The Plant Journal (2010) 61, 11071121 doi: 10.1111/j.1365-313X.2010.04161.

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
1
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 cell-
wall 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 impor-
tant 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
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 struc-
tural 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 load-
bearing structure in the cell wall, and typically constitutes
approximately 3040% of the wall mass. The term hemicel-
luloses covers a diverse group of polysaccharides, which

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1108 Aaron H. Liepman et al.
generally exhibit limited solubility and interact with cellu-
lose 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 back-
bones 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 polysac-
charides are more easily solubilized than hemicelluloses.
About 10% of the genes in Arabidopsis have been esti-
mated 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 polysaccha-
ride 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 (Arabidop-
sis 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 cell-
wall studies as its cell walls resemble those found in many
crop plants and trees. Despite the complexity of plant cell-
wall 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 hydroxycinna-
mate 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 exper-
iments performed in Arabidopsis implicated cellulose
Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
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 involv-
ing seminal experiments that led to elucidation of the
structures of plant cell-wall polysaccharides and character-
ization of the enzymes required to synthesize them; how-
ever, 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 transduc-
tion, 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 func-
tional characterization of enzymes catalyzing various steps
in the biosynthesis of cell-wall polysaccharides and arabi-
nogalactan proteins, and the insights that these discoveries
have provided into the function of particular cell-wall
components in regulating both cell growth and wall struc-
tural 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 mem-
brane, 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 approxi-
mately 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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 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 mem-
brane 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, sug-
gesting 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 References

CESA family Presumed catalytic subunits

of cellulose synthase
CESA1 At4G32410 rsw1 Subunit (position 1) Arioli et al. (1998)
CESA2 At4G39350 Subunit (position 3) Desprez et al. (2007), Persson et al. (2007b)
CESA3 At5G05170 ixr1, eli1, cev1 Subunit (position 2) Cano-Delgado et al. (2003), Ellis et al. (2002),
Scheible et al. (2001)
CESA4 At5G44030 irx5, nws2 Secondary wall Hernandez-Blanco et al. (2007), Taylor et al. (2003)
CESA5 At5G09870 Subunit (position 3) Desprez et al. (2007)
CESA6 At5G64740 ixr2, prc1 Subunit (position 3) Desprez et al. (2002)
Fagard et al. (2000)
CESA7 At5G17420 irx3, fra5, mur10 Secondary wall Bosca et al. (2006), Taylor et al. (1999), Zhong
et al. (2003)
CESA8 At4G18780 irx1, lew2, ern1 Secondary wall Chen et al. (2005), Hernandez-Blanco et al. (2007),
Taylor et al. (2000)
CESA9 At2G21770 Subunit (position 3) Persson et al. (2007b)
CESA10 At2G25540 Subunit (position 1)
COBRA family
COBRA At5G60920 cob Primary wall cellulose defects Benfey et al. (1993), Schindelman et al. (2001)
COBL4 At5G15630 irx6 Secondary wall cellulose defects Brown et al. (2005)
COBL9 At5G49270 mrh4 Root hair development Jones et al. (2006)
KOR1 At5G49720 irx2, kor1 Endo-1,4-b-D-glucanase Nicol et al. (1998), Szyjanowicz et al. (2004)
KOB1 At3G08550 kob1, eld1 Lertpiriyapong and Sung (2003), Pagant et al. (2002)
TED6 At1G43790 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.

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1110 Aaron H. Liepman et al.
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 Arabid-
opsis 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 for-
mation, 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 glycosyl-
phosphatidylinositol 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
Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
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 transmem-
brane 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). Arabidop-
sis 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 approxi-
mately 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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 Arabidopsis cell-wall biosynthetic enzymes 1111

