances in the cell walls also affects the ion concentrations and pH in apoplast. Cation exchange capacity of the pectic substances serves a reservoir function of ions, especially K + and Ca ++ (Grignon and Sentenac 1991). About 50% of the fixed anionic charges in the cell walls of Horse bean and Yellow lupine were protonated at pH 5.0, although pure galacturonic acid and non-esterified pectin showed a pK of 3.2 (Sentenac and Grignon 1981). Therefore pK of the cell walls is bout 5.0. Since the apoplastic pH, at least in a limited tissue or cells, can be immediately changed in response to light, water deficit, IAA, and nitrogen dose, pH in the free space of apoplast is dynamically regulated (Grignon and Sentenac

]42 N. Sakurai

1991). The precise mechanism of homeostasis for phosphate ion

in the apoplast has been recently found (Mimura 1995). Since the available phosphate is present at very low concentration in the soil, plants always suffer phosphate deficient. When plant can uptake phosphate from the soil, it stores in the vacuoles of cells, especially in the old tissues. The stored phosphate is translocated to young tissues for cell division and elongation growth on phosphate starvation. Phosphate concentration in the cytoplasm is regulated in a narrow range by an effective phosphate homeostasis in which the phosphate in the vacuoles acts as buffer (Mimura et al. 1990). The analysis of transportation of phosphate from an old to young barley leaf revealed that the phosphate concentration of apoplast was maintained at 1 mM (Mimura 1995). If the apoplastic phosphate concentration exceeds this level, the vacuoles seems to uptake the extra phosphate in the apoplast to maintain the apoplast concentration at 1 mM. The reason for the homeostatic regulation of phosphate in the apoplast remains to be answered.

Adhesion (cell to cell)

Adhesion of cell to cell is indispensable for constructing a multicellular tissue, but little is known about the responsible substance. Classically, pectin is said to be localized on middle lamella. When fruit softening was believed to be caused by pectin degradation, the existence of pectin in the middle lamella seemed to be reasonable, since the pectic degradation might lead to separation of cells. Unequivocal evidence, however, for the pectin localization is still lacking. Quick-freeze, deep-etch electron microscopic observation revealed that the treatment of carrot parenchyma tissues with polygalacturonase lost granular substances in the middle lamella, but there still remained a meshwork structure (Tamura and Senda 1992), suggesting that the middle lamella does not consist solely of pectin. Heterogeneous composition of many cell types in the plant tissues made it difficult to study the cell adhesion. Using suspension-cultured cells, Kikuchi et al. (1996) disclosed that the size of cell cluster of carrot cells was strongly and positively correlated with the ratio of arabinose to galactose of neutral sugar chain of pectic fraction. The result suggests that not the calcium bridge between acidic rhamnogalacturonans but the bran- ched arabinogalactan side-chain participates in the adhesion of cells.

In animal cells, many types of transmembrane proteins and extracellular matrix have been found to mediate cell-cell adhesion. So far, there is only one report about the detec- tion of such a protein, analogous to integrin, in root tips of Arabidopsis and Chara by immunofluorescence microscopy (Katembe eta/ . 1997). The authors suggested the involvement of the integrin-like proteins in gravity perception.

Air-space

Humiditiy control in the air space of apoplast is essential for the water economy. The range of humidity control must

be very narrow, 98--.100% in apoplast space. When the relative humidity is 98%, negative water potential of the cell wall surface reaches --2.8 MPa, corresponding to 1.25 M of osmotic potential that exceeds the potential of most plant cells. It is well known that absicic acid closes stomata, but it seems that this regulation does not work daily. Lu et al. (1997) proposed a mechanism where sucrose in the guard- cell wall is a physiological signal that regulates stomatal aperture. They showed that mesophyll-derived sucrose in guard-cell walls was sufficient to close stomatal opening by ca. 3/z m. Increase in the level of sucrose in the apoplast is caused either by high transpiration or high efflux of sucrose from mesophyll. The possible involvement of osmotic potential and solutes of apoplast in the regulation of cell expansion has been also pointed out (Grignon and Sentenac 1991, Canny 1995).

