Plant and Soil 247: 71–80, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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The functions of cell wall polysaccharides in composition and architecture
revealed through mutations
Nicholas C. Carpita1,3 & Maureen C. McCann2
1 Department
2 Department
author∗
of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155, U.S.A.
of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, U.K. 3 Corresponding
Received 19 September 2001. Accepted in revised form 25 February 2002
Key words: Arabidopsis, cellulose, cell wall, FTIR spectroscopy, mutants, pectins
Abstract
The plant cell wall is a highly organized composite of many different polysaccharides, proteins and aromatic substances. These complex matrices define the shape of each individual cell, and ultimately, they are the determinants
of plant morphology. The fine structures of the major angiosperm cell wall polysaccharides have been characterized, but it is not well understood how these polysaccharides are assembled into a metabolically active architecture.
Cell wall biogenesis and remodeling may be partitioned into six major stages of development (precursor synthesis,
polymerization, secretion, assembly, rearrangement and disassembly), and to date, a handful of mutations have
been identified that affect the composition and structure in each of these stages. To greatly augment this collection,
we have initiated a program to use Fourier transform infrared spectroscopy as a high through-put screen to identify
a broad range of cell-wall mutants of Arabidopsis and maize. We anticipate that such mutants will be useful to probe
the impact of the individual components and their metabolism on basic processes of plant growth and development.
The structures of dicot and grass walls, the identification of representative cell wall mutants, and the use of a novel
spectroscopic screen to identify many more cell wall mutants, are briefly reviewed.
Abbreviations: FTIR – Fourier transform infrared; GAX – glucuronoarabinoxylan; HGA – homogalacturonan;
RG – rhamnogalacturonan; XET – xyloglucan endo-transglycosylases; XyG – xyloglucan
Introduction
The primary cell walls of plants are made up of two,
and sometimes three, structurally independent but interacting networks. The fundamental framework of
cellulose microfibrils and cross-linking glycans lies
embedded in a second network of matrix pectic polysaccharides (Carpita and Gibeaut, 1993; McCann and
Roberts, 1991). The third independent network consists of the structural proteins or a phenylpropanoid
network. Briefly, Arabidopsis (Zablackis et al., 1995)
and other dicots and the non-commelinoid monocots
possess ‘Type I’ walls (for review, see Carpita and
Gibeaut, 1993), which contain about equal amounts
∗ FAX No: +1-765-494-0363.
E-mail: carpita@btny.purdue.edu
of cellulose and cross-linking xyloglucans (XyGs) and
various minor amounts of arabinoxylans, glucomannans and galacto-glucomannans. The cellulose-XyG
framework is embedded in a pectin matrix that controls several physiological properties, such as wall
porosity, charge density and microfibril spacing. The
two major pectins are homogalacturonans (HGAs) and
rhamnogalacturonan I (RG I). Some HGAs are crosslinked into junction zones by Ca2+ , whereas other
HGAs and RGs are cross-linked by ester linkages to
other pectins or polymers held more tightly in the wall
matrix and can only be released from the wall by deesterifying agents (for review, see Willats et al., 2001).
An unusual polysaccharide called RG II, a relatively
minor pectic component, has the richest diversity of
sugars and linkage structures known. Neutral poly-
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mers (arabinans or galactans) are pinned at one end
to the RG I pectic backbone, but extend into, and
are highly mobile in, the wall pores. Some Type I
walls contain several types of structural proteins that
may interact with the pectin network. The various
structural proteins could form intermolecular bridges
with other proteins without necessarily binding to the
polysaccharide components.
The walls of cereals and related monocots are
different
Maize and other commelinoid monocots possess a different kind of primary wall, a ‘Type II’ wall (Carpita,
1996; Carpita and Gibeaut, 1993). They contain cellulose microfibrils of the same structure as those of the
Type I wall, but glucuronoarabinoxylans (GAXs) are
the principal polymers that interlock the microfibrils.
