Bot. Maq.

Tokyo 104: 235-251, 1991 The Botanical Magazine, Tokyo

9 by The Botanical Society of Japan 1991
I n v i t e d Article

Cell Wall F u n c t i o n s in G r o w t h and D e v e l o p m e n t

--A P h y s i c a l and C h e m i c a l Point of V i e w -

NAOKI SAKURAI*

Department of Environmental Studies, Faculty of Integrated

Arts & Sciences, Hiroshima University, Hiroshima 730

The plant cell changes its cell wall architecture during growth and development
through synthesis and degradation of wall polysaccharides. Changes of chemical
components in the cell wall include not only the synthesis and degradation but also the
shift of molecular-weight distribution of certain species of the component polysacchar-
ides. The changes in chemical structure, in turn lead to alteration of physical properties
of the cell wall. Changes of physical parameters of cell walls obtained by a physical
method accord with the biochemical degradation of polysaccharides. The changes in
chemical structures of the cell wall are regulated by plant hormones, stress signals and
gene expression. The physical and chemical studies of the cell wall have disclosed that
degradation and/or depolymerization of wall polysaccahrides causes decrease in viscos-
ity of the cell wall, leading further extension of the cell wall even under the unchanged
osmotic relation. Furthermore, cell walls of outer and inner tissues play different
regulatory roles in tissue growth and stem strength was governed by the number of
cellulose molecules in the cell wall.

Key words : ABA - - Auxin-- Cell wall - - Cellulose - - fl-1, 3 : 1, 4-Glucan - -

Xyloglucan

Cell walls function in m a n y facets of p l a n t growth and development. The cell

wall has four main functions ; regulation of the growth, strengthening the plant tissues
to support the p l a n t body, softening of fruits, and protection to microbial attack.
Protective role of cell walls in microbial invasion has been extensively studied, and
advanced recently in terms of signal transduction b y cell wall fragments t h a t have
elicitor activities (Yoshikawa 1983, R y a n 1987, H a h n and Cheong 1990). This review
will deal with chemical changes in wall polysaccharides associated with physical
changes in cell wall architecture which lead to the promotion of growth, strengthening
and softening of p l a n t tissues.

A. T u r g o r P r e s s u r e o r Cell Wall L o o s e n i n g ?

There was a long debate for the mechanism of cell elongation : one argued t h a t the
driving force is turgor pressure, the other claimed t h a t cell wall loosening caused cell
elongation (Cosgrove 1986, Masuda 1990). B u t these statements are not alternative.

* Recipient of the Botanical Society Award for Young Scientists, 1990.

 236 N. SAKURAI

If it were not for the turgor pressure that presses the cell wall, plant cell would not
elongate. Without cell wall loosening, plant cell would not change its volume. The
statement of cell wall loosening prerequisite for cell elongation inevitably predicts that
turgor pressure reduces as cell elongates, because cell wall loosening causes reduction
of cell wall pressure of which absolute value is rigidly equal to turgot pressure.
Reduction of turgor pressure does not deny the role of turgor pressure in cell elongation
at all. Turgor pressure may be increased by an active solute uptake (Katou and
Furumoto 1986a, b).
The next problem is what extent the turgor pressure or cell wall loosening
contributes to the cell elongation. If the turgor pressure remained constant during cell
elongation, one could conclude that the turgor pressure plays a main role in the process.
In this case, the cell should have absorbed f~om apoplast the solution whose concentra-
tion is equal to that in symplast or vacuoles. Generally speaking, the turgor pressure
always decreases during cell elongation, especially in auxin-induced elongation.
When the osmolarity of young cells is compared with that of old cells, the former is
always higher than the latter (Sakurai and Kuraishi 1988). But the calculated
amount of solutes of old cells is always greater than that of young cells (Miyamoto and
Kamisaka 1988). Plant cell absorbs not only water but also solutes. Absorbed
solutes are accumulated inside the cell, even some of them are consumed by respiration,
there still be a net increment of solutes. This increment, however, never exceed nor
compensate the water income. Therefore it can be concluded that turgor pressure does
not alone cause cell elongation.
On the contrary, it has been clearly demonstrated that chemical components of
cell wall and the mechanical properties of the cell wall are dynamically changed
during cell elongation. The chemical changes associated with the mechanism of cell
wall loosening is divided into two categories. One is predominantly involved in the
degradation of some species of polysaccharides and the other predominantly in their
synthesis. There is a clear picture how the degradation causes cell elongation. The
degradation of wall polysaccharides decreases viscosity or increases fluidity of the cell
wall, leading to extension of the cell wall under the unchanged osmotic relation.
There, however, is not still a concrete idea how the synthesis of polysaccharides
contributes to cell elongation.

