J 1365-313X 2010 04161 X

The Plant Journal (2010) 61, 11071121
doi: 10.1111/j.1365-313X.2010.04161.x
ARABIDOPSIS: A RICH HARVEST 10 YEARS AFTER COMPLETION OF THE GENOME SEQUENCE
Arabidopsis a powerful model system for plant cell wall

research
Aaron H. Liepman1,*, Raymond Wightman2, Naomi Geshi3, Simon R. Turner2 and Henrik Vibe Scheller4,5
Biology Department, Eastern Michigan University, 316 Mark Jefferson Building, Ypsilanti, MI 48197, USA,
2
Faculty of Life Science, University of Manchester, Oxford Road, Manchester M13 6AY, UK,
3
VKR Research Centre, Department of Plant Biology and Biotechnology, University of Copenhagen,
DK-1871 Frederiksberg C, Denmark,
4
Feedstocks Division, Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA, and
5
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Received 1 November 2009; revised 13 January 2010; accepted 15 January 2010.

*For correspondence (fax +1 734 487 9235; e-mail aliepman@emich.edu).
SUMMARY
Plant cell walls are composites of various carbohydrates, proteins and other compounds. Cell walls provide
plants with strength and protection, and also represent the most abundant source of renewable biomass.
Despite the importance of plant cell walls, comparatively little is known about the identities of genes and
functions of proteins involved in their biosynthesis. The model plant Arabidopsis and the availability of its
genome sequence have been invaluable for the identification and functional characterization of genes
encoding enzymes involved in plant cell-wall biosynthesis. This review covers recent progress in the
identification and characterization of genes encoding proteins involved in the biosynthesis of Arabidopsis cellwall polysaccharides and arabinogalactan proteins. These studies have improved our understanding of both
the mechanisms of cell-wall biosynthesis and the functions of various cell-wall polymers, and have highlighted
areas where further research is needed.
Keywords: cellulose, hemicellulose, pectin, arabinogalactan proteins, glycosyltransferases, glycan synthases.
INTRODUCTION
The cells walls surrounding plant cells serve many important functions. The composition and architecture of cell
walls contribute to the remarkable morphological and
functional diversity of plant cell types. For example, cell
walls strengthen plant cells. Most cells use this strength to
resist positive pressure generated within the cell; by
harnessing this turgor pressure, plants are able to stand
upright. In contrast, thick cell walls of water conducting
vessels and tracheids enable these cells to resist substantial
negative pressure; they do so by depositing a thick cell wall.
Plant cell walls also provide the first line of defense against
invading micro-organisms and abiotic stresses.
There are many types of cell walls; however, most may
be grouped into one of two functional classes: primary
walls or secondary walls. Primary walls are synthesized
during cell expansion in growing cells, while secondary
walls are deposited in certain cell types after cell
2010 The Authors
Journal compilation 2010 Blackwell Publishing Ltd
expansion has ceased. Although cell walls are strong

composites deposited outside the plasma membrane, they
are sufficiently flexible to allow cell expansion, and their
composition is modulated in response to the physiological
needs of the plant.
Cell walls are composed of a variety of polymers, which
typically include cellulose, hemicelluloses, pectin and structural proteins. In addition, some cell walls contain lignin,
which is a polymer of monolignols derived from the
phenylpropanoid pathway. Many recent reviews have
described the structure and diversity of plant cell-wall
polymers as well as their biosynthesis (see, for example,
Somerville, 2006; Mohnen, 2008; Popper, 2008; Vogel, 2008;
Scheller and Ulvskov, 2010). Cellulose is the main loadbearing structure in the cell wall, and typically constitutes
approximately 3040% of the wall mass. The term hemicelluloses covers a diverse group of polysaccharides, which
1107
1108 Aaron H. Liepman et al.

generally exhibit limited solubility and interact with cellulose microfibrils. The hemicelluloses (i.e. xyloglucans,
xylans, mannans and mixed-linkage b-glucans) have
1,4-b-linked backbones of sugars in an equatorial linkage
configuration; short sidechains are appended to the backbones of some of these polymers. Occasionally, other
polymers are considered to be hemicelluloses, but we
consider that a clear definition based on common structural
features is more useful (Scheller and Ulvskov, 2010). Pectins
are very complex acidic polysaccharides with backbones
that are rich in galacturonic acid (see below); these polysaccharides are more easily solubilized than hemicelluloses.
About 10% of the genes in Arabidopsis have been estimated to be involved in the various aspects of cell-wall
metabolism, including polymer biosynthesis, transport,
deposition, remodeling, turnover, and regulation of these
processes (McCann and Carpita, 2008). The problems of
trying to purify membrane-bound enzymes that use complex
substrates using conventional biochemical approaches have
been well-documented. Consequently, this has made the
identification of enzymes that catalyze cell-wall polysaccharide biosynthesis very difficult, and traditional biochemical
methods have led to identification of only a few genes
encoding biosynthetic enzymes, despite extensive efforts for
many years. The Arabidopsis genome sequence (Arabidopsis Genome Initiative, 2000) has provided an invaluable tool
for efficient identification and systematic functional analysis
of genes encoding cell-wall polysaccharide biosynthetic
enzymes. Arabidopsis is an excellent model plant for cellwall studies as its cell walls resemble those found in many
crop plants and trees. Despite the complexity of plant cellwall structure and biosynthesis, forward and reverse genetics
and other functional genomic strategies in Arabidopsis have
led to the identification of many genes encoding proteins
involved in cell-wall biogenesis. These studies have had a
major impact on our understanding of the synthesis of plant
cell-wall polysaccharides, as well as the function of these
polymers within the cell wall. We have also gained a better
understanding of the roles of plant cell walls in regulating
processes such as plant growth and interactions with the
environment. There are, however, some notable differences
between the cell walls found in Arabidopsis and those found
in other plants (Vogel, 2008). Because grass cell walls contain
mixed-linkage b-glucans and xylan-bound hydroxycinnamate esters, which are not found in Arabidopsis, orthologous
genes involved in these biosynthetic pathways may be
absent from the Arabidopsis genome, necessitating the use
of other model systems to understand these pathways. Many
of the other differences between cell walls in grasses and
plants such as Arabidopsis may be quantitative rather than
qualitative, and hence orthologous genes may be present. In
some cases, the absence of orthologous sequences has
proven advantageous. For example, gain-of-function experiments performed in Arabidopsis implicated cellulose
synthase-like F (CSLF) and cellulose synthase-like H (CSLH)