Unbranched GAXs can hydrogen bond to cellulose
or to each other. The attachment of arabinose and
glucuronic acid side-groups to the xylan backbone
of GAXs prevents the formation of hydrogen bonds,
diminishing the extent of cross-linking between two
unbranched GAX chains or GAX to cellulose. In general, grasses are pectin-poor, but with the exception
of the lack of fucose, the pectins they do contain
are similar in structure to those of dicots. When
grass cells begin to elongate, they accumulate mixedlinked (1→3),(1→4)β-D-glucans in addition to GAX.
The mixed-linkage β-glucans are unique to the Poales (Smith and Harris, 1999) and are an example of
a developmental-stage-specific polysaccharide associated with cell expansion (Carpita, 1996).
Grasses, which have very little structural protein
compared with dicots and non-commelinoid monocots, have extensive interconnecting networks of
phenylpropanoids that form primarily when cells stop
expanding (Iiyama et al., 1994). Their richness in
phenolic substances gives species with Type II walls
the distinguishing property of autofluorescence in their
primary walls (Rudall and Caddick, 1994). In the
non-lignified walls, the principal hydroxycinnamate is
ferulic acid, whereas in the lignified walls, both ferulic and p-coumaric acid are found. Ferulic acid is
esterfied to the C-5 of arabinofuranose sidechains of
arabinoxylans and they can dimerize, either via a photochemical reaction leading to cyclodimers (Ford and
Hartley, 1989) or via radical-mediated dimerization
which gives rise to diferulate dehydrodimers (Ralph
et al., 1995).
Cell-wall mutants will help plant biologists to
deduce gene function
Over 17% of the 25 498 Arabidopsis genes have signal
peptides, and over 400 proteins have been identified
that reside in the wall (Arabidopsis Genome Initiative,
2000). If just one-half of the proteins with signal peptides function in the biosynthesis, assembly and modification of the walls, then well over 2000 genes are
likely to participate in wall biogenesis and remodeling
during plant development. If all the cytosolic proteins
that function in substrate generation are included, the
number increases significantly. Some integral plasma
membrane-associated proteins that function in wall assembly, such as cellulose synthase, do not contain
signal peptides. Thus, it is likely that some 15% of the
Arabidopsis genome is dedicated to cell wall biogenesis and modification. Only a few gene families that
encode wall-relevant genes have been characterized,
and it is estimated that over one-half of wall-relevant
genes have yet to be annotated.
The major steps in wall biogenesis and modification can be divided into six specific stages: (1)
the synthesis of monomer building blocks, such as
nucleotide-sugars and monolignols; (2) the biosynthesis of oligomers and polysaccharides at the plasma
membrane and ER-Golgi apparatus; (3) the targeting
and secretion of Golgi-derived materials; (4) the assembly and architectural patterning of polymers; (5)
dynamic rearrangement during cell growth and differentiation; (6) wall disassembly and catabolism of
polymers.
For some of these stages, such as the generation
of known substrates, complete knowledge of the biochemical pathways has led to discovery of many of
the genes encoding the enzymes involved in the catalysis. For other stages, such as wall assembly, we can
only speculate on the nature of the proteins that might
participate. Recently, forward (Reiter et al., 1997;
Turner and Somerville, 1997) and reverse (Reiter,
1998) genetic approaches have provided new insights
on wall-relevant genes. Forward genetic approaches
have historically been hampered by difficult technical
problems associated with characterization of polymer
synthesis in vitro and of higher order architectural
assembly and rearrangement during growth.