B. D e g r a d a t i o n of P o l y s a c c h a r i d e s in M o n o c o t s

B-I fl-L 3 : 1, 4-Glucans in monocots

The facts that auxin accelerates a turnover of hemicellulosic polysaccharides have
been reported in both monocots and dicot (Masuda 1990, Labaviteh 1982). A specific
degradation of fl-glucans of non-cellulosic polysaccharides due to auxin action was first
reported in Avena coleoptiles (Loescher & Nevins 1972). The decrease, however, was
not caused by hydrogen ion which promotes elongation of A v e n a coleoptiles (Sakurai
et al. 1977a). It was demonstrated that auxin action causes depolymerization of/?-1,
3 : 1, 4-glucans of the hemicellulose (Yamamoto et al. 1980). The decrease was also
 Cell Wall Functions in Growth 237

found in barley (Sakurai & Masuda 1978a) and rice (Zarra and Masuda 1979)
coleoptiles treated with auxin and the non-cellulosic fl-glucan content decreased
during the normal growth of barley (Sakurai & Masuda 1978b) and maize coleoptiles
(Luttenegger and Nevins 1985). The depolymerization of fl-1, 3:1, 4-glucans was
turgor-dependent (Loescher and Nevins, 1973, Sakurai and Masuda, 1977b), implying
that glycanases for the depolymerization is secreted from cytoplasm to the cell wall by
exocytosis. Using inhibitors, this depolymerization of fl-1, 3 : 1, 4-glucans was found
to be mediated through de novo syntheses of mRNA and proteins (Sakurai & Masuda
1979).
Huber and Nevins (1981a) purified endo- and exo-fl-glucanase with tool wt of
26,000 and 60,000, respectively. In vitro translation study revealed that one hour
treatment of barley coleoptiles with auxin exhibited de novo synthesis of mRNA
which encoded a protein with tool wt of 26,500 (Sakurai unpublished data). The
endo-fl-glucanase degraded high molecular weight of fl-1, 3 : 1, 4-glucans (1,000,000 > )
into 10,000 (Huber and Nevins 1980). When auxin was added to Arena coleoptiles,
the high mol wt of fl-1, 3 : 1, 4-glucans were degraded, but the intermediate size of
polymer could not be detected (Sakurai et al. 1979). The results suggest that the
auxin-induced degradation is mediated by at least the combination of endo- and
exo-fl-glucanase. Antibodies against wall-bound proteins inhibited auxin-induced
elongation of maize eoleoptiles (Huber and Nevins 1981b, Hoson and Nevins 1989a,b).
Antibodies against fragments of fl-1, 3:1, 4-glucans also inhibited auxin-induced
elongation of maize coleoptile segments as well as the fl-1, 3 : 1, 4-glucan degradation
(Hoson and Nevins 1989c). Furthermore, more specific antibodies against purified
exo-and endo-fl-glucanase from maize cell walls inhibited the auxin-induced elonga-
tion as well as auxin-induced degradation of fl-1, 3 : 1, 4-glueans (Inouhe and Nevins
1991), These studies with antibodies clearly indicate that the specific degradation of
fl-1, 3 : 1, 4-glucans is prerequisite for the auxin-induced elongation.
The degradation of fl-1, 3 : 1, 4-glucans is not only caused by exogenously applied
auxin, but also regulated by endogenous auxin. Inouhe et al. (1982) reported that a
semi-dwarf barley strain has one fourth level of endogenous auxin when compared
with a corresponding normal strain which differs only in a single dwarf gene.
Molecular weight of the hemicelluloses determined by intrinsic viscosity was low in the
elongation zone and high in the mature zone of the normal coleoptiles, suggesting that
low molecular weight of fl-1, 3 : 1, 4-glucans was predominant in the elongation zone,
while a semi-dwarf mutant with less endogenous levels of auxin contained high mole
wt of the polymer even in the elongation zone, suggesting that endogenous auxin is
necessary for the degradation (Sakurai & Kuraishi 1984).
B-II Physical meaning of fl-1, 3 : 1, 4-glucan degradation
Is the degradation of fl-1, 3 : 1, 4-glucans directly involved in the cell wall loosen-
ing ? Physical measurement of the cell walt afforded a clue. Yamamoto et al. (1970)
developed a stress-relaxation analysis for measuring the mechanical properties of the
cell wall. Since the method is strictly based on the Maxwell visco-elastic model, four
 238 N. SAKUR~

