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J. Plant Res. 111: 159-166, 1998
Journal of Plant Research
by The Botanical Society of Japan 1998
JPR Symposium
Construction and Restructuring of the Cellulose-Xyloglucan
Framework in the Apoplast as Mediated by the XyloglucanRelated Protein Family--A hypothetical scheme
K a z u h i k o Nishitani
Biological Institute, Graduate School of Science, Tohoku University, Sendai, 980 8578 Japan
The cellulose-xyloglucan framework functions as the
load-bearing structure of the cell wall and cor~rains cell
shape in plants. Xyk)glucan cross-links which underpin the
framework structure can be modified by transferases and
hydrolases encoded by xyloglucan-related protein (XRP)
family genes, These enzymes are considered to play
critical roles in the construction and restructuring of t~e
three-dimensional structure of the plant cell wall. Although
analyses of their protein structures and gene-expression
profiles for individual members of XRPs have disclosed their
potentially divergent roles in plants, the biochemical reactions catalyzed by individual XRPs and their biochemical
implications remain to be clarified. This review focuses on
the XRP-catalyzed chemical processes occurring in the
apoplast and considers the biochemical steps involved in
the construction and restructuring of the cellulose-xyloglucan framewod(, an ensemble of chemical reactions that
are more complicated than commonly supposed.
Key words: Apoplast-- Cell wall-- Cellulose-- Endoxyloglucan bansferase (EXGT)--Molecular grafting m
Xyloglucan m Xyloglucan related protein (XRP)
The plant cell wall is a dynamic structure composed of a
three-dimensionally interwoven network of cellulose microfibrils embedded in several different classes of matrix polymers and give the apoplast a supermolecular framework.
Xyloglucans are the major component of the matrix and
serve as cross-links between microfibrils to form a cellulosexyloglucan framework (Hayashi 1989). This framework functions as load bearing structure of the cell wall. Gaps within
and around this framework are filled with pectic polysaccharides which form another type of framework in the plant
cell wall (Carpita and Gibeaut 1993, McCann and Roberts
1994). These two major frameworks, coupled with other
subsidiary ones such as glycoproteins and polyphenols, give
the cell wall its characteristic viscoelastic properties (Sakurai
1991, Cosgrove 1997).
The cell wall also has various types of functional molecules including enzymes, some of which are responsible for
its construction and modification (Fry 1995, Robertson et al.
1997). With the aid of these enzymes, the cell wall undergoes drastic structural changes which permit both wall
deposition and extension during plant growth and differentiation (Nishitani 1995).
Two classes of enzymes are known to be involved in the
metabolism of xyloglucans in muro: endo 1, 4-/%glucanase
(EGase) and endo-xyloglucan transferase (EXGT). The
former is a hydrolase capable of cleaving 1, 4-~-glucosyl
linkages in several glucans, including xyloglucans and carboxymethylcellulose, and has also been termed cellulase
because of its potential activity toward cellulose (Wong et al.
1977, Lashbrook e t a / . 1994). EXGT is a transferase that
catalyzes molecular grafting between xyloglucan molecules
and can thereby mediate interchange between xyloglucan
cross-links in the framework (Nishitani and Tominaga 1992,
Fry et a/. 1992, Fanutti et al. 1993). Analyses of cDNAs
encoding EXGT and its sb'ucturally related proteins have
revealed that these enzymes constitute a large multi-gene
family termed xyloglucan related proteins (XRPs) (Nishitani
1997). Data obtained to date suggest the likelihood of
different members of XRP being preferentially expressed in
different tissues (Rose e t a / . 1996, Xu e t a / . 1996) and
potentially exerting divergent enzymatic activities toward
xyloglucans (Nishitani and Tominaga 1992, Fanutti et al. 1996,
Rose e t a / . 1996, Tabuchi et aJ. 1997). In this review,
attention will be paid to the versatile enzymatic activities of
XRPs and their physiological implications for the dynamics of
the cellulose-xyloglucan framework in growing plant cells.
Structural Features of the Cellulose-Xyloglucan
Framework Underplning Cell Wall Architecture
Before describing the roles of XRPs, let us briefly consider
the structural features as well as the synthetic pathway of
the cellulose-xyloglucan framework.
