Molecular Plant • Volume 2 • Number 5 • Pages 1025–1039 • September 2009 RESEARCH ARTICLE

The CELLULOSE-SYNTHASE LIKE C (CSLC) Family ofBarley Includes Members that Are IntegralMembrane Proteins Targeted to the PlasmaMembrane

Fenny M. Dwivanya,d, Dina Yuliaa,e, Rachel A. Burtonb, Neil J. Shirleyb, Sarah M. Wilsona,Geoffrey B. Fincherb, Antony Bacica,c, Ed Newbigina,1 and Monika S. Doblina

a Plant Cell Biology Research Centre, School of Botany, University of Melbourne Victoria 3010, Australiab Australian Centre for Plant Functional Genomics, School of Agriculture and Wine, University of Adelaide, South Australia 5064, Australiac Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne Victoria 3010 Australiad Present address: Department of Biology, Institut Teknologi Bandung, Bandung, Indonesiae Present address: Stem Cell and Cancer Institute, Jl. Jend. Ahmad Yani No.2, Pulo Mas, Jakarta 13210, Indonesia

ABSTRACT The CELLULOSE SYNTHASE-LIKE C (CSLC) family is an ancient lineage within the CELLULOSE SYNTHASE/CEL-

LULOSE SYNTHASE-LIKE (CESA/CSL) polysaccharide synthase superfamily that is thought to have arisen before the diver-

gence of mosses and vascular plants. As studies in the flowering plant Arabidopsis have suggested synthesis of the (1,4)-

b-glucan backbone of xyloglucan (XyG), a wall polysaccharide that tethers adjacent cellulose microfibrils to each other, as

a probable function for the CSLCs, CSLC function was investigated in barley (Hordeum vulgare L.), a species with low

amounts of XyG in itswalls. Four barley CSLC geneswere identified (designatedHvCSLC1–4). Phylogenetic analysis reveals

three well supported clades of CSLCs in flowering plants, with barley having representatives in two of these clades. The

four barley CSLCs were expressed in various tissues, with in situ PCR detecting transcripts in all cell types of the coleoptile

and root, including cells with primary and secondary cell walls. Co-expression analysis showed that HvCSLC3 was coor-

dinately expressed with putative XyG xylosyltransferase genes. Both immuno-EM and membrane fractionation showed

that HvCSLC2was located in the plasmamembrane of barley suspension-cultured cells andwas not in internal membranes

such as endoplasmic reticulum or Golgi apparatus. Based on our current knowledge of the sub-cellular locations of poly-

saccharide synthesis, we conclude that the CSLC family probably contains more than one type of polysaccharide synthase.

Key words: Cellulose synthase-like family C; plant cell wall biosynthesis; xyloglucan; cellulose; glycosyltransferase.

INTRODUCTION

Plant cell walls are highly organized composites that consist of

polysaccharides and provide plants with a skeletal framework

and the intercellular cohesion necessary for structural integrity

(Bacic et al., 1988; Carpita and Gibeaut, 1993; Somerville,

2006). Cell walls are also highly dynamic and complex struc-

tures that give rigidity to the plant overall while providing

the flexibility needed during the processes of cell expansion

and differentiation. In addition, walls form a physical barrier

to plant pathogens but still allow nutrients, gases, and various

intercellular signals to reach the plasma membrane.

Identifying the genes and enzymes responsible for synthe-

sizing cell wall polysaccharides is a major activity in plant

research (Farrokhi et al., 2006; Lerouxel et al., 2006; Scheible

and Pauly, 2004; Somerville et al., 2004). Synthesis of most

b-linked cell wall polysaccharides requires the activity of a proc-

essive-type glycosyltransferase (GT) to produce the linear back-

bone, with current evidence suggesting that these enzymes

are encoded by genes from one of two large gene families,

the Cellulose Synthase/Cellulose Synthase-Like (CESA/CSL)

gene family and the Glucan Synthase-Like (GSL) gene family.

The CESAs code for proteins that make the (1,4)-b-glucan,

1 To whom correspondence should be addressed. E-mail edwardjn@unimelb.

edu.au, fax 61-3-9347-1071, tel. 61-3-8344-4871.

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial

Office in association with Oxford University Press on behalf of CSPP and

IPPE, SIBS, CAS.

doi: 10.1093/mp/ssp064, Advance Access publication 24 August 2009

Received 13 May 2009; accepted 13 July 2009

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cellulose, and the GSLs for proteins that make callose, a (1,3)-

b-glucan (Brownfield et al., 2007; Delmer, 1999; Li et al., 2003).