parameters obtained from the measurement predict the physical changes in chemical
components of the cell wall (Masuda and Yamamoto 1985). Minimum stress-
relaxation time (To) and maximum stress-relaxation time (Tm) predict the size of
molecules which contribute to visco-elastic properties of the cell wall (Fig. 1).
Relaxation rate (R) predicts the number of component of visco-elastic properties.
Residual stress (C) predicts the most elastic properties of the cell wall. In almost all
 Cell Wall Functions in Growth 239

the case when auxin was applied to p l a n t tissues, it decreased To and R. The decrease
in To means t h a t auxin increases fluidity or decreases viscosity. The decrease in R
means t h a t auxin increases the n u m b e r of components.
When polymers with high mol wt is dissolved in high concentration, it shows high
viscosity. Friction of the surface of polymers and even entanglement of neighboring
polymers in concentrated solution results in high viscous nature of solution (Doi and
Edwards 1978). Degradation of the polymers decreases viscosity and increases
fluidity of the solution. I t is evident t h a t the degradation of the p o l y m e r b y an
endowise manner decreases viscosity much faster t h a n t h a t by an exowise manner.
I n the primary cell wall of monocot plants, cellulose content is a b o u t 5 0 o of the
total a m o u n t of cell wall polysaccharides, b u t its spacing volume in the cell wall is
much less t h a n 50%, because a b o u t 50-70% of the cellulose molecules exists in a
crystalline state (Trip 1970, Phillips and Arthur 1985). Therefore most bf the space
of the cell wall is occupied b y the non-cellulosic polysaccharides. T a b l e 1 shows the
content of each polysaccharides in maize cell walls. There is almost no information
of the physical state of non-cellulosic polysaccharides in the cell wall as to whether or
not, for example, the polysaccharides are completely dissolved in the cell wall.
Nevertheless, the measurement of mechanical properties of the cell wall clearly reveals
t h a t the p l a n t cell has elastic and viscous nature at the same time. Since the p l a n t cell
shows m u c h smaller Young's modulus (about 4 • 107 N / m 2) (Sakurai et al. 1984) t h a n
the cellulose molecules (2.5 • 109 N / m 2) (Jeronimidis 1980), cellulose molecules do not
rigidly constrain the cell wall extensibility. Low Young's modulus of p l a n t cell walls
rather suggests t h a t microfibrils are not covalently bonded each other and m a t r i x
polysaccharides other t h a n cellulose are the main components of the visco-elastic
properties of the cell wall.

Fig. 1. Stress-relaxation curve, stress-relaxation parameters and Maxwell visco-elastic

components for plant cell wails. (a), stress-relaxation curve. When a plant specimen
is stretched and the strain is fixed, stress produced by the specimen decreases. The
stress changes are plotted against time in logarithmic scale. The curve is described by
a following equation ;
S/S0 • 100(%) = R Loge(t + Tm)/(t + T0) + C
where So is the initial stress. To is the time at the initial decay of stress, Tm the time
that stress decay stops, R relaxation rate (%) and C the residual stress ; (b), Maxwell-
viscoelastic components. To corresponds to a Maxwell component with the lowest
viscosity, Tm to a component with the highest viscosity, reciprocal of R (I/R) to the
number of components per unit volume, and C to the highest elastic component. (c),
distribution and amount of the components. The amount of component with different
tool wt can be drawn as a distribution of the components. Ordinate is arbitral. (d),
Maxwell components after auxin action. When auxin induces cell elongation, To and
R decrease. The decrease in To corresponds to the new component with lower viscosity
than the lowest viscosity shown in (b). Consequently, the number of components
increases, which is represented by a decrease in R. Depolymerization of polysacchar-
ides in the cell wall results in an increase in the number of molecules and in a decrease
of the wall viscosity ; (e), distiribution of the components after auxin action. Distri-
bution of the components is shifted to lower tool wt when To and R decrease.
 240 N. SAKURAI

Table I. Species, contents and tool wt of polysaccharides in monocots, primary cell

walls.

Polysaccharides Amount (~ Mol wt Ref.