one or more members of the AtCSLC family encode XyG

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 glycosyl-
transferase (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
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. pasto-
ris of AtCSLC4 with an Arabidopsis 1,6-a-xylosyltransfer-
ase (AtXXT1, see below), suggesting interaction between
these two enzymes, and (iii) coordinated expression of
AtCSLC4 and AtXXT1 in Arabidopsis. Because the Arabid-
opsis 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 galacto-
mannan 1,6-a-galactosyltransferase from fenugreek (Trigo-
nella 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 xylosylat-
ing 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 xylosy-
lation (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 Mutant has altered trichome papillae (Perrin et al.,
1999; Vanzin et al., 2002)
XXT1 At3G62720 GT34 1,6-a-xylosyltransferase xxt1 and xxt2 single mutants have no observed
XXT2 At4G02500 GT34 1,6-a-xylosyltransferase phenotype, xxt1 xxt2 double mutant has aberrant root hairs
and significantly altered cell-wall mechanical properties
(Cavalier and Keegstra, 2006; Cavalier et al., 2008)
MUR3 At2G20370 GT47 1,2-b-galactosyltransferase Mutant has aberrant trichome papillae (Madson et al., 2003)
CSLC4 At3G28180 GT2 1,4-b-glucan synthase Cocuron et al. (2007)

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1112 Aaron H. Liepman et al.
proper xylosylation of XyG to occur. Perhaps the most
surprising revelation resulting from studies of xxt1 xxt2
double mutants is their relatively mild phenotypic abnor-
malities; many models of primary walls predict a prominent
structural role of XyG, so it may be necessary for the cell-
wall 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 charac-
terized by a significant reduction in the fucose content of its
cell walls (Reiter et al., 1997), and aberrant XyG galactosy-
lation (Madson et al., 2003). Positional cloning of MUR3
revealed that this gene encodes a xyloglucan b-1,2-galacto-
syltransferase (CaZY family GT47) that specifically galac-
tosylates 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 appropri-
ate glycosylation site for XyG a-fucosyltransferase. Interest-
ingly, 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-galactosyltransfer-
ase (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 fucosyl-
transferase from pea was used to identify the Arabidopsis
AtFUT1 gene, which encodes the XyG a-1,2-fucosyltransfer-
ase 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
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).
Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
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 cell-
wall 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 fer-
ment efficiently. Xylan may also limit the digestibility of
other cell-wall polymers by blocking the access of cellulose-
degrading enzymes, providing further impetus to under-
standing 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-O-
methyl 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 oligo-
saccharide sequence (Figure 2) had been identified at the
reducing end of xylan from both birch and spruce wood
(Johansson and Samuelson, 1977; Andersson and Samuel-
son, 1983); however, the significance of this sequence was
not recognized until its recent identification and character-
ization 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 con-
tributed 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-b-
xylan 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
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Table 3 Characterization of xylan biosynthesis genes and corresponding mutants
Name(s) AGI code Oligosaccharide
IRX7/FRA8 (ortholog F8H) At2G28110 Reduced
At5g22940
IRX8/GAUT12 At5G54690 Reduced
IRX9 At1G27600 Present
IRX10 (ortholog IRX10L) At1G27440 Present
At5g61840
PARVUS/GATL1/GLZ1 At1G19300 Reduced
IRX14 At4G36890 Present
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 oligosaccha-
ride, 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 resi-
dues; however, this processive addition of sugars is pre-
ceded 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
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Arabidopsis cell-wall biosynthetic enzymes 1113
Xylan Possible
synthase Chain CaZY family/ transferase
activity length mechanism activity
Unaltered Variable GT47/inverting Xyl/Rha
Unaltered Variable GT8/retaining GalA
Reduced Reduced GT43/inverting Xyl
Reduced Reduced GT47/inverting Xyl
Unaltered Variable GT8/retaining GalA
Reduced Reduced GT47/inverting Xyl
produce the xylan backbone and reducing-end oligosaccha-
ride (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 back-
bone biosynthesis. Although it has been possible to dem-
onstrate 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 concentra-
tions of artificial acceptors (York and ONeill, 2008). Simi-
larly, 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 methylglucu-
ronic acid side chains exhibit a particular pattern. A notable
aspect of xylan side-branching is the fact that most xylan-
deficient 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 repre-
sents 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
1114 Aaron H. Liepman et al.
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 explana-
tion 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 ho-
mopolymers of mannosyl residues), glucomannans (1,4-b-
linked heteropolymers containing mannosyl and glucosyl
residues) and 1,6-a-galactosylated species of these carbo-
hydrates (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-glu-
cose-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 UDP-
galactose-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
Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
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, atc-
sla3 and atcsla9 was required for complete disruption of
glucomannan accumulation in inflorescence stems. Loss of
glucomannan in this triple mutant caused no other detect-
able mechanical or developmental defects (Goubet et al.,
2009). The phenotypes of some csla mutants, expression
patterns of particular CSLA genes, and accumulation pat-
terns 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 embryo-
genesis (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 galactoglu-
comannan 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 galactosyltransfer-
ase (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). Link-
age 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 excep-
tion 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, methyltransfe-
rases and acetyltransferases are required to synthesize
pectin. The first pectin biosynthetic enzyme to be identified
was GAUT1, a homogalacturonan 1,4-a-galacturonosyl-
2010 The Authors