The first broad screen for cell-wall mutants was
conducted by Wolf-Dieter Reiter in the laboratory
of Chris Somerville, where they selected 11 loci responsible for certain cell-wall monosaccharides to be
over- or under-represented when compared to wild-
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type (Reiter, 1998; Reiter et al., 1997). Among these
mutants, called ‘mur’ (from the Latin murus, meaning ‘wall’), were three in which fucose was either
completely absent (mur1) or under-represented by
about 50% (mur2 and mur3). The mur1 mutation was
mapped to a gene that encoded a 4,6-dehydratase that
functions in the conversion of GDP-Man to GDPFuc (Bonin et al., 1998). Because the mutation lies
in a gene that is responsible for the synthesis of the
substrate for every polymer containing fucose, all
polymers lack this sugar. For XyG, a majority of
the galactosyl extensions of the xylosyl residues that
would normally contain L-Fuc are left naked, but
about 20% of them contain L-Gal in place of L-Fuc
(Zablackis et al., 1996). The MUR4 gene is thought
to encode a C4 epimerase that interconverts UDPXyl and UDP-Ara (Burget and Reiter, 1999). The
mur4 mutant has approximately one-half of the normal arabinose content in its leaf cell walls (Reiter
et al., 1997), and an alternative pathway to UDPAra may provide the limited amount of arabinose in
these plants. With several candidate mutations of the
nucleotide-sugar interconversion pathway, the wildtype phenotype can be restored by feeding plants with
the deficient sugar, which can then form the nucleotide sugar by salvage pathways involving C-1 kinases
and NDP-pyrophosphorylases. Such is the case with
mur1 (Reiter et al., 1993) and mur4 (Burget and Reiter,
1999).
Mutants with altered cellulose deposition have
been identified directly by loss of birefringence, for
example in trichomes (Potikha and Delmer, 1995).
Other cell-wall mutations have appeared serendipitously in the course of screens for developmental phenotypes, such as a temperature-sensitive radial cellswelling mutant (rsw1) that was partially cellulosedeficient (Baskin et al., 1992) or plants with a
collapsed xylem phenotype (Turner and Somerville,
1997). Delmer et al. (Pear et al., 1996) identified
a cDNA produced exclusively during secondary wall
formation in cotton fibers that is most likely a candidate for the catalytic subunit of cellulose synthase
(CesA). Arioli et al. (1998) restored normal cellulose synthesis in the Arabidopsis rsw1 mutant by
genetic complementation with a CesA gene. Similarly, the irregular xylem mutants irx1 and irx3, which
have reduced cellulose content in the secondary walls
of tracheary elements (Turner and Somerville, 1997)
have been traced to defects in CesA genes (Taylor et
al., 1999, 2000). A survey of the Arabidopsis database
revealed several dozen related genes that could func-
tion in the synthesis, not only of cellulose, but perhaps
also of many other cross-linking glycans that comprise the plant cell wall. Synthases of all cross-linking
glycans containing (1→4)β-D-glucosyl, mannosyl, or
xylosyl backbones must overcome the steric problem
of flipping one sugar almost 180◦ with respect to
each neighboring sugar (Carpita and Vergara, 1998;
Gibeaut and Carpita, 1994). One of the conserved
features of the CesA and cellulose-synthase-like (Csl)
gene families (Richmond and Somerville, 2001) are
four motifs responsible for UDP-sugar binding and
catalysis in β(1→4)-linked polymers. Such motifs in
large and diverse families may mean that identification of the cross-linking glycan backbone polymerases
is close at hand. Mutants may also reveal unpredictable roles for known enzymes and proteins. For example, a unique membrane-associated endo-glucanase
(Brummell et al., 1997) appears to be involved in
cellulose synthesis, as judged by a loss-of-function
mutant called korrigan in Arabidopsis (Nicol et al.,
1998).
The complex polysaccharides of the plants’ extracellular matrix are not random mixtures of sugars and
sugar linkages but assemblies of discrete unit structures. For example, the unit structure of the predominant XyG in flowering plants consists of a unit structure
of a cellotetraose unit to which xylose units are added to the O-6 position of three contiguous glucosyl
residues (Figure 1). To about one-half of these subunits, an α-L-fucosyl-(1→2)β-D-galactosyl-(1→2)side-group is added specifically to the xylose residue
closest to the reducing end of the subunit. In Arabidopsis and other dicots, about one-half of these
fucogalacto-side-chains also contain an unfucosylated
β-D-galactosyl-(1→2)- extension on the middle xylose unit. Less frequently, an α-L-arabinosyl unit is
added to the O-2 position of the glucan chain specifically at the xylosyl-bearing unit closest to the reducing
end (Figure 1).