The CSLs are currently subdivided into nine families that are

designated CSLA to CSLH and CSLJ (Fincher, 2009; Hazen

et al., 2002). Consistent with the suggestion that the CSLs

are processive GTs, heterologous expression studies have

shown that proteins from the CSLA, CSLC, CSLF, and CSLH fam-

ilies are able to make the b-linked backbones for various

non-cellulosic polysaccharides (heteromannans, XyGs and

(1,3;1,4)-b-glucans, respectively) found in primary walls

(Burton et al., 2008, 2006; Cocuron et al., 2007; Dhugga

et al., 2004; Doblin et al., 2009; Liepman et al., 2007, 2005;

Suzuki et al., 2006). The functions of the other CSL families

are unknown, although the CSLDs are suggested to be in-

volved in the synthesis of a non-crystalline form of cellulose

(Bernal et al., 2007; Doblin et al., 2001; Manfield et al., 2004).

The proposed function of the CSLCs is synthesizing the XyG

backbone (Cocuron et al., 2007). XyG is a major class of wall

polysaccharide present in the primary walls of most land plants

and its backbone is composed of (1,4)-b-D-glucosyl residues to

which a-D-xylosyl residues and other sugars are attached.

Cocuron et al. (2007) found that long and short chains of

(1,4)-b-glucan accumulated in Pichia pastoris cells that were

co-expressing Arabidopsis CSLC4 (AtCSLC4) and Arabidopsis

XXT1 (AtXXT1), the latter being a xylosyl transferase that adds

the first side-chain xylosyl residue onto XyG (Cavalier and

Keegstra, 2006; Cavalier et al., 2008; Faik et al., 2002). Al-

though no detectable xylose was present on the b-glucan

chains, this was presumably because yeast cells lack UDP-

xylose, which is the AtXXT1 substrate. Since the yeast cells

produced an unbranched b-glucan, it was possible that

AtCSLC4 was involved in synthesizing either cellulose or the

XyG backbone. However, Cocuron et al. (2007) argued that

other evidence pointed most strongly to a role in XyG biosyn-

thesis. This evidence included the highly correlated expression

of AtCSLC4 and AtXXT1 and the likely targeting of stably

expressed AtCSLC4 to the Golgi of BY-2 tobacco cells. The pres-

ence of AtCSLC4 in Golgi is consistent with a role in synthesizing

XyG, which is believed to be synthesized in this compartment

(Cosgrove, 2005). Cellulose synthesis, on the other hand, occurs

at the plasma membrane (Delmer, 1999).

Although CSLC genes are found in all flowering plant

genomes, XyGs are not abundant components of all flowering

plant cell walls. In particular, the commelinoid monocots, of

which the Poaceae or grass family is the best studied example,

have a primary cell wall that is characterized by relatively low

levels of XyGs (Carpita, 1996; Fry, 1989; Harris et al., 1997;

Hayashi, 1989; O’Neill and York, 2003). The amount of XyG

in Poaceae walls is generally lower (1–5% of wall dry weight)

than that in non-commelinoid monocots, gymnosperms, and

eudicots, which have cell walls containing 10–20% XyG. In com-

melinoid monocots, glucuronoarabinoxylans (GAXs) are pro-

posed to be the principal polymers interlocking cellulose

microfibrils (Bacic et al., 1988; Carpita and McCann, 2000; Smith

and Harris, 1999).

The CSLC genes of barley (Hordeum vulgare) were studied in

order to understand the function of CSLCs in the Poaceae,

where the amount of XyG in walls is low. By a combination

of bioinformatic searches and gene-cloning, four barley CSLCs

were identified (HvCSLC1–4). As all the barley CSLC ESTs iden-

tified to date are derived from these four genes, HvCSLC1–4

represent the most actively transcribed members of this family

in the barley genome. CSLCs were expressed in cells with pri-

mary and secondary walls and at various stages of the barley

lifecycle. In sub-cellular fractions of barley suspension culture

cell membranes, HvCSLC2 preferentially partitioned into frac-

tions enriched in plasma membrane and was barely detectable

in other membrane-containing fractions. HvCSLC2 was also

detected in the plasma membrane by immuno-electron micros-

copy (immuno-EM) with an antibody raised against HvCSLC2.