Cellulose microfibrils
Abbreviations: EGase, endo-1,4-#-glucanase; EXGT, endoxyloglucan bansferase; XRP,xyloglucan related protein; XG, xyloglucan
E-mail: [email protected]
Cellulose is polyrnerized and crystallized to form microfibrils by the cellulose-synthesizing machinery called terminal
complex located on the plasma membrane (Brown et al.
160
K. Nishitani
1994, Delmer and Amor 1995) (cf. Fig1). The cellulose
microfibril is a long crystalline rod composed of several
dozen 1, 4-/3-glucan chains bound tightly to each other by
means of both intra- and inter-molecular hydrogen bonds.
In seed plants, its width is estimated to be about 5-12 nm
(McCann et al. 1990, Itoh and Ogawa 1993). The crystalline
core of each microfibril is surrounded by an amorphous layer
of 1, 4-/%glucans, which plays a role in the association with
matrix polymers (Talbott and Ray 1992a). Microfibrils are
laid down in a single layer covering the plasma membrane,
with individual microfibrils spaced regularly 13-40 nm apart
(McCann et al. 1990, Itoh and Ogawa 1993). The cell wall
consists of several stacked layers of these lamellae.
There are two distinct models for the mode of deposition
of microfibrils: the multi-net model and the helicoidal model.
The former states that new cellulose microfibrils are deposited on the inner surface of the wall in a predominantly
transverse orientation to the growth axis of the cell. The
transversely oriented lamellae permit the entire wall to
expand longitudinally along the cell axis. As the cell elongates, each sequential lamella becomes stretched and
extended longitudinally relative to the growth axis so that the
microfibrils in successively older layers become progressively
reoriented in the longitudinal direction. While this is occurring, additional microfibrils are laid down transversely on the
inner surface. As a result, the microfibrils on the innermost
lamellae are likely to show a transverse orientation, whereas
those on the outermost lamellae would be oriented randomly
or longitudinally (Preston 1982). This passive reorientation
model for the celt wall microfibrils is essentially applicable to
most parenchymatous and collenchymatous cells.
According to the helicoidal model, the cell wall is deposited on successive lamellae, each containing microfibrils
oriented in parallel arrays, with the orientation angle shifting
successively from lamella to lamella in a clockwise manner
(Levy 1991). This model applies to the epidermal cell walls
of several plants, in which the microfibrillar orientations in the
neighboring lamellae display a regular and successive rotation of about 90 degrees (Takeda and Shibaoka 1981).
Both models envisage newly synthesized microfibrils
becoming attached to the preexisting framework of the cell
wall and that this framework being continuously reoriented
relative to each other as the lamellae extend. Thus, the
precondition for these models is the existence of some way
in which the cellulose microfibrils can become spatially
rearranged within the cell wall.
Matrix polymers
In seed plants, except for Poaceae, xyloglucans are the
major component of the cell wall matrix and are characterized by a cellulose-like 1, 4-8 glucan backbone with o~xylosyl side chains attached at the 6 - 0 position of some of
the glucosyl residues (Vincken et al. 1997). Some xylosyl
side chains are further substituted with a 1, 2-/5'-gaiactosyt or
1,2-~-fucosyl-l,2-/%galactosyl side chain (York e t a / .
1995). The length of the xyloglucan molecules, which is
estimated to be 30-400 rim, is long enough to bind to two or
more cellulose microfibrils (McCann et al. 1992). Although
the mechanism by which the association between cellulose
and xyleglucans is established still remains unclear, hydrogen bondings between glucosyl residues in both polymers
are considered to be essential and the side chains of
xyloglucans seem to affect the binding ability (Levy et al.
1997).
Electron microscopic observation of xyloglucans by means
of a negative staining technique coupled with immunogold
labeling procedures showed that most of the polysaccharide
was distributed between cellulose microfibrils as well as on
them (Baba et al. 1994). In terms of solubility in alkaline
solution, xyloglucans in the cell wall exist in two forms, one
being soluble in 4% potassium hydroxide solution containing
8 M urea and the other in 24% potassium hydroxide solution,
the concentration which can disrupt the crystalline structure
of the cellulose microfibrils (Nishitani and Masuda 1983).