sequences in mixed-linkage b-glucan biosynthesis (Burton
et al., 2006; Doblin et al., 2009; Fincher, 2009).
This review describes the current state of knowledge with
regard to the identities of genes encoding proteins involved
in the biosynthesis of cell-wall polysaccharides and
arabinogalactan proteins in Arabidopsis. Much progress in
Arabidopsis was made possible by pioneering work involving seminal experiments that led to elucidation of the
structures of plant cell-wall polysaccharides and characterization of the enzymes required to synthesize them; however, space constraints prevent detailed discussion of many
of these experiments. As many other processes are vital for
proper cell-wall biogenesis, including precursor synthesis
and transport, transcriptional regulation, signal transduction, transport of cell wall polymers to the apoplast, and
integration of these polymers into the cell wall, we refer you
to other recent reviews covering these topics (Zhong and Ye,
2007; Hematy and Hofte, 2008; Reiter, 2008; Reyes and
Orellana, 2008; Vogel, 2008). The focus of this review is to
highlight ways in which the availability of the Arabidopsis
genome sequence has enabled the identification and functional characterization of enzymes catalyzing various steps
in the biosynthesis of cell-wall polysaccharides and arabinogalactan proteins, and the insights that these discoveries
have provided into the function of particular cell-wall
components in regulating both cell growth and wall structural integrity.
CELLULOSE
Cellulose is composed of unsubstituted 1,4-b-glucan chains,
and is a major load-bearing and ubiquitous constituent of
plant cell walls. The plasma membrane localized machinery
that performs cellulose synthesis is a large multi-protein
complex, termed the cellulose synthase complex. This
complex simultaneously synthesizes the 1836 glucan
chains that are found bonded together in a single microfibril.
This complex resides within the plane of the plasma membrane, where it is able to access intracellular UDP-glucose
and to extrude microfibrils outside the cell, directly into the
cell wall (for recent reviews, see Somerville, 2006; Taylor,
2008).
The cellulose synthase complex has been visualized in
freeze fracture studies as a six-lobed structure of approximately 30 nm in diameter, known as a rosette (Mueller and
Brown, 1980). The first genes found to be involved in
cellulose synthesis encoded the cellulose synthase (CESA)
proteins. These were first identified as ESTs isolated from
developing cotton fibers based on their sequence homology
to bacterial genes that encode proteins involved in cellulose
biosynthesis and the ability of the corresponding protein to
bind to UDP-glucose (Pear et al., 1996). Subsequent analysis
has shown that the Arabidopsis genome encodes a large
family of proteins that show homology to the bacterial
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Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
Arabidopsis cell-wall biosynthetic enzymes 1109

cellulose synthases (CESA superfamily; Richmond, 2000;
Richmond and Somerville, 2000); these proteins have been
implicated in the biosynthesis of a variety of plant cell-wall
polysaccharides (see below). The first functional proof that
CESA proteins were essential for cellulose synthesis came
from studies of the temperature-sensitive Arabidopsis
mutant radial swelling 1 (rsw1), which exhibited decreased
cellulose content when grown at the restrictive temperature
due to a defect in a member of the CESA gene family
(AtCESA1). rsw1 mutants also show a loss of rosette
structures from the plasma membrane at the restrictive
temperature, demonstrating a clear connection between
CESA proteins, cellulose synthesis and the requirement for
rosettes in the plasma membrane, and providing functional
evidence that the rosette structures visualized were indeed
the protein complexes required for cellulose synthesis
(Arioli et al., 1998). Forward genetic screens have identified
a number of other cellulose-deficient mutants resulting from
mutations in CESA genes, including ixr1, ixr2/prc1, irx1, irx3
and irx5 (Arioli et al., 1998; Taylor et al., 1999, 2000, 2003;
Fagard et al., 2000; Scheible et al., 2001; Desprez et al., 2002)
(see Table 1). Sequencing of the Arabidopsis genome
revealed a family of ten CESA genes (Richmond, 2000).
The irx1 (CESA8), irx3 (CESA7) and irx5 (CESA4) mutants
specifically exhibit secondary wall cellulose defects (Taylor
et al., 1999, 2000, 2003), while the remainder have a range of
phenotypes consistent with primary wall cellulose defects.
Comparative analyses of Arabidopsis CESA sequences with

those from other higher plants revealed similar sets of
CESA orthologs. For example, studies of the rice brittle culm
mutants have identified three rice CESA genes that appear
to be direct functional analogs of the Arabidopsis CESA4,
CESA7 and CESA8 genes (Tanaka et al., 2003). All three
secondary wall-associated CESA members in Arabidopsis
are required for both complex formation and plasma membrane localization (Gardiner et al., 2003; Taylor et al., 2003).
This indicates that cellulose synthase contains combinations
of three unique positions each occupied by different CESA
subunits, and, in the case of the secondary wall CESA
proteins, no subunit can substitute for another. The situation
is more complex for the remaining CESA genes. Both CESA1
and CESA3 encode subunits that permanently occupy two of
the three positions, with the remaining position being filled
by CESA2, CESA5, CESA6 or CESA9 (Desprez et al., 2007;
Persson et al., 2007b). These four proteins exhibit partial
redundancy and non-overlapping expression profiles, suggesting that they are subtly suited to slightly different
functions in different plant tissues. Indeed, when the CESA6
promoter is used to drive the expression of CESA2, the
encoded protein can occupy the CESA6 position within the
cellulose synthase complex in CESA6-expressing tissues;
however, this same construct does not fully rescue the prc1
(cesA6) mutant (Persson et al., 2007b). The function of CESA10,
the remaining member of this family, remains unclear.
Table 1 Genes involved in cellulose biosynthesis

Gene name
AGI code
Mutant alleles
Function
Presumed catalytic subunits
of cellulose synthase
Subunit (position 1)
CESA family
CESA1
CESA2
CESA3
At4G32410
At4G39350
At5G05170
rsw1
CESA4
CESA5
CESA6
At5G44030
At5G09870
At5G64740
irx5, nws2
ixr2, prc1
Secondary wall
CESA7
At5G17420
irx3, fra5, mur10
Secondary wall
CESA8
At4G18780
irx1, lew2, ern1
Secondary wall
CESA9
CESA10
COBRA family
COBRA
COBL4
COBL9
KOR1
KOB1
TED6
ixr1, eli1, cev1
At2G21770
At2G25540
References
Arioli et al. (1998)

Desprez et al. (2007), Persson et al. (2007b)
Cano-Delgado et al. (2003), Ellis et al. (2002),
Scheible et al. (2001)
Hernandez-Blanco et al. (2007), Taylor et al. (2003)
Desprez et al. (2007)
Desprez et al. (2002)
Fagard et al. (2000)
Bosca et al. (2006), Taylor et al. (1999), Zhong
et al. (2003)
Chen et al. (2005), Hernandez-Blanco et al. (2007),
Taylor et al. (2000)
Persson et al. (2007b)
At5G60920
At5G15630
At5G49270
cob
irx6
mrh4
Primary wall cellulose defects

Secondary wall cellulose defects
Root hair development
Benfey et al. (1993), Schindelman et al. (2001)

Brown et al. (2005)
Jones et al. (2006)
At5G49720
At3G08550
At1G43790
irx2, kor1
kob1, eld1
Endo-1,4-b-D-glucanase
Nicol et al. (1998), Szyjanowicz et al. (2004)

Lertpiriyapong and Sung (2003), Pagant et al. (2002)
Endo et al. (2009)
References are listed for mutant alleles with the exception of TED6, for which RNAi lines were generated. We have designated CESA10 as
occupying position 1 due to its sequence similarity to CESA1, but this has not yet been demonstrated experimentally.
2010 The Authors