Cellulose 45
fl-1, 3 : 1, 4-Glucan 14 1,000,000 < A
Glucuronoarabinoxylan2 25 200,000 B
Other polysaccharides 16
Rhamnogalacturonann C
fl-1, 4-Galactan D
fl-1, 3-Glucan 70,000 E
Xylogluean 120,000 F
Arabinogalactan 100,000 G
1, personal communication (Y. Kato). ~, Thirty to seventy ~ of polymer are fer-
uloylated (Nishitani and Nevins 1990). A, Kato and Nevins, 1984c, Sakurai et at.
1979, Kato and Nevins, 1986; B, Kato and Nevins, 1984d; C, Kato and Nevins
1989; D, Kato and Nevins, 1984b; E, Kato and Nevins, 1985; F, Inouhe et al.
1984 ; G, Kato and Nevins, 1984a.

fl-1, 3 : 1, 4-Glueans have no ability to make hydrogen bonds inside the molecules,
since the insertion of a fl-1, 3-linkage between two fl-1, 4-linkages would hinder the
intramolecular hydrogen bonding (Fig. 2). Therefore, fl-1, 3 : 1, 4-glucans exist in the
cell wall as a soluble form. Huber and Nevins (1980) first assumed that there are two
or three contiguous fl-1, 3-linkages in the glucan which were confirmed later (Kato and
Nevins 1984b). Endo-fl-glucanase activity found in maize coleoptile cell walls
(Huber and Nevins 1981a) is possibly an endo-fl-1, 3-glucanase which is expressed in
response to auxin, and attacks the contiguous region of fl-1, 3-linkages, leading to
decrease its molecular weight from 1,000,000 to 10,000 in the cell wall. The specific
endo-fl-1, 3-glueanase, however, has not yet been found. Extended regions of contigu-
ous fl-l,4-1inkages were found in the fl-1, 3:1, 4-glucans of barley cell walls
(Woodward et al. 1983), and the region was proposed to be close to the region of
contiguous fl-1, 3-linkages (Hatfield and Nevins 1986). Therefore, the endo-enzyme
responsible for depolymerization of fl-1, 3 : 1, 4-glucans into 10,000 could have an
activity of endo-fl-1, 4-glucanase.
It is quite difficult to asses the spacing size of polymer molecules in the cell wall.
If, however, one assumes that a fl-1, 3 : 1, 4-glucan with tool wt of 1,000,000 is dissolved
and forms a random coil, the effective radius can be calculated to 37 nm (Masuda et
al. 1982) using the measured intrinsic viscosity (Wada and Ray 1978, Sakurai et al.
1979, Sakurai and Kuraishi 1984) according to Flory's theory (Flory 1953). Arabinox-
ylans, on the other hand, have a tool wt of 200,000 (Wada and Ray 1978) to 600,000
(Sakurai et al. 1979). The estimated radius is about 4.4-6.4 nm. There is a hole with
a radius of 5.8 nm among three fl-1, 3 : 1, 4-glucan molecules contacting with each
other. There is another hole with a radius of 1 nm among three arabinoxylan
molecules contacting with each other. The cell wall pore radii of 2 nm (Carpita et al.
 Cell Wall Functions in Growth 241

M o l wt (10,000) --I

Putativecontiguous
~ ~ -1,3-linkages(a)
- ~ \~~:tl:ii'~i:::::i::b;US
<

1
0 [3-1,3-1inkedglucose 0 [3-1,4-1inkedglucose
Fig. 2. Structure of fl-1, 3 : 1, 4-gluean fl-1, 3 : 1, 4-glucan is mainly composed of two units ;
trisaccharide, 3-O-fl-eellobiosyl-D-glucose and tetrasaechaxide, 3-O-fl-cellotriosyl-D-
glucose. The molecule can be rotated at fl-1, 3-linkages, but fixed at fl-1, 4-linkages.
More than three contiguous fl-1, 3-linkages are supposed to be present at every 50-60
glucose residues (mol wt about 10,000). At least four contiguous fl-1, 4-linkages are
supposed to be present close to the regions of contiguous fl-1, 3-linkages. Contiguous
fl-1, 3-linkages kink the polymer. Native fl-1, 3:1, 4-glucan consists of about 100
turns of subunits with mol wt 10,000. Putative endo-fl-glucanase responsible for
degrading fl-1, 3 : 1, 4-glucan may cleave either contiguous fl-1, 3-linkages or fl-1, 4-
linkages.