These results indicate the presence of two types of interactions between xyloglucans and cellulose microfibrils, namely,
a relatively weak interaction formed at the surface of cellulose microfibrils by hydrogen bondings and a strong interaction by which xyloglucans are entrapped within the crystalline core of the cellulose (Hayashi 1989, Edelmann and Fry
1992). The two different interactions seem to form the basis
of cross-links between cellulose microfibrils and xyloglucans, which function as load-bearing molecules in the cell
wall and constrain cell shape. The biological implication of
the cellulose-xyloglucan framework is that the cross-link
must be broken or rearranged for the cell wall to expand.
Enzymes Involved in Modification
of Xyloglucan-Cross Links
endo- 1, 4- ~-glucanase (EGase)
When auxin induces the cell extension growth of pea
epicotyl sections, it promotes the metabolic turnover of
xyloglucan in the cell walls (Labavitch and Ray 1974). In
epicotyl sections of azuki bean (Vigna angularis), the molecular size of xyloglucans in the cell wall decreases during the
auxin- and acidic pH- induced cell extension (Nishitani and
Masuda 1981,1982). Similar changes in xyloglucan molecules have been observed in various plant species (Inouhe et
al. 1984, Wakabayashi et al. 1993, Talbott 1992b, Revilla and
Zarra 1987, Lorences and Zarra 1987). Furthermore, in azuki
bean epicotyls, a fucose-binding lectin and antibodies raised
against xyloglucans inhibited the xyloglucan breakdown and
also significantly suppressed the auxin-induced cell wall
expansion, suggesting a causal relationship between degradation of xyloglucan cross-links and cell wall expansion
(Hoson 1991,1993). These observations offer strong evidence to support the hypothesis that cleavage of xyloglucan
cross-links is the rate-limiting step in cell wall extension.
As candidates for enzymes responsible for the degradation
of xyloglucans in plant cell walls, endo-1, 4-/~-glucanases
(EGase) capable of hydrolyzing 1, 4-/~-glucosidic linkages
have been considered (Wong et al. 1977, del Campillo et al.
1988, Hayashi and Ohsumi 1994, Wu et al. 1996). EGases
have been found in several plant species (Lashbrook 1994,
Nakamura et al. 1995, Ohmiya et al. 1995, Brummell et al.
Xyloglucan Related Proteins in the Apoplast
1997a) as well as in the microbial world. Plant EGases
isolated thus far are of the E-type, which is encoded by a
large single multi-gene family. In tomato, seven genes
coding for members of this family have been cloned. The
different members of this family show different expression
profiles between tissues. Expression of tomato Cell is
associated with abscission, whereas tomato Cel2 is expressed in ripening fruit. The expression of tomato Cel7 is
restricted to elongating hypocotyls and is up-regulated by
auxin (Catala et al. 1997). Most of the type E endo 1, 4-/~glucanases possess signal sequences destined for initial
transfer to the endoplasmic reticulum and final secretion to
the apoplast. Recently, Brummell eta/. (1997b) showed that
a member of this family, Cel3, is membrane-anchored and
localized in Golgi and plasma membranes, the site of synthesis of xyloglucans. Although EGases exhibit hydrolytic
activity toward the 1, 4-/~-glucosidic linkage in carboxymethylcellulose, they do not always act efficiently toward xyloglucans (Hayashi and Ohsumi 1994). Thus, the real roles of
EGases in xyloglucan metabolism are not clear.
Xyloglucan-specific 1, 4-/~'-glucanase purified from germinating nasturtium (Tropaeolum majus) efficiently and specifically degrades storage xyloglucans in the cotyledonary
cell wall (Edwards et al. 1985). This glucanase is structurally
distinct from the EGase and belongs to the EXGT-related
gene family (or XRP), which will be described in the following
section.