To date, the cellulose synthase complex has proven
intractable to biochemical analysis. The precise interactions
among the three subunits within the large rosette structure
are unknown, but recent data show that CESA proteins are
able to form oligomers (Atanassov et al., 2009; Timmers
et al., 2009). Until very recently, no biochemical data had
shown any other proteins to be associated with the complex;
however, TED6, a plasma membrane-localized protein of
unknown function, has been demonstrated to interact with
the CESA7 subunit of the secondary wall complex (Endo
et al., 2009). TED6 appears to have a general role in
secondary cell-wall formation in vessels, but whether it
has a specific function in cellulose deposition is unclear.
Many recent advances in our understanding of cellulose
synthesis have come from the field of cell biology. The
cellulose synthase complex was studied using electron
microscopy and freeze fracture long before the first plant
CESA subunit was identified (Giddings et al., 1980; Mueller
and Brown, 1980). In more recent years, Arabidopsis has
been used for studying the complex within living tissue.
Live-cell imaging of fluorescent protein fusions to Arabidopsis CESA subunits has permitted the observation of a
number of processes, including movement of the complex
within the plasma membrane (presumably during glucan
chain formation), delivery of the complex to the plasma
membrane, and its relationships to the cortical cytoskeleton
(Paredez et al., 2006, 2008; DeBolt et al., 2007a,b; Wightman
and Turner, 2008; Wightman et al., 2009).
In addition to the CESA proteins, a number of other
proteins have been implicated in cellulose microfibril formation, but none of them have been found to be associated
with the cellulose synthase complex. COBRA (COB) was
originally discovered in a screen of mutants with cell
expansion abnormalities in the root (Benfey et al., 1993).
COB encodes a small protein that is anchored to the
extracellular face of the plasma membrane by a glycosylphosphatidylinositol anchor and contains a motif with some
similarities to a cellulose-binding domain (Roudier et al.,
2002). cob mutants exhibit defects in anisotropic expansion
as a result of altered cellulose microfibril orientations;
however, the exact role of this protein in polymerization or
cell-wall deposition is unclear (Schindelman et al., 2001;
Roudier et al., 2002, 2005). Within the Arabidopsis genome,
there are a further 11 members of the COBRA-LIKE (COBL)
family. The cobl4 mutant exhibits secondary wall cellulose
defects, and, as for CESA proteins, probably represents a
secondary wall-specialized enzyme (Brown et al., 2005).
Gene network analysis placed COBL6 in a sub-network with
CESA9 (Ma et al., 2007); like CESA9, COBL6 may therefore
be involved in cellulose synthesis in pollen and anthers
(Persson et al., 2007b). COBL9 has been implicated in root
hair development, specifically tip-directed growth (Parker
et al., 2000; Jones et al., 2006). From their very specific gene
expression profiles, both COBL10 and COBL11 are probably
involved in aspects of flower development (Brady et al.,

2007).
Another key player in cellulose biosynthesis is KORRIGAN
(KOR1), an endo-1,4,-b-D-glucanase with a single transmembrane spanning sequence (Nicol et al., 1998). Several kor
mutant alleles have been reported and result in a marked
reduction in cellulose synthesis. Interestingly, it was found
that defects in this single glucanase result in perturbations in
both primary and secondary wall formation, as well as
cytokinesis (Lane et al., 2001; Sato et al., 2001; Szyjanowicz
et al., 2004). It has been hypothesized that KOR1 functions in
removing the growing cellulose chain from some type of
primer or initiator (Peng et al., 2002). Alternative hypotheses
propose that KOR1 alters the crystallization properties
within microfibrils (Szyjanowicz et al., 2004; Takahashi
et al., 2009) or that it is involved in the release of completed
cellulose microfibrils from cellulose synthase complexes
(Szyjanowicz et al., 2004). The effect of KOR1 activity upon
the growing cellulose microfibril is further supported by
recent data that show lower velocity of the cellulose
synthase complex in kor mutants (Paredez et al., 2008).
The KOBITO1 (KOB1) gene encodes another protein that
appears to be involved directly in cellulose biosynthesis. The
KOB1 protein appears to be either membrane-localized or to
exist in the cell wall; there are no recognizable motifs that
indicate its molecular function (Pagant et al., 2002;
Lertpiriyapong and Sung, 2003). Mutants of other genes
have indirect or subtle effects upon cellulose deposition. Of
particular interest are those encoding sterol biosynthetic
enzymes, thus demonstrating a link between sterols and
cellulose formation (Schrick et al., 2004).
The next major goal is to identify the precise molecular
functions of each of the encoded activities of the above genes
and to decipher where along the cellulose biosynthetic
pathway each activity occurs. This will probably involve
reconstituting cellulose synthesis in an in vitro system.
XYLOGLUCAN
Xyloglucan (XyG; Figure 1) consists of a 1,4-b-linked glucan
backbone with regular substitutions of 1,6-a-linked xylosyl
residues or side chains composed of xylosyl, galactosyl and
fucosyl residues (Hayashi, 1989; Fry et al., 1993). Arabidopsis XyG is of the characteristic XXXG type (Fry et al., 1993;
Vincken et al., 1997; Lerouxel et al., 2002; Cavalier et al.,
2008) that is typical of many other dicot plants. In
Arabidopsis, XyG is the most abundant hemicellulosic
polysaccharide in primary cell walls, representing approximately 20% of leaf cell walls (Zablackis et al., 1995). XyG
associates non-covalently with cellulose microfibrils, and
this celluloseXyG network has been proposed to contribute
to the load-bearing capacity of the primary wall, to regulate
cell expansion, and to prevent self-association of cellulose
microfibrils (McCann et al., 1990; Carpita and Gibeaut, 1993;
Pauly et al., 1999; Pena et al., 2004; Somerville et al., 2004).
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XXXG
XXLG
XXFG
= D-glucose
= D-galactose
= D-xylose
= L-fucose
Figure 1. Schematic representation of a segment of Arabidopsis xyloglucan.

Using the nomenclature conventions of Fry et al. (1993), the identities of
oligosaccharides that would be released by cellulase treatment, which results
in cleavage only at unsubstituted glucose residues, are indicated. See text for
details on the positions and anomeric configuration of each linkage.
At least four types of enzymatic activities are needed

to synthesize this complex carbohydrate (Faik et al.,
2002): (i) UDP-glucose-dependent 1,4-b-glucan synthase to
assemble the glucan backbone, (ii) UDP-xylose-dependent
1,6-a-xylosyltransferase to attach xylosyl residues to
selected glucosyl residues of the glucan backbone, (iii)
UDP-galactose-dependent 1,2-b-galactosyltransferase to
attach galactosyl residues to specific xylosyl residues,
and (iv) GDP-fucose-dependent 1,2-a-fucosyltransferase to
attach fucosyl residues to selected galactosyl residues. One
or more genes encoding each type of biosynthetic enzyme
needed for XyG biosynthesis have been identified and
characterized (Table 2).
The Arabidopsis CELLULOSE SYNTHASE-LIKE C4
(CSLC4) gene, encoding a member of the CaZY glycosyltransferase (GT) family GT2 (Cantarel et al., 2009), has
been implicated as a 1,4-b-glucan synthase involved in
XyG backbone biosynthesis (Cocuron et al., 2007). CSLC4
was identified as the most closely related Arabidopsis
sequence to a CSLC gene from nasturtium (Tropaeolum
majus) that is highly expressed at a stage of seed
development during which mass deposition of storage
XyG occurs. Although confirmation of function requires
the characterization of XyG in CSLC mutant plants, several
compelling lines of evidence support the prediction that
one or more members of the AtCSLC family encode XyG