1979) and 3.5 nm (Tepfer and Taylor 1981) fall into the above range.
The above estimation predicts that the noncellulosic polysaccharides such as fl-1,
3 : 1 , 4-glucans and arabinoxylans are intensively packed in the cell wall and the
surfaces of the polysaccharides contact with each other. Such a close contact will
result in great friction, leading to afford the viscous nature to the cell wall. When
high molecular weight of fl-1, 3 : 1, 4-glucan is degraded b y an endo-type glucanase, the
cell wall viscosity will decrease, leading to so-called wall loosening.
The close contact of polysaccharides with each other also makes a chance of
entanglements among the polysaccharides. The entanglement serves as a cross link
between two polysaccharides. The cross links in turn contribute to the elastic
properties of the cell wall. Therefore, elastic properties are also expected to decrease,
when fl-1, 3 : 1, 4-glucan is degraded. The fl-1, 3 : 1, 4-glucan seems to serve as one of
the main visco-elastic component of the monocot, coleoptile cell wall. The degrada-
tion of fl-1, 3 : 1, 4-glucans results in decrease in viscosity and increase in the number
 242 N. SAKURA/

of components. This is predicted by the decreases in To and R values (Fig. 1).

C. Degradation of polysaccharides in dicots

C-I Xyloglucans in dicots
Xyloglucan turnover has been reported in dicot plant cell walls (Hayashi, 1989,
Fry 1989). Since cell walls of dicot plants have no fl-1, 3 : 1, 4-glucans, the turnover
of xyloglucans has been thought as a good candidate for cell wall loosening in dicot cell
elongation. Auxin promoted a release of fragments of xyloglucans in pea epicotyl
segments (Labavitch and Ray 1974, Terry and Bonner, 1980), decreased an average
molecular weight of xyloglucans in azuki epicotyl segments (Nishitani and Masuda
1983) and squash hypocotyl segments (Wakabayashi et al. 1991b). Lectin which
binds fucose residue of xyloglucans was found to inhibit auxin-induced elongation of
azuki bean epicotyls (Hoson and Masuda 1991). Furthermore, antibodies against
hepta- and octa-saccharides of xyloglucans could inhibit auxin-induced elongation of
azuki bean epicotyls but not of oat coleoptiles (Hoson et al. 1991), suggesting that the
degradation of xyloglucan is prerequisite for auxin-induced elongation of dicot plants.
Recently Nishitani and Tominaga (1991) reported that an apoplastic enzyme
obtained by a centrifugation method changed xyloglucans of medium size in mol wt
into those of the lower and higher size in azuki bean epicotyls, and the enzyme activity
was higher in the upper juvenile than in the lower non-elongating regions of the
epicotyl and it was optimum at pH 5.4. They suggested that the transglycosylation
of xyloglucans was involved in cell elongation, and in acid-induced cell elongation.
Low and high molecular weights of xyloglucans were reported ; low ~nol wt (40 x
10') extracted by a weak alkaline solution and high tool wt (106x 104) by a strong
alkaline solution in azuki (Nishitani and Masuda, 1983) and in squash hypocotyls, low
mol wt (20 x 104) extracted from inner tissues and high tool wt (48 x 104) from outer
tissues (Wakabayashi et al. 1990). Auxin decreased an average tool wt of xyloglucans
with higher mol wt in azuki and in outer tissues of squash hypocotyls. A specific
enzyme for degradation of xyloglucans (endo-fl-1, 4-glucanase) was isolated from pea
epicotyls (Hayashi et al. 1984). Fragments of xyloglucan resulted from the action of
endo-fl-1, 4-glucanase is thought to promote xyloglucan degradation (Hayashi 1991).
Auxin-induced degradation of xyloglucans may be restricted in outer tissue of stems,
where high tool wt of xyloglucans is predominant. Lower tool wt of xyloglucans
found in the inner tissue of squash hypocotyls implies that the cell wall is more
loosened than the outer tissue so as to produce inner tissue tension (Masuda 1990).
C-II Physical meaning of Xyloglucan degradation
Is xyloglucan degradation directly involved in cell wall loosening in dicots ?
Xyloglucans are thought to be bound to cellulose molecules by hydrogen bonds
(Hayashi 1989). Xyloglucan has a rigid fl-1, 4-glucan backbone. It is likely that
xyloglucan exists in the cell wall as an extended form. Length of xyloglucan
molecules can be calculated as 200 nm for oat xyloglucan (mol wt 12,000, Inouhe et al.
 Cell Wall Functions in Growth 243