Endoxyloglucan transferase (EXGT)
Simple cleavage of load-bearing xyloglucan cross-links
will result in disassembly of the cellulose-xyloglucan framework; the degradation processes occurring in fruit ripening,
organ abscission and degradation of storage tissues in
germinating seeds. These processes can be explained
solely by the actions of hydrolytic enzymes. In the case of
most growing tissues, the cell wall polymers are continuously
synthesized, and the nascent cellulose microfibrils together
with newly secreted xyloglucans are integrated into a preexisting cellulose-xyloglucan framework. Thus, in these tissues, partial or localized disassembly of the cellulose-xyloglucan framework is continuously restored so that the wall's
physical properties remain almost unchanged. Furthermore,
the molecular weight of xyloglucans in the cell wall is fairly
precisely controlled during the cell extension process (Nishitani and Masuda 1982, Talbott and Ray 1992b, Wakabayashi et al. 1993). These findings suggest the presence of a machinery by which the xyloglucan cross-links
can be continuouly processed so as to rearrange the framework structure in muro. These processes apparently
require enzymes capable of cutting and pasting xyloglucan
chains.
EXGT-V1 (formerly Vigna EXT) is the first enzyme identified
that catalyzes endo-type transfer of the xyloglucan molecule
and thereby mediates molecular grafting reactions between
xyloglucans. The EXGT-mediated molecular grafting reaction consists of two steps: (1) endo-type cleavage of unsubstituted 1, 4-/~-Iinked glucosyl residues in the xyloglucan
molecule and (2) rejoining of the newly generated reducing
]61
terminus of the split xyloglucan to the 4 - 0 position of the
glucosyl residue at the nonreducing terminus of other xyloglucan chains (Nishitani and Tominaga 1992).
This transferase belongs to a fairly large gene family
termed xyloglucan-related proteins (XRP) (Nishitani 1997).
Based on the phylogenetic trees produced from the alignment of their deduced amino acid sequences, the XRP family
is classified into three subfamilies. Vigna EXGT-V1 belongs
to subfamily I, whereas NXG1, which encodes nasturtium
xyloglucan specific p-l, 4-glucanase, is a member of subfamily II1. With respect to Arabidopsis genes, subfamilies I,
II, and III contain two, four and three members, respectively
(Nishitani 1997, Xu et al. 1996). It is noteworthy that each
subfamily includes two or more XRP members for a single
plant species. Each member shows potentially different
expression profiles with respect to tissue specificity and
responsiveness to hormones and other signals. For example, Arabidopsis Meri-5, which belongs subfamily II, is preferentially expressed in meristematic tissues (Medford et al.
1991, Verica and Medford 1997), whereas Arabidopsis EXGTA1 (subfamily I) and TCH4 (subfamily II) are predominantly
expressed in tissues with massive deposition activity of the
cell walls (Xu et al. 1995, Nishitani 1997).
Different members of XRP can exhibit potentially different
enzymatic actions towards xyloglucans (Rose et al. 1996,
1997). Recombinant proteins of soybean BRUl(Zurek and
Clouse 1994) and Arabidopsis TCH4 (Xu et al. 1995) as well
as tomato tXET-B1 (de Silva et al. 1994) exhibit endo-xyloglucan transferase. On the other hand, the purified nasturtium NXG1 shows hydrolytic activity (de Silva et al. 1993).
The hydrolysis and the group transfer reactions differ in only
one step. The transferase transfers the split end of xyloglucan to the hydroxyl group on the non-reducing terminus of
another xyloglucan molecule, whereas the hydrolase transfers it to a water molecule. Consequently, the transferase
always accomplishes molecular grafting reaction (Fanutti et
al. 1996, Nishitani 1997). It is likely that there are divergent
enzymatic reactions between the two extremes. Recently,
Tabuchi et al. (1997) isolated, from azuki bean epicotyls,
isozymes for EXGTs with distinct catalytic activity. These
isozymes exhibited hydrolytic activity toward high Mr xyloglucan, whereas it hardly cleaved xyloglucans with Mr smaller
than 60 kDa. This seems to be an example of an enzyme
between the two extremes.
Potential Roles of XRP in Construction and
Restructuring of the CelluloseXyloglucan Framework
Formation of the cellulose-xyloglucan complex, a unit structure for construction of the framework
The process by which xyloglucans become associated to
cellulose microfibrils, probably in two distinct ways, is one of
the difficult problems to solve in order to understand the
construction process of the cellulose-xyloglucan framework.