b-glucan synthase, including: (i) accumulation of soluble
cellodextrins upon heterologous expression of AtCSLC4
and TmCSLC in Pichia pastoris, (ii) production of longer,
insoluble b-glucan chains upon co-expression in P. pastoris of AtCSLC4 with an Arabidopsis 1,6-a-xylosyltransferase (AtXXT1, see below), suggesting interaction between
these two enzymes, and (iii) coordinated expression of
AtCSLC4 and AtXXT1 in Arabidopsis. Because the Arabidopsis CSLC family contains five members, disruption of
expression of multiple members of the family may be
necessary to perturb XyG biosynthesis. There is also
evidence that some CSLC sequences are involved in the
synthesis of other polysaccharides (Dwivany et al., 2009).
Several genes encoding glycosyltransferases involved in
the addition of xylosyl residues to the glucan backbone of
XyG have been identified (Faik et al., 2002; Cavalier and
Keegstra, 2006; Cavalier et al., 2008; Zabotina et al., 2008).
Each of these 1,6-a-xylosyltransferases is a representative of
a seven-membered CaZY GT34 family in Arabidopsis.
AtXXT1, the first functionally characterized representative
of this family in Arabidopsis (Faik et al., 2002), was identified
based on biochemical and sequence similarities to galactomannan 1,6-a-galactosyltransferase from fenugreek (Trigonella foenum-graecum) (Edwards et al., 1999). Cavalier and
Keegstra (2006) characterized recombinant AtXXT1 and its
closest homolog, AtXXT2, and observed that these enzymes
are biochemically similar, each being capable of xylosylating cellohexaose in vitro. These enzymes preferentially
xylosylate cellohexaose at the fourth glucosyl residue from
the reducing end, and will also produce doubly and triply
xylosylated products. The AtXXT5 gene encodes another
putative xylosyltransferase, which, when disrupted, yields
plants with decreased XyG quantity and degree of xylosylation (Zabotina et al., 2008), but its activity has not been
confirmed. Reverse genetic analysis of Arabidopsis xxt1 and
xxt2 single mutants indicated that these plants exhibit no
significant morphological perturbations, suggesting genetic
redundancy of these sequences. In contrast, xxt1 xxt2
double mutant plants lack detectable XyG (Cavalier et al.,
2008). The apparent absence of XyG in xxt1 xxt2 double
mutants suggests that XXT1 or XXT2 is required in order for
Table 2 Biochemically characterized sequences involved in XyG biosynthesis

Gene name(s)
AGI code
CaZY family
Function
Notes/reference(s)
FUT1/MUR2
At2G03220
GT37
1,2-a-fucosyltransferase
XXT1
XXT2
At3G62720
At4G02500
GT34
GT34
1,6-a-xylosyltransferase
1,6-a-xylosyltransferase
MUR3
CSLC4
At2G20370
At3G28180
GT47
GT2
1,2-b-galactosyltransferase
1,4-b-glucan synthase
Mutant has altered trichome papillae (Perrin et al.,

1999; Vanzin et al., 2002)
xxt1 and xxt2 single mutants have no observed
phenotype, xxt1 xxt2 double mutant has aberrant root hairs
and significantly altered cell-wall mechanical properties
(Cavalier and Keegstra, 2006; Cavalier et al., 2008)
Mutant has aberrant trichome papillae (Madson et al., 2003)
Cocuron et al. (2007)
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proper xylosylation of XyG to occur. Perhaps the most
surprising revelation resulting from studies of xxt1 xxt2
double mutants is their relatively mild phenotypic abnormalities; many models of primary walls predict a prominent
structural role of XyG, so it may be necessary for the cellwall research community to reconsider the roles of XyG.
Genetic screening has proven powerful as a tool for
identifying genes encoding cell-wall biosynthetic enzymes
in Arabidopsis. A screen that examined the monosaccharide
composition of cell walls among mutant plants resulted in
identification of numerous murus (mur) loci that affect the
composition of cell-wall polysaccharides (Reiter et al., 1993,
1997). Among these mutants was mur3, a mutant characterized by a significant reduction in the fucose content of its
cell walls (Reiter et al., 1997), and aberrant XyG galactosylation (Madson et al., 2003). Positional cloning of MUR3
revealed that this gene encodes a xyloglucan b-1,2-galactosyltransferase (CaZY family GT47) that specifically galactosylates the third xylosyl residue from the non-reducing
end of XXXG, forming XXLG (Madson et al., 2003); the
reduction in fucose content observed in walls of the mur3
mutant therefore resulted from the absence of an appropriate glycosylation site for XyG a-fucosyltransferase. Interestingly, the second xylosyl residue from the non-reducing end
of XXXG was excessively galactosylated in the mur3 mutant,
indicating the contribution of at least one additional XyG
b-galactosyltransferase in XyG biosynthesis (Madson et al.,
2003). One or more members from a group of MUR3-like
genes probably encode this additional b-galactosyltransferase (Li et al., 2004).
Identification of the AtFUT1 gene made this the first
characterized sequence encoding an enzyme involved in
XyG biosynthesis. Biochemical purification of the fucosyltransferase from pea was used to identify the Arabidopsis
AtFUT1 gene, which encodes the XyG a-1,2-fucosyltransferase that terminally fucosylates XyG (Perrin et al., 1999). A
previously identified mutant plant (mur2) (Reiter et al.,
1997), exhibiting a deficiency in cell-wall fucose content,
was later shown to contain a lesion in the AtFUT1 gene,
resulting in a loss of XyG fucosylation (Vanzin et al., 2002).
Although the FUT1 protein is a member of a family of ten
related CaZY GT37 members in Arabidopsis, the absence of
detectable fucosylated XyG in plants with lesions in the
FUT1 gene (Vanzin et al., 2002; Perrin et al., 2003) supports
the prediction that the other AtFUT sequences do not
fucosylate xyloglucan, but rather other molecules (Sarria

et al., 2001).
XYLAN
Until recently, xylan biosynthesis has not received as much
attention as other plant cell-wall polymers. This changed
dramatically with the increased interest in using plant cellwall material as a potential source of biofuels. As a major
component of many woody secondary cell walls, xylan has
enormous potential as a source of sugar, but is composed
predominantly of pentoses that may prove difficult to ferment efficiently. Xylan may also limit the digestibility of
other cell-wall polymers by blocking the access of cellulosedegrading enzymes, providing further impetus to understanding and manipulating xylan biosynthesis (Yang and
Wyman, 2004; Jeoh et al., 2007).
Glucuronoxylan found in Arabidopsis is composed of a
1,4-b-linked xylan backbone with glucuronic acid and 4-Omethyl glucuronic acid side chains (Figure 2). This structure
is similar to that found in glucuronoxylan from a variety of
dicot species, including trees such as birch. A short oligosaccharide sequence (Figure 2) had been identified at the
reducing end of xylan from both birch and spruce wood
(Johansson and Samuelson, 1977; Andersson and Samuelson, 1983); however, the significance of this sequence was
not recognized until its recent identification and characterization in Arabidopsis (Pena et al., 2007). By analogy with
xylan from other species, it is likely that some sugars are
modified by the addition of acetate groups, but the exact
nature of these substitutions in Arabidopsis has not yet been
reported.
The Arabidopsis genome sequence has significantly contributed to recent advances in our understanding of xylan
biosynthesis, most notably by facilitating the use of
genome-wide expression data. Co-expression analysis,
using marker genes for secondary cell-wall biosynthesis,
identified six glycosyltransferases (IRX7, IRX8, IRX9, IRX10,
IRX14 and PARVUS) with a role in xylan biosynthesis (Brown
et al., 2005, 2007, 2009; Persson et al., 2005, 2007a; Zhong
et al., 2005; Pena et al., 2007; Wu et al., 2009). A mutation in
any one of these sequences results in a reduction in 1,4-bxylan content. In the case of IRX10, this reduction in xylan
content is much greater when a closely related gene
(IRX10L) is also mutated (Brown et al., 2009; Wu et al.,
2009). An allele of IRX7 was identified independently using
Figure 2. Structure of Arabidopsis xylan.