These findings are discussed in the context of the diversity

that exists within the CSLC family and the possibility that it

contains more than one type of polysaccharide synthase.

RESULTS

Cloning and Preliminary Characterization of Barley CSLC

cDNAs and Genes

Barley CSLC genes were identified by iterative searching of

cDNA and BAC libraries with CSLC-derived gene probes and

databases with CSLC EST sequences. Library searching began

with the sequence of a barley pre-anthesis spike EST (accession

no. BE455720) that encodes a putative CSLC. Primers to this

sequence were used to amplify a fragment from barley pre-

anthesis spike cDNA, and this was used to screen a barley

(cv. Schooner) cell suspension culture cDNA library. From this

screen, cDNAs for two different CSLC genes (designated

HvCSLC1 and HvCSLC2) were identified. Sequence analysis

indicated that the HvCSLC1 cDNA was full-length (2.8 kb)

but that about 1.7 kb of sequence was missing from the 5’

end of the HvCSLC2 cDNA. An EST (accession no. AV836446)

that overlapped the HvCSLC2 cDNA was used to extend the se-

quence of this gene in the 5’ direction. The continuity of these

two sequences was confirmed by amplifying an overlapping

DNA fragment from suspension-cultured cell cDNA.

A fragment amplified from the HvCSLC1 cDNA was used to

screen a barley (cv. Morex) BAC library. After screening over

184 000 colonies (estimated to be roughly a three-fold cover-

age of the barley genome), 14 positive BAC clones were iden-

tified that sequence analysis showed included the two HvCSLC

genes already identified, as well as two new HvCSLC genes

(designated HvCSLC3 and HvCSLC4). A 2.9-kb BAC fragment

that included almost all of HvCSLC3 except for ;500 bp from

the 5’ end of the open reading frame, and a 1-kb BAC frag-

ment that contained the central portion of HvCSLC4, were se-

quenced. A near full-length HvCSLC4 was produced using ESTs

that extended the sequence in both the 5’ and 3’ directions.

Most of the intron/exon boundaries predicted by FGENESH

(www.softberry.com) in the HvCSLC1 and HvCSLC4 genomic

sequences were confirmed from EST data.

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A schematic representation of the four polypeptides

encoded by the HvCSLC genes is shown in Figure 1. The sequen-

ces for HvCSLC1–4 have been deposited in GenBank with acces-

sion numbers GQ386981 to GQ386984. All 50 barley CSLC ESTs

in GenBank (as of April 2009) are derived from these four

genes (Supplemental Table 1). HvCSLC1 is predicted to encode

a 698-amino-acid polypeptide with a molecular weight of

78.2 kDa and six transmembrane domains (two at the NH2-

terminus and four at the COOH-terminus). Amongst the rice

and Arabidopsis CSLC proteins, HvCSLC1 is most similar to

OsCSLC7 (86.2% identity, 92.1% similarity) and AtCSLC12

(66.8% identity, 80.0% similarity). The partial sequences of

HvCSLC2, HvCSLC3, and HvCSLC4 are predicted to encode pro-

teins of 535, 597, and 530 amino acids, respectively, that have

the same predicted membrane topology as HvCSLC1, as de-

duced by comparison to their putative rice orthologs OsCSLC9,

OsCSLC3, and OsCSLC1 (Figure 2). All four genes encode pro-

teins with the D,D,D,QQHRW motif within homology (H)

domains 1–3. This motif is also found in all the CSLCs from rice

(Oryza sativa), poplar (Populus trichocarpa), the moss Physco-

mitrella patens, and in the majority of CSLCs from sorghum

(Sorghum bicolor), grapevine (Vitis vinifera), and four of the

five Arabidopsis CSLCs (AtCSLC6 has QQYRW instead).

Figure 2 shows a Neighbour Joining tree of the CSLC family

as currently proscribed (Hazen et al., 2002; Richmond and

Somerville, 2000; Roberts and Bushoven, 2007). This tree

was produced from an alignment of the four barley CSLC poly-

peptides and full-length CSLC sequences from a number of

monocots and eudicots, the moss Physcomitrella, the lyco-

phyte Selaginella moellendorffii, and the green alga Chara

globularis (Supplemental Figure 1). Trees with similar se-

quence groupings were produced when other phylogenetic

methods were used and when only the H1-3 regions were used

(data not shown). The CSLCs receive strong bootstrap support

as a separate clade within the CESA/CSL superfamily and the

Figure 1. Domain Structure of Barley CSLC Proteins Compared toArabidopsis AtCSLC4.