Although the XyG polymer may be assembled
from various combinations of these basic subunits, the
reason for such specificity of linkage structure is inherent in the composition of the synthase complex that
makes the polymer. One may estimate that at least
eight unique enzyme activities are needed to synthesize a fully decorated XyG unit (Figure 2). Because
of the difficulties that investigators have experienced
in purifying such complexes from their membrane
locations in an active form, molecular biological approaches have now been employed to identify the
genes that encode the components of the XyG syn-
Figure 1. The fundamental building block of (fucogalacto)xyloglucan is a cellotetraose unit to which xylose units are added to the O-6 position of the three contiguous glucosyl residues.
Oligomers containing these unit structures are generated by hydrolysis of xyloglucan by a Trichoderma cellulase that is restricted to cleavage of the reducing end of unbranched glucosyl
residues (arrows). A single-letter nomenclature, referring to the terminal subtending sugar or G for glucose in the backbone, is shown for two subunit structures (Fry et al., 1993). To about
one-half of these fundamental subunits (XXXG), an α-L-fucosyl-(1→2)β-D-galactosyl-(1→2)- extension is added to the xylose residue closest to the reducing end (XXFG). In Arabidopsis
and other dicots, about one-half of these fucogalacto-extended subunits also contain a β-D-galactosyl-(1→2)- extension on the middle xylose unit. Less frequently, an α-L-arabinosyl unit is
added to the O-2 position of the glucan chain specifically at the xylosyl-bearing unit closest to the reducing end.
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Figure 2. An estimated minimum of eight enzymes is required to
construct a xyloglucan. A glucan synthase (1) that synthesizes the
backbone is coordinated with three distinct xylosyl transferases (2,
3 and 4). It is doubtful that a single xylosyl transferase adds all three
residues because of spatial and symmetry considerations. A galactosyl transferase (5) extends the first xylosyl residue, and a fucosyl
transferase (6) extends the side-group further. Mutations have been
identified that are defective in the activities of these enzymes. A
second galactosyl transferase (7) adds this residue to the middle
xylosyl residue. This activity is enhanced in mutants that fail to
produce the galactosyl transferase (5). An arabinosyl transferase (8)
adds this residue to the glucan chain, precisely at the O-2 position
of the glucosyl residue containing the trisaccharide side-group.
thase. The most powerful means of gene identification
is the characterization of mutants that are missing
one or more of the components of synthesis. Mur2
and mur3 are mutations in genes that enclose enzymes associated with XyG synthase specifically; the
MUR2 gene encodes the XyG-specific fucosyl transferase (Vanzin et al., 2002) and MUR3 encodes the
pre-requisite galactosyl transferase that initially extends the xylosyl residue (Madson et al., personal
communication).
The mur1 mutation causes plants to be slightly
stunted and results in a loss of tensile strength of the
floral stem (Reiter et al., 1993). The loss in tensile
strength was attributed to the loss of fucose from
XyG, because modeling studies had suggested that
the fucosylated trisaccharide plays a role in physically
straightening the glucan backbone to facilitate binding
to cellulose microfibrils (Levy et al., 1991). Hence,
loss of fucose meant loss of this function, and a compromised XyG-cellulose binding would be expected
to result in loss of cell wall strength. However, mur2
and mur3 plants, despite complete loss of the trisaccharide function, are phenotypically indistinguishable
from wild-type in every respect, and tensile strength
of the floral stalk is comparable to wild-type (Madson
et al., personal communication). These findings indicate that the loss of wall strength in the mur1 plants
may be a consequence of a loss of fucose from pectic
polysaccharides.
The synthesis of XyGs is relatively simple when
compared to the synthesis of some pectic polysaccharides. Synthesis of HGA requires a minimum of two enzymes, as the monomeric backbone GalA residues are
subsequently methyl esterified (Doong and Mohnen,
1998) and synthesis of RG I requires three: two
to assemble the α-L-Rha-(1→2)-α-D-GalA-(1→4)backbone unit, with additional acetyl esters on some
of the GalA residues. Several neutral sugar chains
are added to the rhamnosyl units at certain stages of
cell development, notably the arabinans, galactans,
and arabinogalactans (Carpita and Gibeaut, 1993).