1984) and 600 nm for squash xyloglucan in outer tissue of squash hypocotyls (mol wt
400,000, Wakabayashi et al. 1991b), provided that the xyloglucans are composed of
repeating units of a combination of hepta- and nona-saccharides (Hayashi 1989). The
lengths are comparable to those of the cellulose molecules in primary cell wails, 250-
1,000 nm for DP=500-2,000 (Blaschek et al. 1982).
Xyloglucan is not extracted by 4% KOH from the cell wall (Hayashi 1991).
Addition of 8 M urea to 4% KOH could extracted 40% of xyloglucan from dicot cell
wails, but the extractable xyloglucans had a smaller tool wt than those extracted by
24% KOH, and furthermore, the smaller xyloglucans were not depolymerized by auxin
action (Nishitani and Masuda 1983). The extent of decrease in average tool wt of
xyloglucans by auxin action was 26% in azuki (Nishitani and Masuda 1983) and 230/0
in squash (Wakabayashi et al. 1991b), and in both cases the auxin action depolymer-
ized the higher mol wt of xyloglucans. The data suggest that some smalle.r tool wt of
xyloglucans which is not involved in auxin-induced elongation is bound go cellulose
molecules.
Xyloglucan backbone is similar to that of cellulose molecules, fl-1, 4-glucans. It
is unlikely that the fl-1, 4-glucan backbone in the xyloglucans serves as hydrogen
bonding to cellulose molecules, since (1-*6)-linked-a-D-xylose residues hinder such
bonding by steric hindrance and even by the occupation of xylose residue at an oxygen
atom (C6) of glucose, which would serve as a hydrogen bonding to cellulose molecules
ran in parallel with xyloglucans. Conformational study of xyloglucans revealed that
the residues of xylose and a-L-fucosyl-(1-~2)-fl-D-galactose covered the fl-1, 4-glucan
backbone (Ogawa et al. 1990). A molecular docking study to ascertain the contribu-
tion of these side chains to hydrogen bonding is also necessary.
In spite of the above uncertainty, the side chains of xyloglucan may cause friction
between cellulose molecules and other non-cellulosic polysaccharides in the cell wall,
provided that the xyloglucan is bound to cellulose. The degradation of xyloglucans
may result in a decrease in the friction, leading to decrease viscosity of the cell wall.
Whether or not the decrease in average tool wt of xyloglucans by only 25o/o is enough
to cause a significant decrease in the wall viscosity is open to be answered.

D. Computer simulation of cell elongation

If the mechanical properties of the cell wall are fully described by the four
parameters obtained by the stress-relaxation method and they regulate cell elongation,
one can simulate the cell elongation by computation. Plant cell elongation is a creep
process within a short time range ; the strain changes under the constant stress. The
Maxwell visco-elastic model can be inputted with a known set of four parameter values
into a computer, and one can calculate the strains under even changing stress
(Yamamoto and Sakurai 1990). When epidermis isolated from pea segments was
stretched in vitro by a constant stress, the epidermal cell wall showed a typical creep
process, which was precisely reproduced by the computer calculation (Yamamoto
1986). When lower To and R were introduced into the calculation, faster and longer
 244 N. SAKURAI

creep was obtained (Yamamoto and Sakurai 1990). The in vitro creep process,
however, does not mimic the in vivo cell elongation at all. Within a very short time
range (in a few second), almost all the strains of in vitro creep was completed.
Therefore, in vivo creep experiments were developed and compared with the computer
calculation. Pea stem segments were incubated for 2 hr in mannitol solution, then
transferred to a plain buffer solution. The experiment was carried out at 4~ to reduce
and avoid the complex involvement of metabolic activities in chemical structure of the
cell wall during the experiment. The elongation after transferring segments to a plain
buffer was designated as the in vivo creep process. The in vivo creep showed much
slower than the in vitro creep. It is evident that mannitol in apoplast did not
immediately diffuse to the bathing medium. Amount of mannitol diffusing into
bathing medium was measured by a gas-liquid chromatography and radioactivity.
The diffusion pattern was exponential as reported (Cosgrove and Cleland 1983). The
results demonstrate that turgot recovery increased exponentially. When the
exponential increase in stress was introduced into the computer, the calculated creep
process accorded with the observed in vivo creep (Yamamoto and Sakurai, unpub-
lished data).
The above computer simulation of the cell elongation clearly revealed that the
mechanical properties governs the cell elongation, and the water potential difference
between apoplast and symplast (turgot pressure) governs it as well. At room tempera-
ture, the in vivo creep continued more and longer. This means that the metabolic
activities are involved in modification or turnover of cell wall architecture and even
solute uptake, and thus contribute to a long term growth of plant tissues.