Predicted HvCSLC1–4 proteins with AtCSLC4 protein as comparison,drawn to scale as boxes. Length of amino acid sequence is shown atthe end of each box. Dashed vertical lines at the NH2-terminus in-dicate incomplete proteins. Proteins are divided into the sevendomains defined by Pear et al. (1996). H-1, H-2, and H-3 (gray boxes)are homology domains; CR-P, plant conserved region; HVR, hyper-variable region; N and C refer to the NH2- and COOH-terminaldomains, respectively. Black boxes define the U1-4 regions contain-ing amino acid residues of the conserved D,D,D,QQHRW motif asdefined by Pear et al. (1996). Black bars underneath boxes indicatethe location of trans-membrane helices predicted by WoLF PSORT.The hashed box beneath HvCSLC2 indicates the antigen region usedfor polyclonal antibody production.

Figure 2. Phylogenetic Analysis of the Plant CSLC Family.

A distance cladogram using Neighbour Joining clustering showingthe majority consensus of 1000 bootstrap replicates (as a percent-age) of the CSLC proteins of barley (Hordeum vulgare) and full-length sequences from Arabidopsis thaliana (At), Oryza sativa(Os), Sorghum bicolor (Sb), Populus trichocarpa (Pt), Vitis vinifera,(Vv), Medicago truncatula (Mt), Solanum lycopersicum (Sl), Tro-paeolum majus (Tm, nasturtium), Zea mays (Zm), Physcomitrellapatens (Pp), Chara globularis (Cg), and Selaginella moellendorffii(Sm). The tree was rooted with the Chara CSLC sequence. The fourCSLC clades (I–IV) are indicated and the sub-clades of Clade I arelabeled a–c. Barley sequences are marked in bold underline, Arabi-dopsis sequences in gray italics. * indicates AtCSLC4, shown to have(1,4)-b-glucan synthase activity and a likely Golgi location (Cocuronet al., 2007). ^ indicates HvCSLC2 shown to be located at the PM(this study).

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most divergent member of this group, the CSLC from Chara,

was chosen as the root of the tree shown in Figure 2. Tree to-

pology is characterized by four well supported groups (clades

I–IV), with three of these groups (clades I–III) being part of

a polytomy. All the CSLCs from Physcomitrella and Selaginella

are in clade III, which suggests taxonomic relationships are one

source of tree structure. However, clades I and II contain mono-

cot and eudicot CSLCs, suggesting functional specialization as

another possible source of tree structure. HvCSLC1, HvCSLC2,

and HvCSLC4 are in clade I and HvCSLC3 is in clade II. Clade IV

contains AtCSLC6 and CSLCs from grapevine, Medicago trun-

catula, and poplar.

HvCSLC Transcript Levels and Correlations with Other

Genes

Quantitative RT–PCR (QPCR) was used to determine the pattern

and transcript level of barley CSLCs. The normalized transcript

levels for each HvCSLC across a range of barley tissues and sus-

pension-cultured cells are shown in Figure 3. In nine of the 12

tested tissues, HvCSLC2 transcript levels were either the highest

or equal highest of the four genes, in agreement with the high

level of representation of HvCSLC2 sequences among barley

CSLC ESTs (Supplemental Table 1). In coleoptile, HvCSLC1 and

HvCSLC4 transcript levels were higher than those of HvCSLC2

and, in root tip, HvCSLC1 and HvCSLC3 transcript levels were

higher (Figure 3A). Root tip was the only tissue to accumulate

significant levels of HvCSLC3 transcript, although low levels of

this transcript were present in several other tissues.

The four HvCSLC genes are present on the Affymetrix 22K

Barley1 GeneChip with the sequence IDs listed in Supplemen-

tal Table 2. The Barley1 microarray contains at least 21 439

genes and has been used in a number of experiments for which

the datasets are publically available through both BarleyBase/

PLEXdb (Shen et al., 2005; Wise et al., 2007) and ArrayExpress

(Parkinson et al., 2006). In an experiment in which various

vegetative and floral tissues were sampled across plant devel-

opment in two barley cultivars (Druka et al., 2006), the expres-

sion pattern of the HvCSLCs in common tissues is generally

consistent with those found using QPCR (Supplemental Figure

2A). For example, HvCSLC2 transcripts are present at relatively

high levels in most tissues, whereas HvCSLC3 transcripts are

generally low except in root tips.