The mur5 and mur6 mutations also result in reduced
amounts of cell-wall arabinose (Reiter, 1998), but they
cannot be rescued by feeding of arabinose, and therefore, the genes affected encode enzymes involved in
downstream events in wall biogenesis.
One of the most complex polysaccharides ever discovered in nature is RG II, with at least 21 enzymes
required for its synthesis (Figure 3). The function of
each monosaccharide in the structure is far from understood, but one of the essential functions of the RG
II polymer is to cross-link to form boron di-diester
dimers, which limits pore size (Fleischer et al., 1998)
and increases tensile strength. Walls swell when plants
are grown on boron-deficient media (Ishii et al., 2001).
Identification of all the genes involves in the synthesis
and assembling of RG II will be a daunting task.
Secretion mutants related to vesicle targeting have
been well characterized in yeast, but comparable defects in plants have not (Staehelin and Moore, 1995).
Judging from the yeast paradigm, such mutations
would be predicted to result in a general loss in wall
mass with concomitant accumulation of cell-wall containing vesicles in the cytoplasm. A single secretion
mutant, called knolle, with a defective syntaxin gene,
does not complete formation of the cell plate and cells
are not correctly partitioned (Lukowitz et al., 1996).
Wall assembly mechanisms are not understood at
all, and we cannot predict the impact of defects to
Figure 3. At least 21 enzymes are required to assemble a rhamnogalacturonan II molecule. This is one of the most complex biopolymers in Nature and contains several rare sugars, including
3-deoxy-D-manno-2-octulosonic acid (KDO), 3-deoxy- D-lyxo-2-heptulosonic acid (DHA), aceric acid (3-C -carboxy-5-deoxy-L-xylose), apiose, 2-O-methyl xylose, and 2-O-methyl fucose.
The molecule dimerizes to form boron di-diesters with the first apiose residues (2). This dimerization is associated with increases in tensile strength and decreased porosity.
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these processes. Important mutants to identify are the
enzymes and proteins that function in wall rearrangement during growth, such as expansins and xyloglucan
endotransglycosylases (XETs). Exogenous expansin
can alone trigger leaf primordium formation in the
epidermal cells of an apical dome (Fleming et al.,
1997) and so mutants might be expected to have
severe developmental phenotypes. Since many hydrolases function as transglycosylases, enzymes such
as XET may have a strict transglycosylase activity
(Nishitani, 1995). Such activities may serve roles in
wall assembly rather than in wall dynamics and disassembly (Thompson et al., 1997). One member of
the XET family is touch-inducible (Xu et al., 1995).
Dwarf and miniature mutants represent a visible class
of mutants, whose basis may lie in defects in wall
dynamics caused by lack of XETs (Akamatsu et al.,
1999; Verica and Medford, 1997).
Hydrolysis of polymers during wall disassembly
results in production of several monosaccharides that
are salvaged back into the nucleotide-sugar pool (Reiter and Vanzin, 2001). Defects in these pathways
would result in an inability to recycle sugars and may
lead to reduced content of those sugars in the wall.
Many of these sugars are also phytotoxic. In fact,
an arabinose C-1 kinase mutant was discovered because the plants are unable to grow in the presence
of the high concentrations of arabinose (Dolezal and
Cobbett, 1991).
Antisense inhibition experiments have been of
equivocal value in assessing the role in growth and
development of cell-wall related proteins because of
the spectrum of functions that individual members
of a gene family may possess. Two additional reasons for the difficulty in interpreting the phenotypes
of antisense plants stem from either excess enzyme
levels such that pathways can proceed normally with
as little as 1% of the normal level, or genetic redundancy. Meaningful studies may need to await discovery
of activity-controlling proteins revealed as developmental mutants. Many other developmental mutants
exist whose defects are likely to be traced to cell
wall components (Benfey et al., 1993; Lolle et al.,
1997; Reynolds et al., 1998; Smith et al., 1996), although their functions in wall architecture remain to
be elucidated.