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 se-
quences 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-arabinosyltransfer-
ase, 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-b-
galactan. No data are available to suggest which glyco-
syltransferases might be involved in synthesizing this
important structural component.
Xylogalacturonan is a modified form of homogalacturo-
nan containing b-xylosyl residues linked to O3 of the
backbone galacturonosyl residues. Xylogalacturonan is
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Arabidopsis cell-wall biosynthetic enzymes 1115
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 exo-
genous 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 struc-
ture from that found in leaves, and indicates that there must
be additional xylosyltransferases involved in xylogalacturo-
nan 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 forma-
tion 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
1116 Aaron H. Liepman et al.
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 glucuron-
osyltransferase involved in RG-II biosynhesis (Iwai et al.,
2002). However, studies of the closest Arabidopsis homo-
logs, 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 ubiqui-
tous 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 com-
plex arabinogalactans. AGPs often contain a glycosylphos-
phatidyinositol (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 com-
plex post-translational modifications, including cleavage of
an N-terminal signal sequence, hydroxylation of proline
residues, GPI anchor modification, and arabinogalactosyla-
tion 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 hydroxy-
proline-rich glycoproteins in cell walls (Fincher et al., 1983),
but recent genomics and proteomics research has sug-
gested 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 struc-
ture, including AGPs containing Lys-rich domains and
Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
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 non-
contiguous hydroxyproline residues tend to be arabinoga-
lactosylated (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 pep-
tides (Borner et al., 2003). Essentially any peptide sequence
containing a secretion signal and clustered non-contiguous
hydroxyproline residues can be considered AGP-like, includ-
ing extensin-, COBRA-, glycerophosphodiesterase-, SKU5-
and receptor-like proteins (Borner et al., 2003).
Prolyl hydroxylation of AGPs is catalyzed by prolyl-4-
hydroxylases (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-Pro-
Val-Pro repeat were only 60% hydroxylated in Arabidopsis
(Estevez et al., 2006).
Many AGPs are considered as GPI-anchored in Arabidop-
sis 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-GlcN-
inositylphosphoceramide lipid (Oxley and Bacic, 1999). The
structure was consistent with that found in animals, proto-
zoans 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 deficien-
cies 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 polysaccha-
ride composition as well as developmental abnormalities
(Gillmor et al. 2005).
Most of the glycan structure of AGPs consists of a 1,3-b-
galactan 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 Nothna-
gel, 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 spe-
cific disaccharide as acceptor and creates a specific glyco-
sidic linkage, an arabinogalactan structure added to a
hydroxyproline residue of a synthetic (Ala-Hyp)51 sequence
expressed in tobacco would require at least 11 glyco-
syltransferases (Tan et al., 2004). Bioinformatics analysis
using animal 1,3-b-galactosyltransferase sequences sug-
gested 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 unambig-
uously assigned a function in xylan biosynthesis. It is clear,
however, that identifying these genes is an excellent first
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Journal compilation 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 11071121
Arabidopsis cell-wall biosynthetic enzymes 1117
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 biosyn-
thesis have been identified. The availability of this informa-
tion 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 abnormal-
ities, 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 gener-
ated 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 biosyn-
thesis 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 synthe-
sis 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 bio-
mass 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-
1118 Aaron H. Liepman et al.
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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