Correlations were sought between the transcript profiles

of individual HvCSLCs and those of other genes on the array

(Figure 4). As a standard by which to assess the significance

of these correlations, the lowest pairwise correlation coeffi-

cient (0.89) of the transcript profiles of the three barley

primary wall CESAs (HvCESA1, HvCESA2, and HvCESA6) was

used, as it is known that their expression is highly correlated

(Burton et al., 2004; Supplemental Figure 2C). By this criterion,

none of the HvCSLC genes showed significant co-expression

with another CSLC or with another member of the CESA/CSL

superfamily, with the most highly correlated pair being

HvCSLC1 and HvCSLC3 (0.77). Supplemental Table 3 lists the

top 20 correlations for each HvCSLC gene.

Part of the evidence linking the CSLCs to XyG production is

the coordinate expression of AtCSLC4 and AtXXT1 (Cocuron

et al., 2007). To determine whether the barley CSLCs were co-

ordinately expressed with barley orthologs of AtXXT1, puta-

tive barley homologs were identified by iterative database

searches. This yielded a total of 39 ESTs (Supplemental Table

4) that sequence alignments showed represented the partial

sequences of five genes that we provisionally named

HvGT1–5 (for H. vulgare glycosyl transferase). HvGT1 has the

longest sequence and encodes a protein of 296 amino acids

that covers the COOH-terminal half of AtXXT1 (Supplemental

Figure 3). HvGT1 is also the most closely related of the five

HvGTs to AtXXT1 and AtXXT2 (82.6 and 84.9% amino acid

identity, respectively). All five HvGT genes are present on

the Barley1 microarray and the contig IDs for these genes

are listed in Supplemental Table 2. Figure 4 shows where

the five HvGT genes occur in the correlation distributions of

Figure 3. Transcript Abundance of HvCSLC1–4 as Determined byQPCR.

Normalized transcript levels of HvCSLC1–4 in 12 vegetative and flo-ral tissues (A) and developing endosperm (2–11 d after pollination(DAP)) (B). Control genes for normalization in (A) were GAPDH,cyclophilin and a-tubulin; in (B), cyclophilin, a-tubulin and EF1a.Error bars indicate SD.

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each HvCSLC gene. The transcript profiles of HvGT3 and

HvCSLC3 were highly correlated (correlation coefficient =

0.99), as transcripts for both predominantly accumulate in

root tips (Supplemental Figure 2A and 2B). The next highest

correlation coefficient, 0.82, for the transcript profiles of

HvGT1 and HvCSLC1, was below the level considered signifi-

cant. The transcript profiles of 83 other genes were more

highly correlated to HvCSLC1 than HvGT1.

To study the cell-type specificity of HvCSLC expression, in situ

RT–PCR (Koltai and Bird, 2000) was carried out on barley

coleoptiles and root tips that had been harvested 3 d after ger-

mination. As expected, 18S rRNA transcripts (positive control)

were detectable in most root and coleoptile cells (Figure 5C

and 5F) and no signal was detected when primers were omit-

ted (Figure 5B and 5E). In root and coleoptile, HvCSLC1 label-

ing was seen in all cell types and was particularly apparent in

the vascular bundles (Figure 5A and 5D), indicating that tran-

scripts for this gene accumulate in cells with a primary wall as

well as cells with a secondary wall. While less labeling was seen

in cortical cells, this was probably due to their low content of

cytoplasm. Similar results were obtained for the other three

CSLC genes, with signal strength reflecting QPCR transcript lev-

els (Supplemental Figure 4).

Transient Xyloglucan Deposition during Early Endosperm

Development

Figure 3B shows the normalized expression of three HvCSLC

genes (HvCSLC1, 2, and 4) during early endosperm development.

HvCSLC3 transcripts were barely detectable in this tissue.

HvCSLC4 transcript levels were the highest and, early in develop-

ment, were well above those of any barley CSLC gene in

Figure 4. Ranked Correlation Plots for HvCSLC1–4 from the Barley122K Affymetrix GeneChip Probe Sets in Experiment BB3: TranscriptPatterns during Barley Development (Druka et al., 2006).