E. Synthesis of wall polysaccharides

It is quite evident that synthesis of wall polysaccharides is necessary for in vivo
cell wall elongation (Labaviteh 1982, Masuda 1990, Taiz 1984). The involvement of
wall synthesis in cell elongation has been well demonstrated by inhibitors of polysac-
charide synthesis; eoumarin inhibited GA-induced growth of lettuce hypoeotyls as
well as the hemicellulose and cellulose syntheses (Kawamura et al., 1976) ; monnensin
inhibited IAA-induced elongation of pea stem (Brunmell and Hall 1985) and squash
hypocotyls (Wakabayashi et al. 1989a) as well as hemieellulose synthesis; IAA-
induced growth of monocots coleoptiles was inhibited by galactose (Yamamoto et al.
1981, 1988) and that of dicot stem tissues by 2-deoxygalactose (Inouhe and Yamamoto
1991) as well as hemicellulose and cellulose synthesis through the reduction of
UDP-glucose level (Inouhe et al. 1986, Inouhe and Yamamoto 1991).
When squash seedlings were grown under a water deficit condition, the hypocotyl
cell elongation and cell wall synthesis (especially hemicelluloses and cellulose) were
substantially inhibited (Sakurai et al. 1987a,b). The stress caused 3.5 fold increase in
endogenous ABA level in the hypocotyls 6 hr after the treatment, when the growth was
not still inhibited by the stress (Sakurai et al. 1985). The results strongly suggested
that the increase in ABA level was a primary, endogenous factor for inhibiting cell
 Cell Wall Functions in Growth 245

elongation. ABA exogenously applied to the excised segments of squash hypocotyls

inhibited the elongation of the segments and the synthesis of hemicellulose and
cellulose only in outer tissues of the segments (Wakabayashi et al. 1989a). This
inhibition was not caused by the reduction of UDP-glucose levels (Wakabayashi et al.
1991a) nor through the myo-inositol passway (Wakabayashi et al. 1989b), suggesting
that ABA impeded some polymerization process for cellulose and hemicelluloses.
Although wall synthesis is necessary for sustaining the segment growth, how the
syntheses of wall polysaccharides regulate cell elongation is still unclear. Newly
synthesized polymers may serve as a new visco-elastic component of the cell wall.
Precise mechanism of wall synthesis will afford a clue. There is a possibility that
newly synthesized polysaccharides in Golgi vesicles are packed in a folded form
because of the hydrophobic environment in the vesicles and after exocytosis the
polysaccharides swell under hydrophilic environment in the cell wall spa'c~, creating
some pressure to extend cell wall within the cell wall.

F. S t r e n g t h of P l a n t T i s s u e s

F- I Stem
Three barley strains of mutants generated by treating a normal cultivar uzu
Akashinriki with ethylene methane sulfonate show brittleness in the culms. The
maximum bending stress when the culm is broken by bending stress was much less in
the mutants (20-50%) than in the normal strains (Kokubo et al. 1989). Since the
maximum bending stress is expressed as a stress per unit cross sectional area, low
maximum bending stress does not depend on culm geometry such as diameter, but
represents an inherent cell wall properties of the mutants. All three brittle mutants
contained less cellulose content and thinner wall thickness than the normal (Koknbo
et al. 1989).
The results were further confirmed with three isogenic lines of barley mutants
(Kokubo et al. 1991). Three gene loci responsible for brittle culm (fs, f s 2 andfs 3) are
located on chromosome seven (Takahashi et al. 1953), five (Takahashi et al. 1966) and
one (Hayashi and Moriya 1985), respectively. The brittle mutants with fs 2 and fs 3
showed less cellulose content and thinner cell wall than the corresponding normal
strains, but degree of polymerization (DP) of cellulose in these mutants were similar
to DP of the corresponding normal strains (Kokubo et al. 1991). Lignin and hemicel-
lulose contents in the mutants were also similar to those of normal strains. The results
revealed that the brittle mutants has less number of cellulose molecules in the cell wall.
Since the maximum bending stress is a stress per unit cross sectional area, the breaking
strength depends on the density of stress-bearing components of the cell wall. If
cellulose molecules or microfibrils are the components, brittleness of the mutant is due
to the low density of cellulose molecules in the cell wall. The reason why the mutant
can not produce an abundant cellulose molecules is open to be answered. The f s 2
seems involved in secondary wall thickening, because the mutants with fs 2 did not
show less cellulose content in their coleoptiles (Kokubo, Sakurai and Kuraishi, unpub-
 246 N. SAKURAI