The reducing-end oligosaccharide referred to in the text is highlighted in bold. The side branch may be either glucuronic acid or 4-O-methyl glucuronic acid. The
position of the side branch is only representative and its position may vary in the wild-type. The degree of polymerization of Arabidopsis xylan is reported to peak at
approximately 110, making n approximately 15. The sugars are all in the dextro (D) configuration, except rhamnose, which is in the laevo configuration (L).
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Table 3 Characterization of xylan biosynthesis genes and corresponding mutants
Name(s)
AGI code
Oligosaccharide
Xylan
synthase
activity
IRX7/FRA8 (ortholog F8H)
At2G28110
At5g22940
At5G54690
At1G27600
At1G27440
At5g61840
At1G19300
At4G36890
Reduced
Unaltered
Variable
GT47/inverting
Xyl/Rha
Reduced
Present
Present
Unaltered
Reduced
Reduced
Variable
Reduced
Reduced
GT8/retaining
GT43/inverting
GT47/inverting
GalA
Xyl
Xyl
Reduced
Present
Unaltered
Reduced
Variable
Reduced
GT8/retaining
GT47/inverting
GalA
Xyl
IRX8/GAUT12
IRX9
IRX10 (ortholog IRX10L)
PARVUS/GATL1/GLZ1
IRX14
Chain
length
CaZY family/
mechanism
Possible
transferase
activity
Oligosaccharide refers to that found at the reducing end; xylan synthase activity and chain length refer to the mutant relative to wild-type.
forward genetics and termed fra8 (Table 3) (Zhong et al.,

2005). Recently, a homolog of IRX7/FRA8 termed F8H has
been shown to be a functional paralog (Lee et al., 2009).
The mutant phenotypes caused by disrupting the genes
encoding xylan biosynthetic enzymes listed above may be
grouped into two classes. The irx7, irx8 and parvus mutants
contain reduced amounts of the reducing-end oligosaccharide, have a more heterogeneous distribution of xylan chain
lengths, and microsomal extracts show no change in xylan
synthase activity. In contrast, mutants within the second
group containing irx9, irx14 and irx10 appear to have
relatively large amounts of the oligosaccharide, shorter
chains and reduced xylan synthase activity (Table 3) (Brown
et al., 2007, 2009; Lee et al., 2007; Pena et al., 2007; Wu et al.,
2009). The reduced abundance of the oligosaccharide
observed among mutants in the first group suggests that
these sequences play a role in its synthesis. To date, there is
no direct evidence to suggest which enzyme catalyzes which
reaction, and tentative inferences can only be made based
upon the activities of homologous enzymes (Table 2). IRX9
and IRX14 are related GT43 enzymes that were originally
proposed to function as the xylan synthase that extends the
xylan backbone (Brown et al., 2007; Pena et al., 2007). The
roles of these sequences are less clear now that IRX10, an
unrelated GT47 enzyme, has also been demonstrated to be
essential for xylan synthase activity (Brown et al., 2009; Wu
et al., 2009). One possibility is that IRX10 might transfer a
single xylosyl residue prior to processive addition of Xyl
residues by IRX9 and IRX14. This would be analogous to
glycosaminoglycan biosynthesis in mammals. In the case of
heparin biosynthesis, the exostosin proteins EXT1 and EXT2
work together to add alternating GlcUA and GlcNAC residues; however, this processive addition of sugars is preceded by the essential addition of a single sugar to a
tetrasaccharide linker by a separate enzyme (EXTL2 or
EXTL3) (Esko and Selleck, 2002).
No candidates for the genes that are required to add the
side branches to the xylan backbone have been reported. It is
also unclear whether additional genes are required to
produce the xylan backbone and reducing-end oligosaccharide (Figure 2). Two additional mutants with altered xylan
synthase activity have been described. qua1 exhibits a
pleiotropic phenotype that includes aberrant cell separation
and reduced xylosyltransferase activity, but comparatively
minor reductions in xylose content (Orfila et al., 2005).
Similarly, mutants in AtCSLD5 exhibit both reduced xylan
synthase and homogalacturonan synthase activities (Bernal
et al., 2007). It is not clear whether xylan biosynthesis is the
primary function of the products of these two genes, but
these examples highlight interconnections between various
aspects of cell-wall biosynthesis.
Questions remain about the direction of the xylan backbone biosynthesis. Although it has been possible to demonstrate the addition of xylosyl residues to the non-reducing
end of a 1,4-b-xylohexaose acceptor, there are potential
problems associated with the use of very high concentrations of artificial acceptors (York and ONeill, 2008). Similarly, the function of the reducing-end oligosaccharide is
unclear; it has been postulated to act either as a primer or as
a terminator (York and ONeill, 2008).
Side chains are present every seventh xylose residue, on
average, along the xylan backbone in Arabidopsis (Brown
et al., 2007). Although MALDI analysis of the digestion
pattern suggests the side branches are not clustered, no
direct information is available on the distribution of these
side branches or whether the glucuronic and methylglucuronic acid side chains exhibit a particular pattern. A notable
aspect of xylan side-branching is the fact that most xylandeficient mutants possess mostly methylglucuronic acid
side chains (Brown et al., 2007, 2009; Pena et al., 2007;
Persson et al., 2007a; Wu et al., 2009). As the frequency
of branching is maintained (Brown et al., 2007), this represents substitution of glucuronic acid by methylglucuronic
acid residues. It has been suggested that this might be the
result of limiting quantities of the methyl donor required for
conversion of glucuronic acid to methylglucuronic acid,
such that, when xylan synthesis is reduced, the available
methyl donor is sufficient to convert all glucuronic acid to
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methylglucuronic acid (Pena et al., 2007), or that there are
two alternative pathways of xylan biosynthesis (Persson
et al., 2007a). Both explanations are difficult to reconcile
with the observation that conversion of glucuronic acid to
methylglucuronic acid is essentially complete in mutants
that have a range of xylan contents. An alternative explanation involves alteration of sugar nucleotide pools (Brown
et al., 2007), but there is no direct evidence to support this
idea.
In many species, especially in grasses, xylans also have
abundant arabinofuranosyl side branches. However, these
structures have not been reported in Arabidopsis.
MANNANS
Mannan polysaccharides are another class of hemicellulose
that is widespread among plant species (Popper, 2008). This
group of carbohydrates includes mannans (1,4-b-linked homopolymers of mannosyl residues), glucomannans (1,4-blinked heteropolymers containing mannosyl and glucosyl
residues) and 1,6-a-galactosylated species of these carbohydrates (galactomannans and galactoglucomannans,
respectively). Mannan polysaccharides serve structural and/
or storage roles in many plants and algae (Frei and Preston,
1968; Meier and Reid, 1982; Maeda et al., 2000). Mannan
polysaccharides exist in a variety of tissues and cell types of
Arabidopsis (Handford et al., 2003; Liepman et al., 2007;
Moller et al., 2007); however, relatively little is known about
which specific mannan polysaccharides are present.
At least two types of enzymes are needed for the
biosynthesis of mannan polysaccharides (Edwards et al.,
1999). The 1,4-b-linked backbones of these carbohydrates
are polymerized by GDP-mannose-dependent 1,4-b-mannan
synthases. Some mannan synthases also have GDP-glucose-dependent 1,4-b-glucan synthase activity, and are
therefore called glucomannan synthases. The addition of
1,6-a-galactosyl side chains to mannosyl residues within
mannan and/or glucomannan chains is catalyzed by UDPgalactose-dependent 1,6-a-galactosyltransferases.
The first mannan synthase-encoding gene sequence, a
member of the CELLULOSE SYNTHASE-LIKE A (CSLA)
family (CaZY family GT2), was characterized from guar
(Cyamopsis tetragonoloba; Dhugga et al., 2004). Members
of the Arabidopsis CSLA family also encode glucomannan
synthases (Liepman et al., 2005, 2007). In Arabidopsis, there
are nine members of the CSLA family; biochemical analysis
of several recombinant Arabidopsis CSLA proteins and a
number of homologs from diverse plant species (Dhugga
et al., 2004; Liepman et al., 2005, 2007; Suzuki et al., 2006)
suggests conservation of (gluco)mannan synthase activity
among all CSLA enzymes. These biochemical studies have
been complemented by studies of one or more mutant
corresponding to each Arabidopsis CSLA gene (Goubet
et al., 2003, 2009; Zhu et al., 2003; Ubeda-Tomas et al.,
2007). Most of these single mutants displayed no obvious
phenotype under laboratory conditions, suggesting genetic