The correlations of HvCSLC1–4 with all 21 439 barley probe sets onthe Barley_1 microarray were ranked highest to lowest and thenumber of probe sets within each 0.05 interval tallied and plotted.The interval marked –1.00 shows the number of probe sets witha correlation of between –1.00 and –0.95, –0.95, between –0.95and 0.90, etc. The correlations (r) of the five putative barley XylTs(HvXT1–5) are given and their rank position indicated by black ver-tical lines. The ranks of any CSLC or GT gene were noted if theyappeared in the top 300 correlated genes. For comparison, therange of correlations between the primary wall CESA genes(HvCESA1, 2, 6; Burton et al., 2004) is 0.895–0.958, between the sec-ondary CESA genes (HvCESA4, 5/7, 8) 0.975–0.981 (SupplementalFigure 2C).(A) HvCSLC1. Correlations with HvGT1 and HvGT3 are ranked at 84and 182, respectively, HvCSLC3 (r = 0.771) at 289. An XET is rankedat 31 (r = 0.847).(B) HvCSLC2. A second HvCSLC2 contig (HvCSLC2b) has an r-value of0.901 and is the 13th most highly correlated gene. Correlation witha third HvCSLC2 contig (HvCSLC2c) was less (r = 0.759), most likelybecause of the significantly lower expression level detected withthis probe set (Supplemental Figure 2A). Three contigs of the sameexpansin were identified in the top 20 correlations (ranked 6, 11,13, r = 0.924, 0.908, 0.904, respectively).(C) HvCSLC3. HvGT3 is the gene most highly correlated gene withHvCSLC3. Two XETs are listed within the top 50 correlated genes(ranked 6 and 43, r = 0.983, 0.928, respectively).(D) HvCSLC4.

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vegetative tissues. HvCSLC4 transcript levels were relatively con-

stant up to 5 d after pollination (DAP) and declined thereafter.

HvCSLC1 and HvCSLC2 transcript levels were lower than HvCSLC4

levels at 2 DAP and were undetectable by 6 DAP.

Detecting CSLC expression in early stages of endosperm de-

velopment raised questions about their proposed role in XyG

synthesis, as XyG has not been detected by chemical analysis in

the walls of mature barley endosperm cells (Bacic and Stone,

1981; Fincher, 1975). Immuno-EM with the LM15 monoclonal

antibody (Marcus et al., 2008) was used to determine whether

XyG was present in immature endosperm walls. The LM15 an-

tibody was used because it recognizes a non-fucosylated XyG-

derived oligosaccharide, whereas other antibodies, CCRC-M1

for example, bind to a fucosylated XyG epitope that is not

present on barley XyG (Gibeaut et al., 2005; Puhlmann

et al., 1994).

To confirm that LM15 could be used to detect XyG in barley,

thin sections of 3-day-old barley coleoptiles, which are known

to have XyG in their walls (Gibeaut et al., 2005), were examined.

LM15 labeling was evident in the thin primary walls of cortical

cells and thick secondary walls of vascular cells (Figure 6A and

6B, respectively). Labeling was abolished in coleoptile

sections pre-incubated with either the non-fucosylated XyG

from Nicotiana plumbaginifolia suspension-cultured cells

(Figure 6C and 6D) or Tamarindus indica (tamarind) seed (data

not shown; Sims and Bacic, 1995; Sims et al., 1996; York et al.,

1990). Labeling was also unchanged when sections were co-

incubated with two derivatives of cellulose (microcrystalline cel-

lulose, carboxymethyl cellulose), cellohexaose (a cellodextrin),

barley flour (1,3;1,4)-b-glucan, and laminarin (Supplemental

Figure 5A–5E, respectively). No labeling was detected when

the LM15 antibody was omitted (Supplemental Figure 5F) or

when the CCRC-M1 antibody was used (data not shown).

At 4 DAP in barley endosperm, cellularization is taking place

and the first anticlinal cell walls can be seen growing out from

the central cell wall between nuclei of the multinucleate syn-

cytium (Figure 6E; Wilson et al., 2006). Light LM15 labeling was

seen in the first-formed anticlinal walls, periclinal walls, and in

the wall of the central cell, as well as in the walls of the sur-

rounding maternal tissues (Supplemental Figure 6A and 6B,

respectively). By 8 DAP, the endosperm is fully cellularized

(Wilson et al., 2006) and LM15 labeling was no longer evident,

although a significantly reduced level persisted in the walls of

the maternal cells, which served as an internal positive control

for antibody labeling (Supplemental Figure 6D).

Figure 5. Distribution of HvCSLC1 Transcripts as Determined by insitu PCR.