lished data).
F- 2 Fruits
Fruit softening process has been extensively investigated in tomato (Huber
1983b). Softening of fruit tissues is demonstrated to result from cell wall degrading
enzymes. In tomato fruits, polygalacturonase (PG) is the best characterized enzyme
associated with ripening events. PG mRNA increased during tomato ripening (Del-
laPenna et al. 1986). PG activity was absent in green tomato fruits but was expressed
during ripening (Tucker and Grierson 1982). Tieman and Handa (1989) demonstrated
that PG does not uniformly appear within the fruit as the fruit ripened, but appeared
first in columella region followed by sequential appearance in the exopericarp and
endopericarp from blossom end to shoulder of the fruit. Pectic polysaccharides
composed of polygalacturonans, and hemicelluloses were degraded during ripening,
and the tool wt of these polymer decreased (Huber 1983a, Koch and Nevins 1990).
Although the tissue softening was measured by a conventional method modified by
Arhens and Huber (1990), the depolymerization of the polysaccharides were confirmed
by a stress-relaxation technique (Kojima et al. 1991). Instead of using a tensile tester
for a usual stress-relaxation method, they inserted a fine conical probe into the various
regions of tomato fruits, and found that To and R decreased during ripening.
Furthermore, decrease in To and R appeared first in columella regions followed by
sequential appearance in exopericarp and endopericarp from blossom end to shoulder
of the fruits.

G. Concluding Remarks
Degradation of noncellulosic polysaccharides such as that of fl-1, 3 : 1, 4-glucans in
monocots and xyloglucans in dicots in response to auxin and of tomato cell wall
polysaccharides during ripening directly causes a physical changes of the cell walls.
The physical changes estimated by a physical method accord with chemical changes of
the wall polysaccharides. Involvement of wall synthesis in the physical changes of
the cell wall, however, is still obscure, partly because the enzyme responsible for
synthesis of wall polysaccharides has not yet been well characterized except for fl-1,
3-glucan synthase (Delmer 1987, ])elmer et al. 1991). Understanding of polysacchar-
ide synthesis will afford a great deal of information of polysaccharide structure as well.
Recent enzymic study of a new type of xylanase which degrades glucuronoarabinox-
ylans in azuki and maize seedlings, revealed that the polymer is composed of definite
repeating units (Nishitani and Nevins 1991b). As for fl-1, 3 : 1, 4-glucans and xylog-
lucans, there is little information of whether or not they are composed of uniform
repeating units. It seems that a chemical analysis has a limitation for characterizing
complex, high-mol wt polysaccharides. Since the structure of polysaccharides is not
directly governed by DNA sequence like that of protein, but by a combination of
glycanases and synthases, one tends to consider that even a certain species of polysac-
charides has a diversity in the structure. Future study of enzymes involved in the
 Cell Wall Functions in Growth 247

d e g r a d a t i o n a n d s y n t h e s i s o f p o l y s a c c h a r i d e s w i l l disclose w h e t h e r or n o t the p o l y s a c -
c h a r i d e s t r u c t u r e is r e g u l a t e d in t h e f u z z y a n d e q u i v o c a l m a n n e r .

I w i s h to express m y sincere t h a n k s to P r o f . Y. M a s u d a of T e z u k a y a m a College for

his c r i t i c a l r e a d i n g o f t h i s m a n u s c r i p t a n d his c o n t i n u o u s e n c o u r a g e m e n t t h r o u g h o u t
m y w o r k s a n d to Prof. S. K u r a i s h i of H i r o s h i m a U n i v e r s i t y for his i n v a l u a b l e sugges-
t i o n s to d e v e l o p m y scientific interests. I d e e p l y t h a n k Prof. R. Y a m a m o t o o f Tezu-
k a y a m a College for his i n v a l u a b l e discussion a n d c r i t i c i s m a n d Dr. Y. K a t o for his h e l p
of m o d e l i n g fl-1, 3 : 1, 4-glucans.

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Received June. 14, 1991