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 wallresearch

Aaron H. Liepman1,*, Raymond Wightman2, Naomi Geshi3, Simon R. Turner2 and Henrik Vibe Scheller4,5

1Biology Department, Eastern Michigan University, 316 Mark Jefferson Building, Ypsilanti, MI 48197, USA,2Faculty of Life Science, University of Manchester, Oxford Road, Manchester M13 6AY, UK,3VKR Research Centre, Department of Plant Biology and Biotechnology, University of Copenhagen,

DK-1871 Frederiksberg C, Denmark,4Feedstocks Division, Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA, and5Physical 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 [email protected]).

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 30–40% of the wall mass. The term ‘hemicel-

luloses’ covers a diverse group of polysaccharides, which

ª 2010 The Authors 1107Journal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 61, 1107–1121 doi: 10.1111/j.1365-313X.2010.04161.x

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

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 18–36 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

1108 Aaron H. Liepman et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 1107–1121

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 subunitsof 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 familyCOBRA 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 asoccupying position 1 due to its sequence similarity to CESA1, but this has not yet been demonstrated experimentally.

Arabidopsis cell-wall biosynthetic enzymes 1109


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

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 cellulose–XyG 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).



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

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. 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 observedphenotype, xxt1 xxt2 double mutant has aberrant root hairsand significantly altered cell-wall mechanical properties(Cavalier and Keegstra, 2006; Cavalier et al., 2008)

XXT2 At4G02500 GT34 1,6-a-xylosyltransferase

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)

= D-glucose

XXXG XXLG XXFG

= 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.



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

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

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).



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

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 O’Neill, 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 O’Neill, 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

Table 3 Characterization of xylan biosynthesis genes and corresponding mutants

Name(s) AGI code Oligosaccharide

Xylansynthaseactivity

Chainlength

CaZY family/mechanism

Possibletransferaseactivity

IRX7/FRA8 (ortholog F8H) At2G28110At5g22940

Reduced Unaltered Variable GT47/inverting Xyl/Rha

IRX8/GAUT12 At5G54690 Reduced Unaltered Variable GT8/retaining GalAIRX9 At1G27600 Present Reduced Reduced GT43/inverting XylIRX10 (ortholog IRX10L) At1G27440

At5g61840Present Reduced Reduced GT47/inverting Xyl

PARVUS/GATL1/GLZ1 At1G19300 Reduced Unaltered Variable GT8/retaining GalAIRX14 At4G36890 Present Reduced Reduced GT47/inverting Xyl

‘Oligosaccharide’ refers to that found at the reducing end; xylan synthase activity and chain length refer to the mutant relative to wild-type.



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

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-



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

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

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.



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

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 10–13 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 GPI–transamidase complex involved in

the transfer of D-GlcNAc to phosphatidylinositol, specifically



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

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 number2007-03542 from the United States Department of AgricultureCooperative State Research, Education and Extension Service. N.G.and R.W. are supported by the 7th Framework Program of theEuropean Union, project number 211982, ‘Renewall’. H.V.S. issupported by the US Department of Energy, Office of Science, Officeof Biological and Environmental Research, through contractDE-AC02-05CH11231 between Lawrence Berkeley National Labora-



tory and the US Department of Energy. A.H.L. acknowledgesDr Cory Emal (Eastern Michigan University, Department ofChemistry) and Dr David Cavalier (Michigan State University, PlantResearch Laboratory) for helpful discussions.

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