A new high through-put method to identify
cell-wall mutants
Given the enormous range of cellular, developmental,
and molecular events that the six stages of cell-wall
biogenesis and remodeling comprise, and the paucity
of cell-wall mutants in hand, we need to augment the
mutant collection substantially. We have developed a
powerful method to do this. Fourier transform infrared
(FTIR) microspectroscopy is an extremely rapid, noninvasive vibrational spectroscopic method that can
quantitatively detect a range of functional groups (McCann et al., 1992, 1997; Séné et al., 1994). We have
optimized FTIR as a high through-put screen for cell
wall mutants (Chen et al., 1998). The advantages of
FTIR spectroscopy for a mutant screen are many-fold.
The time taken to acquire a spectrum of a sample is
roughly 30 s. It is non-destructive and does not require
derivatization of the sample, so further assays can be
applied to the same sample once potential mutants are
identified. Several functional groups absorb infrared
radiation at characteristic frequencies, making assignments of some specific wall components possible. In
particular, frequencies corresponding to carboxylic
and phenolic esters and the carboxylate stretches of
uronic acids are clearly resolved in the spectrum (Figure 4A). As chemically different specimens can give
rise to visually similar spectra, or may have very
subtle differences, we have used chemometric methods to analyze these multivariate data. By using data
compression methods such as Principal Component
Analysis, we can transform a data set comprising a
large number of inter-correlated variates into a new
set of a small number of variables (Figure 4B). The
derived variables are known as ‘loadings’, and are uncorrelated and ordered such that the first few account
for the majority of the variance that was spread across
all of the original variates (Kemsley, 1998). Use of this
algorithm enables one to analyze very large populations of spectra (Figure 4B). Additional chemometric
methods can be applied, including Linear Discriminant Analysis, which tests if all members of a given
population vary significantly from all members of a
wild-type population, and other algorithms that test if
a specimen is a member of the ‘wild-type’ class at the
95 or 99% probability level (Kemsley, 1998).
We are also using reverse-genetic approaches to
identify cell wall biogenesis-related genes in Arabidopsis and maize. Cloning of these genes is simplified because of the use of insertional mutants, either
transposon-tagged maize or T-DNA transformed Ara-
78
Figure 4. How Discriminant Analysis of Fourier transformed infrared spectra is used to differentiate cell walls of different composition. (A)
Spectra are shown of ten samples each of maize walls from control and those sequentially treated with acidic sodium chlorite to oxidize phenolic
substances and 0.1 m NaOH to remove mostly highly substituted GAXs. A difference spectrum shows that ester (1238 and 1734 cm−1 ) and
phenolic residues account for the major differences revealed in a difference spectrum of the averaged spectra of these two groups. (B) Principal
Components 1 and 2 account for over 80% of the total variation among spectra of both samples (30 each) and completely resolve them. The
‘loading’ of Principal Component 2, which is the primary discriminator, illustrates the differences are primarily due to ester linkages and
phenolic residues lost in the treated walls.
bidopsis. A major practical goal is to generate plants
with genetically defined variation in composition and
architecture to permit assessment of modifications on
wall properties and plant development. Several of
these mutants may be useful to determine the role
of the apoplasm in regulating inorganic nutrient uptake. As the mutations we identify by spectroscopy
are characterized, the plant biology community will
be informed of them through a web site, and a system will be devised to provide seeds and clones to the
community through established centers.
Acknowledgements
Characterization of the mur2 and mur3 mutants is
supported by a grant from the U.S. Department of
Agriculture-National Research Initiative Competitive
Grants Program (to N.C.C.) and the joint program to
develop the use of FTIR spectroscopy to identify cellwall mutants is supported by grants from the U.S.
National Science Foundation Genome Research Program (to N.C.C.) and the U.K. Biotechnological and
Biological Science Research Council (to M.C.M.).
M.C.M. is grateful for a University Research Fellowship fom the Royal Society. Journal paper No. 16,
xxx of the Purdue University Agricultural Experiment
Station.
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