redundancy among members of the AtCSLA family (Goubet
et al., 2009). In fact, mutation of all three genes atcsla2, atcsla3 and atcsla9 was required for complete disruption of
glucomannan accumulation in inflorescence stems. Loss of
glucomannan in this triple mutant caused no other detectable mechanical or developmental defects (Goubet et al.,
2009). The phenotypes of some csla mutants, expression
patterns of particular CSLA genes, and accumulation patterns of mannan polysaccharides suggest other potential
roles of these carbohydrates during plant growth and
development. For example, a mutant with a lesion in the
csla7 gene has defects in pollen tube growth and embryogenesis (Goubet et al., 2003), and another mutant (rat4) with
a defective CSLA9 gene is resistant to transformation by
Agrobacterium tumefaciens (Zhu et al., 2003). Furthermore,
mannan polysaccharides and transcripts of CSLA genes are
relatively abundant in Arabidopsis floral tissues (Liepman
et al., 2007). These observations underscore important, yet
poorly understood, roles of mannan polysaccharides in
Arabidopsis, and probably other plants.
It is not yet clear whether galactomannan or galactoglucomannan is present in Arabidopsis, and therefore whether
genes encoding galactomannan 1,6-a-galactosyltransferase
exist in this plant. The two Arabidopsis genes most closely
related to the fenugreek galactomannan galactosyltransferase (Edwards et al., 1999) are the AtGT6 and AtGT7
sequences (Faik et al., 2002); the activity of the proteins
encoded by these genes remains to be determined.
PECTIN
Arabidopsis pectin appears to have a composition similar to
that found in other plants (Figure 3). The primary cell walls
of Arabidopsis typically contain about 40% pectin, including
homogalacturonan, rhamnogalacturonan I (RG-I) and
rhamnogalacturonan II (RG-II) (Zablackis et al., 1995). Linkage analysis has shown the presence of branched arabinans
and 1,4-b-galactans in approximately equal proportions
(Zablackis et al., 1995). Xylogalacturonan is also abundant in
Arabidopsis walls (Zandleven et al., 2007). With the exception of some terminal structures, Arabidopsis RG-II is
structurally nearly identical to RG-II from a wide variety of
other plant species (Glushka et al., 2003). It is worth noting
that most studies of Arabidopsis pectin structure have
focused on leaves, and that different tissues and cell types
differ in their pectin composition. For example, the mucilage
of Arabidopsis seeds is rich in a largely unsubstituted form
of RG-I (Penfield et al., 2001; Arsovski et al., 2009).
Due to the complex structure of pectin, a large number of
enzymes must be required for its synthesis. Mohnen (2008)
has estimated that 67 glycosyltransferases, methyltransferases and acetyltransferases are required to synthesize
pectin. The first pectin biosynthetic enzyme to be identified
was GAUT1, a homogalacturonan 1,4-a-galacturonosyl-
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Figure 3. Schematic representation of pectin (Scheller et al., 2007).

The four domains of pectin that are found in Arabidopsis and other plants are illustrated.
transferase (Sterling et al., 2006). This enzyme appears to be

present in a complex with the homologous GAUT7 protein
(Mohnen, 2008). The GAUT proteins form a sub-group
within the CaZY GT8 family, and this sub-group contains
15 members in Arabidopsis. Although all 15 GAUT sequences may be a-galacturonosyltransferases, so far this
activity has only been demonstrated for GAUT1. Another
member of the GAUT family, GAUT9 (QUASIMODO1), is
apparently involved in pectin biosynthesis, as the qua1
mutant has a pectin-deficient phenotype (Bouton et al.,
2002). However, the mutant shows pleiotropic effects,
including defects in xylan deposition (Orfila et al., 2005).
The biosynthesis of RG-I must require many enzymes, but
these remain largely unknown despite extensive forward
and reverse genetics studies. The Arabidopsis mutant arad1
is specifically deficient in 1,5-a-linked arabinan side chains of
RG-I (Harholt et al., 2006). Thus ARAD1, a member of the
CaZY GT47 family, is most likely a 1,5-a-arabinosyltransferase, and is so far the only glycosyltransferase known to be
involved in RG-I biosynthesis. Activity of the heterologously
expressed ARAD1 protein remains to be demonstrated. It is
hypothesized that the sub-family of CaZY GT47 to which
ARAD1 belongs contains additional proteins required for
synthesizing the various linkages in arabinan. The other
major type of RG-I side chain, besides arabinan, is 1,4-bgalactan. No data are available to suggest which glycosyltransferases might be involved in synthesizing this
important structural component.
Xylogalacturonan is a modified form of homogalacturonan containing b-xylosyl residues linked to O3 of the
backbone galacturonosyl residues. Xylogalacturonan is
abundant in Arabidopsis leaves, and as much as 30% of