Cellulose microfibrils are generated at the terminal complex
located on the plasma membrane, whereas xyloglucans are
polymerized in the Golgi apparatus and are secreted into the
I62
K. Nishitani
Fig. 1. A hypothetical scheme for the generation of a xylogiucan-cellulose complex. A xyloglucan molecule is indicated as strings with a triangleand fork at the reducing
and nonreducing termini, respectively. Xyloglucans are
secreted intothe apoplastby exocytosis,whereascellulose
microfibrils are synthesizedat the terminal complex 0-C).
In the apoplast,centraldomainof xyloglucansare associated with the cellulose microfibrilseither by strong (broken
line) or weak (wavy line) interactions. PM, plasma membrane; XG, xytoglucan; CM, cellulose microfibril.
cell wall space via exocytosis (Brummell eta/. 1990, White et
al. 1993). Clearly, the two components become associated
after they are secreted into the apoplast. Little or no
experimental results are available about the molecular
processes by which newly secreted xyloglucans are adsorbed into the nascent cellulose microfibrils (Hayashi 1989).
One possible explanation is that some secretory vesicles
containing xyloglucan molecules fuse with the plasma
membrane at the vicinity of the terminal complex and the
xyloglucans are directly drawn into the bundle of crystallizing
microfibrils (Fig.l). This results in the xyloglucans being
intercalated into the microfibrils to form a "strong interaction."
In this complex, xyloglucan chains would be strongly anchored to the microfibrils. Meanwhile, other vesicles containing
xyloglucans may fuse with the plasma membrane away from
the terminal complex, and discharge free xyloglucans into
the cell wall space. These xyloglucans would become
adsorbed to the surface of the microfibrils via a 'Weak
interaction."
Processing of the cellulose-xyloglucan complex
The cellulose-xyloglucan complex, assembled at the
surface of the plasma membrane might be subjected to
Fig. 2. Processingby XRPs of xyloglucan chains associated
with the microfibrils. Chainextension. A free xyloglucan
molecule is cleaved,and the split end is connected to the
nonreducing terminusof an anchored xyloglucan(Reaction
1). This reaction results in extensionof the non-reducing
terminal domain of the xyloglucan chain. The anchored
xyloglucan may be split at the reducing terminal domain
and linked to the free xyloglucan polymer to extend the
reducing terminal domain (Reaction2). Loop formation.
A moleculargrafting reaction between xyloglucan chains
anchored to the same microfibrilwill give rise to a closed
loop of xyloglucan. Solidtrianglesindicatesites of cleavage of donor substrate by XRPs.
XRP-mediated processing of the xyloglucan chains, which
are anchored to the cellulose microfibril. The xyloglucan
chain may be extended by the molecular grafting reaction: If
a free xyloglucan molecule is split at its mid point and the
newly generated reducing end is connected to the
nonreducing terminus end of an anchored xyloglucan, the
chain will be elongated (Fig. 2, Reaction 1) (Nishitani and
Tominaga 1991,1992, Thompson et al. 1997). On the other
hand, the reducing terminal domain of the anchored xyloglucan may be split and become linked to the free xyloglucan
polymer to extend the reducing terminal domain of the
anchored xyloglucan (Fig. 2, Reaction 2). Thus, conceptually, xyloglucan chains on the cellulose microfibril can be
extended in both directions by the molecular grafting reaction.
Molecular grafting reactions between xyloglucan chains
anchored in the same microfibrils will generate a closed Io0p,
which might function as a hook to interact with other
polymers (Fig. 2, Reaction 3). The reverse reaction of the
loop formation results in immobilization of the soluble xyloglucan oligomer or polymer by the molecular grafting reaction.
Xyloglucan Related Proteins in the Apoplast
]63
Fig. 4. XRP-mediated cleavage and interchange. Cleavage
of xyioglucan cross-links is achieved either by hydrolysis
(Reaction6) or group transfer to the free xyloglucan molecule (Reaction7). Both reactions are mediated by members of XRP. For symbols, see Fig. 1 and 2.