Expression of one of the endo-1, 3 : 1, 4-/~-glucanase gene (Ell, see 1.a) is restricted to the aleurone layer of germinated barley grains. The gene for El, however, is transcribed at relatively high levels in young leaves as well as in the scutellum and aleurone layer (Slakeski et a/. 1990). The El expression pattern implies that the isozyme El is involved in the separation of mesophyll cells and creation of air space in leaf. ff each mes0phyU cell was completely separated, there would be no translocation of photoassimilates to phloem. The mechanism of the cell wall degradation with remained junction between two cells at plas- modesm, is intriguing.

Unknown

Extensin, PRPs, GRPs, and AGPs are well known cell wall proteins, and various speculative proposals for their functions are described, but the biological significance is still contro- versial, though 3, 4-dehydroproline, the inhibitor of proryl hydroxylase, inhibits cell-wall assembly and cell division in tobacco protoplast, suggesting that these hydroxyproline- rich proteins are essential for cell growth and development (Cooper et al. 1994).

Several plant lectins induced the production of pisatin, a phytoalexin of pea (Toyoda et al. 1995). Chitin-binding lectins are ubiquitous in plants, and most of the lectins are vacuolar proteins. Chitin-binding lectins, however, of Solanaceae. species, such as potato, thorn apple and tomato, differ from all other chitin-binding lectins in that they are rich in arabinose, and contains the cystein/glycine-rich domain and the hydroxyproline/serine-rich domain (Raikhel eta/. 1993). The second domain is extensively glycosylated and exists as a polyproline helix, a similar secondary structure to extensin or hydroxyproline-rich proteins. Chitin- binding lectin is thought to have antifungal, and insect antinutrient activity.

Ascorbate oxidase is secreted from symplast to apoplast in pumpkin (Esaka 1993) and bound to the cell walls of Vigna angularis (Takahama and Oniki 1994). Analysis of the promoter by transient expression assay in the pumpkin fruit tissues suggested the existence of a cis-acting region 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 unknown, ascorbate free radicals generated by ascorbate oxidase from ascorbic acid, enhanced the root growth of onion (Hidalgo et al. 1991) by increase in the uptake of nutrients into the vacuoles (Gonzales-Reyes et al. 1994). The association of apoplastic ascorbate oxidase with auxin- induced elongation was also reported in Vigna angularis (Takahama and Oniki 1994).

Concluding Remarks

This past decade has witnessed many advances and new directions in apoplast research. Recently Robertson et al. (1997) extracted cell wall-bound proteins from Arabidopsis, carrot, French bean, tomato and tobacco, and 233 proteins were selected on SDS-PAGE for protein sequencing, 146 proteins gave N-terminal data. They found that a signifi- cant proportion of wall proteins (74%) has not been previous- ly described. These data represent a future protein resource and function for apoplast studies.

The volume of apoplast was estimated at about 5--,10% (Cosgrove and Cleland 1983, Sakurai and Kuraishi 1988) in stem tissues and 5---40% in leaves (Kramer 1983). If the volume of vacuoles is estimated as 80% in the tissue, the apoplast volume (10%) is compatible to the cytoplasmic volume (10%). Apoplast is not a small space in the plant body. A space surrounded by a membrane within a cell is toporogically outside. Therefore, the vacuole space is rather outside, similar to the apoplast. If the vacuole is defined as apoplast, the cytoplasm is sandwiched between two apoplasts. In this sense, the vacuole is also an important internal environment in the plant body. The difference between two types of apoplasts is the fact that apoplast is a continuum, while the vacuole is isolated within a cell. The continuum of apoplast suits the signal transduction from environment, while the lar~3e volume of vacuole does buffer- ing the abrupt environmental changes. The coordination of two spaces in plant body should be elucidated more in the future.

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(Received December 20, 1997: Accepted January 19, 1998)

dynamic function and regulation of apoplast in the plant body

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