the xylose in leaf primary cell walls may be associated with
this polysaccharide (Jensen et al., 2008). The Arabidopsis
xgd1 mutant completely lacks xylogalacturonan in leaves
(Jensen et al., 2008). The XGD1 protein is another member
of the CaZY GT47 family, and, when transiently expressed in
tobacco leaves, it was shown to catalyze the transfer of
xylose from UDP-xylose onto endogenous pectin and exogenous oligogalacturonides. While leaves of the xgd1
mutant had no detectable xylogalacturonan, the mutant
retained LM8 xylogalacturonan epitopes in siliques. This
suggests that xylogalacturonan in siliques differs in structure from that found in leaves, and indicates that there must
be additional xylosyltransferases involved in xylogalacturonan biosynthesis in Arabidopsis (Jensen et al., 2008).
RG-II has a highly complex structure, comprising 12
different sugars in 20 different linkages; however, very few
sequences involved in RG-II synthesis have been identified.
Three homologous proteins, RGXT1, RGXT2 and RGXT3,
which are members of GT77, have been shown to specifically
catalyze the transfer of xylose from UDP-xylose onto fucosyl
residues via 1,3-a-linkages (Egelund et al., 2006, 2008). This
enzymatic activity suggests a specific involvement in formation of the A-chain of RG-II, the only cell-wall carbohydrate
structure known to contain a-xylose-(1,3)-fucose. The sugar
composition of RG-II isolated from T-DNA insertional
mutants of either RGXT1 or RGXT2 showed no significant
changes, suggesting genetic redundancy among RGXT
isoforms. However, the RG-II isolated from these mutant
lines functioned as a specific acceptor substrate in an RGXT
biochemical assay. The close linkage between the RGXT1 and
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RGXT2 sequences on chromosome 4 has so far prevented
the generation of a double mutant.
The NpGUT1 protein was identified in a tobacco mutant
deficient in cell adhesion, and suggested to be a glucuronosyltransferase involved in RG-II biosynhesis (Iwai et al.,
2002). However, studies of the closest Arabidopsis homologs, IRX10 and IRX10L, have shown an involvement in
xylan biosynthesis (see above), and failed to confirm any
involvement in RG-II biosynthesis.
All pectic polysaccharides may show methyl esterification
of the carboxyl groups of galacturonic acid residues. The
qua2 mutant is affected in a putative methyltransferase and
has decreased pectin content (Mouille et al., 2007). However,
the biochemical activity of QUA2 has not been confirmed.
Acetylation on O2 or O3 is also found in pectin. Recently, an
Arabidopsis mutant, reduced wall acetylation 2 (rwa2), with
decreased acetylation of wall polysaccharides, has been
identified (Y. Manabe, Feedstocks Division, Joint BioEnergy
Institute, and H.V.S., unpublished results), but it is not yet
clear whether the affected protein is a specific pectin
acetyltransferase.
ARABINOGALACTAN PROTEINS
Arabinogalactan proteins (AGPs) are abundant and ubiquitous proteoglycans on the cell surface in plants, and play
important roles during plant growth and development, and
in interactions with micro-organisms (for a recent review,
see Seifert and Roberts, 2007). AGPs consist of a core protein
backbone decorated heavily by O-glycosylation with complex arabinogalactans. AGPs often contain a glycosylphosphatidyinositol (GPI) lipid anchor. The structure of
Arabidopsis arabinogalactans is poorly characterized.
Characterization has been very difficult due to heterogeneity
of the glycan structures, not only between different AGPs or
depending on developmental stage and tissue type, but
even on the same peptide sequence in the same tissue
(Estevez et al., 2006). Biosynthesis of AGPs involves complex post-translational modifications, including cleavage of
an N-terminal signal sequence, hydroxylation of proline
residues, GPI anchor modification, and arabinogalactosylation of hydroxyproline residues. The initial steps of these
post-translational modifications are known to some degree,
but very little is known about the glycosylation process.
Originally AGPs were found as a sub-group of hydroxyproline-rich glycoproteins in cell walls (Fincher et al., 1983),
but recent genomics and proteomics research has suggested that a much wider variety of proteins are modified by
arabinogalactans. Classical AGPs, which are similar in
structure to the products of the first cloned full-length cDNAs
encoding AGPs (Chen et al., 1994; Du et al., 1994), consist of
a central domain rich in Pro, Ala, Ser and Thr, flanked by an
N-terminal signal peptide and a C-terminal GPI anchor
modification. Many AGPs deviate from this domain structure, including AGPs containing Lys-rich domains and
fasciclin-like AGPs. Sequence analysis of the Arabidopsis

genome predicted 13 classical AGPs, three AGPs containing
Lys-rich domains, approximately 20 fasciclin-like AGPs, and
approximately ten arabinogalactan peptides consisting of
only 1013 amino acid residues (Borner et al., 2002; Schultz
et al., 2002). Of these proteins, eight classical AGPs were
isolated, and the predicted cleavage of the signal sequence
and attachment of the GPI anchor were confirmed (Schultz
et al., 2004).
The consensus sequence motif that leads to glycosylation
with arabinogalactan is not fully understood, but studies
using synthetic peptides suggested that clustered noncontiguous hydroxyproline residues tend to be arabinogalactosylated (Kieliszewski and Shpak, 2001). According to
this hypothesis, 40% of the GPI-anchored proteins isolated
in Arabidopsis (100 of 248) are predicted to be AGPs, of
which only 29 are classical AGPs or arabinogalactan peptides (Borner et al., 2003). Essentially any peptide sequence
containing a secretion signal and clustered non-contiguous
hydroxyproline residues can be considered AGP-like, including extensin-, COBRA-, glycerophosphodiesterase-, SKU5and receptor-like proteins (Borner et al., 2003).
Prolyl hydroxylation of AGPs is catalyzed by prolyl-4hydroxylases (P4H) in the ER. At least six P4H sequences are
present in Arabidopsis, of which P4H-1 and P4H-2 encode
active P4H isoforms with different substrate specificities
(Hieta and Myllyharju, 2002; Tiainen et al., 2005). The
efficiency of prolyl hydroxylation may depend on the
different P4H isoforms, but also on peptide sequences. For
instance, the proline residues in a Ser-Pro-Ser-Pro repeat
were almost fully hydroxylated, whereas those in a Val-ProVal-Pro repeat were only 60% hydroxylated in Arabidopsis
(Estevez et al., 2006).
Many AGPs are considered as GPI-anchored in Arabidopsis based on cleavage by phospholipase C and attachment
to ethanolamine at the C-terminus (Borner et al., 2003;
Schultz et al., 2004). Little is known about the structure of
GPI in plants, but AGP from suspension-cultured pear cells
contained D-Man-(1,2)-a-D-Man-(1,6)-a-D-Man-(1,4)-a-D-GlcNinositylphosphoceramide lipid (Oxley and Bacic, 1999). The
structure was consistent with that found in animals, protozoans and yeast, except for a partial substitution with Gal on
the O4 position of the 6-linked mannosyl residue, and the
presence of a ceramide lipid instead of glycerolipid. For the
conserved portion of the structure, the proteins that catalyze
the reactions within this pathway have been well studied in
other organisms. Little is known about them in plants, but
the Arabidopsis genome contains genes homologous to
several animal and yeast genes responsible for GPI anchor
synthesis. Mutations in these genes cause severe deficiencies in plants. For example, mutations in the SETH1 and
SETH2 genes in Arabidopsis, which are homologs of two
components in the GPItransamidase complex involved in
the transfer of D-GlcNAc to phosphatidylinositol, specifically
2010 The Authors