In situ PCR images of 3-day-old roots (A–C) and coleoptiles (D–F)using probes for HvCSLC1 (A, D), 18S rRNA (positive control) (C,F), and a negative control (no primers) (B, E). Cells in which tran-scripts are detected stain purple to dark brown and cells in whichno transcript is detected stain light brown. Scale bars represent250 lm. Figure 6. XyG Is Present in the Cell Walls of Coleoptiles and Early

Developing Endosperm.

Transmission electron micrographs showing the cell walls of the co-leoptile (A–D)and starchy endosperm 4 and 8 DAP, respectively (E, F)probed with gold-labeled LM15 antibody. (A) and (B) probed withLM15 only, (C) and (D) controls in which the LM15 was co-incubatedwithN.plumbaginifolia-derivedXyG.(A)and(C)showcor-tical cells, (B) and (D) show thick secondary walls of the vascular cells.cw, cell wall; anticlinal cw, acw (arrows). Scale bars represent 0.5 lm.

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An HvCSLC2 Antibody Detects a Protein in the Plasma

Membrane

To determine the sub-cellular location of the CSLCs, a rabbit

antiserum was raised to a peptide covering amino acids

411–466 of HvCSLC2 (Supplemental Figure 7). This region

was chosen because it lacks sequence similarity to other mem-

bers of the CESA/CSL superfamily. It is, however, possible that

this antiserum detects CSLCs other than HvCSLC2. In prelimi-

nary experiments, the anti-HvCSLC2 antiserum recognized

a single protein band of ;80 kDa in Western blots of a deter-

gent-soluble, mixed-membrane (MM) fraction from coleop-

tiles, which is consistent with the expected size of a CSLC

protein (data not shown). To determine which membrane

type contains HvCSLC2, MM (125 000 g pellet) from barley

suspension-cultured cells were fractionated by PEG/DEX

two-phase partitioning (Larsson et al., 1987) into a fraction

enriched in plasma membrane (PM) (the PEG phase) and a

second fraction containing other membrane types and some

PM (the DEX phase). The degree of membrane enrichment

in each fraction (homogenate, MM, PEG, and DEX) was

assessed by Western blot analysis using antisera to proteins

with known sub-cellular locations and biochemical marker

assays (data not shown; Dwivany, 2003).

Figure 7 shows the results of Western blots incubated with

antisera to an Arabidopsis H+-ATPase (a PM marker; Chevallet

et al., 1998) and two Golgi apparatus markers, pea RGP1

(Dhugga et al., 1997) and HvGlyT4 (Farrokhi, 2005). HvGlyT4

is a member of CAZy family GT47 and has highest sequence

similarity to b-glucuronyltransferases (Supplemental Figure

8) that in other systems are located in the Golgi apparatus

(Brown et al., 2009; Iwai et al., 2002; Wu et al., 2009). Consis-

tent with these reports, immuno-EM with the anti-HvGlyT4 an-

tibody detected label in the Golgi apparatus of suspension-

cultured cells (Supplemental Figure 9). The anti-H+-ATPase an-

tiserum detected a ;90-kDa band in the homogenate fraction,

and bands of ;80 and ;60 kDa in the MM, PEG, and DEX frac-

tions. These bands correspond to sizes previously reported for

H+-ATPase, with the lower MW bands presumably arising by

degradation during sample processing (Chevallet et al.,

1998). The H+-ATPase bands were most intense in the PEG frac-

tion and less intense in the DEX, MM, and homogenate frac-

tions (Figure 7B). The anti-RGP1 antiserum detected a protein

of the expected size in the homogenate, MM, and DEX frac-

tions that was much less abundant in the PEG fraction (Figure

7C). A similar pattern of labeling was also observed with the

HvGlyT4 antiserum, with the 55-kDa protein larger than pre-

dicted (41 kDa), suggesting that it is post-translationally mod-

ified (Figure 7D). Collectively, these data indicated that the

PEG fraction was enriched in PM proteins and largely depleted

of proteins from the Golgi apparatus. Enzyme marker assays

done on the same PEG and DEX fractions are consistent with

these findings (data not shown; Dwivany, 2003).

When blots of the fractions were incubated with the anti-

HvCSLC2 antiserum, the ;80-kDa protein was enriched in the

PEG fraction, with trace amounts present in the DEX fraction

and no detectable protein in the homogenate and MM frac-

tions (Figure 7A). Thus, the anti-HvCSLC2 antiserum detected

a low-abundance integral membrane protein that preferen-

tially partitioned into a PM-enriched fraction.