Fig.3. XRP-mediated fom~tion of cross-link. Molecular
grafting reactions in two ways (Reactions4, 5) betweentwo
xyloglucans anchoredto different cellulose microfibrils give
rise to the formation of xyloglucan c~ss-links. Crosslinkir~g between cellulo6e microfibrils is the key step in
constructing the celluloea-xyloglucan framework. In this
scheme, a nascent cellulose-xyloglucan complex (shaded)
is linked to the preexistingcellulose-xylaglucanfram~oW,.
In Reaction4, a xyloglucan associated with the nascent
complex is split, whereas in ReactionS, a xyloglucan
molecule anchored on the preexisisting~amework is split.
For symbols, see Fig. 1 and 2.
In this case, the cetlulose-xyloglucan loop functions like a
flytrap for xyloglucan oligosaccharides. More than 10 years
ago, Baydoun and Fry (1998) noticed the presence of transgJycosylation activity which could mediate the integration of
labelled xyloglucan oligosaccharides into polymeric fractions
in the medium of Spinacia cell cultures. Using the purified
EXGT-V1 and cell wall material derived from azuki bean
epicotyls, the flytrap reaction has been demonstrated to
occur in vitro: When a purified azuki bean EXGT-VI was
incubated with a m)xture of fluorescently labeled xyloglucan
oligomers and isolated cell wall, the oligomer was efficiently
incorporated into the cellulose-xyloglucan framework. This
indicated that immobilized xyloglucans act as donor sub-
stTates and xyloglucan oligomers as acceptor substrates
(Nish~tani 1997). Thus, some XRPs poter~ial{y possess the
capability to trap free xyloglucan oligosaccharides and
thereby regulate their concentrations in the apoplast.
Integration of new XG-cellulose into the framework
The cellulose-xyloglucan complex prefabricated at the
surface of the plasma membrane may be assembled and
integrated into the preexisting cell wall framework in two
ways (Fig. 3). When a xyloglucan cross-link in the preexisting framework is split and the generated reducing terminus is
reconnected to the free end of the xyloglucan molecule in
the nascent cellulose microfibril, the complex will be
attached to the framework (Reaction 4 in Fig. 3). The other
possibility is that the xyloglucans anchored on the nascent
cellulose microfibril are split and connected to the free
reducing end of xyloglucan anchored on the framework
(Reaction 5 in Fig. 3). In addition to these XRP-mediated
integrations, a single free xyJoglucan molecule can unity two
cellulose microfibrils by the weak interactions at their surface
(cf. Fig. 1).
Cleavage of cross-links
The simplest reaction mediated by XRP is the hydrolysis.
Xyloglucans occur in coty}edonary cell walls as the storage
material in many plants. In cotyledons of germinating
nasturtium seedlings, the storage xyloglucan is digested by
164
K. Nishitani
link is interchanged (Fig. 5, Reaction 8). Repetition of the
interchange reaction at a high rate will render the cellulosexyloglucan framework sensitive to mechanical stress, and
the wall will easily be extended if turgor pressure is applied
a process called chemical creep (Cosgrove 1997).
The molecular processes which underlie the construction
and restructuring of the three-dimensional architecture of
the plant cell wall consist of various biochemical steps, most
of which are still poorly understood. XRPs possess divergent enzymatic activities and can potentially play important
roles in xyloglucan metabolism, particularly, in both assembling and disassembling the cellulose-xyloglucan framework.
Molecular dissection of individual steps mediated by the
members of XRP occurring in the apoplast of plants will be
one of the most promising approaches for disclosing the
whole picture of cell wall dynamics in plants.
This work was supported by The Japan Society for the
Promotion of Science (JSPS-RFTF96LO0403) and by a
Grant-in-Aid for Scientific Research (09440269) from the
Ministry of Education, Science, Culture and Sports, Japan.
References
Fig. 5. XRP-mediatedinterchange of cross-links. When the
split end of the cross-link is connectedto the non-reducing
terminus of an anchored xyloglucan molecule, the crosslink will be interchanged (Reaction8). For symbols, see
Fig. 1 and 2.
the action of nasturtium NXG, a member of XRP (Edwards et
In growing vegetative tissues, however, partial and
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increased mobility of the framework and thereby may
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(Received January 8, 1998: Accepted January 19, 1998)