blocked pollen germination and tube growth (Lalanne et al.,
2004). Another example is the Arabidopsis peanut1 mutant
(pnt1), which is defective in a homolog of a mammalian
mannosyltransferase that is essential for GPI biosynthesis,
and displayed numerous changes in cell-wall polysaccharide composition as well as developmental abnormalities
(Gillmor et al. 2005).
Most of the glycan structure of AGPs consists of a 1,3-bgalactan backbone branched at theO6 position, with side
chains mainly composed of arabinose, but also containing
glucuronic acid, rhamnose, xylose and other sugars (Fincher
et al., 1983, Showalter, 1993; Majewska-Sawka and Nothnagel, 2000; Gaspar et al., 2001; Seifert and Roberts, 2007).
Arabinogalactan glycosylation may be initiated in the ER
(Oka et al., 2010), but mainly occurs in the Golgi apparatus
(Kato et al., 2003). Many glycosyltransferases are required to
build an entire arabinogalactan structure. For example,
assuming that every glycosyltransferase recognizes a specific disaccharide as acceptor and creates a specific glycosidic linkage, an arabinogalactan structure added to a
hydroxyproline residue of a synthetic (Ala-Hyp)51 sequence
expressed in tobacco would require at least 11 glycosyltransferases (Tan et al., 2004). Bioinformatics analysis
using animal 1,3-b-galactosyltransferase sequences suggested involvement of the CaZY GT31 family for synthesis
of 1,3-b-galactans present in the arabinogalactan backbone
(Qu et al., 2008).
PERSPECTIVES
The wealth of information gained from the Arabidopsis
genome sequence (Arabidopsis Genome Initiative 2000),
coupled with the powerful tools available to Arabidopsis
researchers (Seki et al., 2002; Alonso et al., 2003; Rhee et al.,
2003), has facilitated much progress within the cell-wall
research community in identification of genes encoding
enzymes involved in cell-wall biosynthesis. The progress
made since completion of the Arabidopsis genome
sequence is illustrated by numerous studies. Within the
genome, there are many gene families containing members
with related function. For example, the characterization of
GAUT1, a GalA transferase involved in pectin biosynthesis,
has resulted in the identification of another 14 genes with
potential roles as GalA transferases, and this information
should facilitate rapid functional characterization of the
remaining family members. Similar situations exist for
members of other families (Sarria et al., 2001; Faik et al.,
2002; Zhong et al., 2003; Liepman et al., 2007).
While the availability of the genome has had a significant
impact on the identification of genes encoding enzymes that
are essential for xylan biosynthesis, there has been a relative
lack of progress in the complementary biochemical analysis.
Consequently, no single gene product has been unambiguously assigned a function in xylan biosynthesis. It is clear,
however, that identifying these genes is an excellent first
step and will facilitate further functional analysis. In the case

of XyG biosynthesis, more progress has been made in
identifying the activities of individual enzymes; sequences
corresponding to at least one representative of each glycan
synthase and glycosyltransferase needed for XyG biosynthesis have been identified. The availability of this information has enabled new experimental approaches to examine
the role of XyG in the cell wall. For example, a mutant lacking
detectable XyG displays only modest phenotypic abnormalities, challenging the validity of many models about the roles
of XyG in cell-wall structure and in regulating growth
(Cavalier et al., 2008). As corresponding mutants are generated for other polysaccharides, it is likely that current models
of cell-wall structure and function will undergo a paradigm
shift.
The utility of Arabidopsis in comparative studies is also
clearly apparent. The structure of Arabidopsis xylan is
broadly similar to that of a range of both gymnosperm and
angiosperm trees. Furthermore, genes with similarities to
IRX7, IRX8, IRX9 and PARVUS have been identified in poplar
and shown to function as true orthologs by complementing
the corresponding Arabidopsis mutant (Zhou et al., 2006,
2007; Kong et al., 2009). In contrast, in a comparison of
Arabidopsis, poplar and rice, no obvious ortholog of IRX8
was found in rice (Caffall et al., 2009), although orthologs of
other genes encoding enzymes involved in xylan biosynthesis clearly exist in monocots and are likely to represent
invaluable starting points for further analysis. It will be
interesting to determine whether xylan biosynthesis in
monocots is essentially a variation of a well-conserved
pathway, or whether alternative pathways for xylan synthesis exist. This illustrates a type of comparative study that
would not have been possible without the availability of the
genome sequence and the tools for functional analysis that
are available in Arabidopsis.
Arabidopsis will continue to play an important role in the
identification of genes and functional analysis of proteins
involved in plant cell-wall biosynthesis. As more steps in
wall biosynthesis are identified, there will probably be many
more discoveries that challenge our current understanding
of cell-wall biosynthesis and the functional roles of various
cell-wall components. Furthermore, knowledge gained from
these studies will have important practical applications,
including enabling the engineering of plant cell-wall biomass tailored for various uses.
ACKNOWLEDGEMENTS
A.H.L. is supported by National Research Initiative grant number
2007-03542 from the United States Department of Agriculture
Cooperative State Research, Education and Extension Service. N.G.
and R.W. are supported by the 7th Framework Program of the
European Union, project number 211982, Renewall. H.V.S. is
supported by the US Department of Energy, Office of Science, Office
of Biological and Environmental Research, through contract
DE-AC02-05CH11231 between Lawrence Berkeley National Labora-
2010 The Authors


tory and the US Department of Energy. A.H.L. acknowledges
Dr Cory Emal (Eastern Michigan University, Department of
Chemistry) and Dr David Cavalier (Michigan State University, Plant
Research Laboratory) for helpful discussions.
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The Plant Journal (2010) 61, 11071121

ARABIDOPSIS: A RICH HARVEST 10 YEARS AFTER COMPLETION OF THE GENOME SEQUENCE

Arabidopsis a powerful model system for plant cell wall

Received 1 November 2009; revised 13 January 2010; accepted 15 January 2010.

expansion has ceased. Although cell walls are strong

1108 Aaron H. Liepman et al.

synthase-like F (CSLF) and cellulose synthase-like H (CSLH)

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Arabidopsis cell-wall biosynthetic enzymes 1109

Comparative analyses of Arabidopsis CESA sequences with

Table 1 Genes involved in cellulose biosynthesis

irx3, fra5, mur10

irx1, lew2, ern1

ixr1, eli1, cev1

Arioli et al. (1998)

Primary wall cellulose defects

Benfey et al. (1993), Schindelman et al. (2001)

Nicol et al. (1998), Szyjanowicz et al. (2004)

1110 Aaron H. Liepman et al.

involved in aspects of flower development (Brady et al.,

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Arabidopsis cell-wall biosynthetic enzymes 1111

Figure 1. Schematic representation of a segment of Arabidopsis xyloglucan.

At least four types of enzymatic activities are needed

one or more members of the AtCSLC family encode XyG

Table 2 Biochemically characterized sequences involved in XyG biosynthesis

Mutant has altered trichome papillae (Perrin et al.,

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1112 Aaron H. Liepman et al.

fucosylate xyloglucan, but rather other molecules (Sarria

Figure 2. Structure of Arabidopsis xylan.

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Arabidopsis cell-wall biosynthetic enzymes 1113

IRX7/FRA8 (ortholog F8H)

forward genetics and termed fra8 (Table 3) (Zhong et al.,

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phenotype under laboratory conditions, suggesting genetic

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Arabidopsis cell-wall biosynthetic enzymes 1115

Figure 3. Schematic representation of pectin (Scheller et al., 2007).

transferase (Sterling et al., 2006). This enzyme appears to be

abundant in Arabidopsis leaves, and as much as 30% of

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fasciclin-like AGPs. Sequence analysis of the Arabidopsis

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Arabidopsis cell-wall biosynthetic enzymes 1117

step and will facilitate further functional analysis. In the case

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Arabidopsis cell-wall biosynthetic enzymes 1119

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1120 Aaron H. Liepman et al.

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Arabidopsis cell-wall biosynthetic enzymes 1121

xyloglucan because of a lesion in fucosyltransferase AtFUT1. Proc. Natl

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