To confirm this location, barley suspension-cultured cells

were prepared for immuno-EM (Figure 8). The anti-HvCSLC2

antiserum gave a light level of labeling (arrowheads) that

was restricted to the PM (Figure 8A, arrows). Little or no label-

ing was seen in intracellular organelles such as the Golgi ap-

paratus and endoplasmic reticulum (Figure 8B).

DISCUSSION

Here, we report the identification and characterization of four

barley genes belonging to the CSLC family of polysaccharide

synthases. Our data show that at least one of the barley CSLCs

resembles the better characterized CESAs in being an integral

membrane protein targeted to the PM. This cellular location is

relevant to discussions of the proposed role of CSLCs as poly-

saccharide synthases and contrasts with previous work on the

Figure 7. Barley CSLCs Are Enriched in the Plasma Membrane Frac-tions of Barley Suspension Culture Cells.

Western blots of barley cell suspension culture membrane fractions(;20 lg protein per lane) probed with antibodies towards HvCSLC2(A), a PM H+-ATPase (B), and the Golgi-located RGP1 (C) andHvGlyT4 (D) proteins. Hm, homogenate; MM, 125 000 g mixedmembrane pellet fraction; upper PEG and lower DEX, the upperand lower fractions from two-phase PEG/dextran separation ofthe mixed membrane fraction, respectively. Numbers to the rightof the figure indicate the sizes of protein markers (kDa).

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Arabidopsis CSLC4 that suggests some of these proteins are tar-

geted to the Golgi apparatus and involved in XyG backbone

synthesis (Cocuron et al., 2007).

The four CSLC genes described here potentially represent the

full complement of functional CSLC genes in the barley ge-

nome, since all barley CSLC ESTs were derived from one or other

of these four genes. However, as Arabidopsis (Richmond and

Somerville, 2000), poplar (Suzuki et al., 2006), grapevine (Jaillon

et al., 2007; www.genoscope.cns.fr/externe/GenomeBrowser/

Vitis/), and sorghum (Paterson et al., 2009) all have five func-

tional CSLC genes, rice has six functional CSLCs and at least

four CSLC-derived pseudogenes (http://waltonlab.prl.msu.edu/

research-cwb.htm), and maize has 12 CSLCs, at least one of

which is a pseudogene (van Erp and Walton, 2009), it is possible

that one or more barley CSLC genes remain to be discovered.

For instance, there is most likely at least one more clade Ic-type

sequence, as there are two rice and two Sorghum CSLC sequen-

ces within this subclade and only one barley sequence, HvCSLC2

(Figure 2). Because they are not represented among the barley

ESTs, any functional CSLC genes still missing are likely to be

expressed at only low levels or only by certain cell types during

particular developmental stages.

Phylogenetic analysis shows the CSLC family contains four

well supported but poorly resolved clusters. One of these clus-

ters (clade III) contains all the moss and lycophyte CSLCs and

the other three clusters all the flowering plant CSLCs (Figure

2). Clades I and II contain both grass and eudicot CSLCs,

whereas clade IV contains only eudicot sequences. One barley

CSLC is in clade II and the other three are in clade I. Within

clade III, the Physcomitrella and Selaginella CSLCs are well sep-

arated, reflecting the separate evolutionary paths taken by the

lycopod and moss lineages (Figure 2). Consistent with previous

work, none of the Physcomitrella CSLCs is basal to any of the

flowering plant clades, indicating that divergence within flow-

ering plant CSLC lineages occurred later than the divergence

of the flowering plants and moss lineages (Roberts and

Bushoven, 2007). A CSLC from Chara globularis lies outside

these clades and further sampling of CSLCs from other non-

flowering plants and charophyte algae is required to resolve

the evolutionary history of this family.

Accumulation of CSLC transcripts in barley is generally quite

low and transcript abundance is usually less than 10% of the

level of any CESA mRNA expressed in the same tissue (Burton

et al., 2004) (compare Supplemental Figure 2A and 2C). Al-

though three of the barley CSLC genes (HvCSLC1, HvCSLC2,

and HvCSLC4) are expressed to varying degrees in most tissues

examined, these genes do not appear to be coordinately reg-

ulated at the transcriptional level. This does not, however, pre-

clude different CSLC isoforms from acting jointly in the

synthesis of a particular polysaccharide. Furthermore, unlike

the CESAs, the CSLCs cannot be classified by expression pattern

into those that function in primary cell wall synthesis and those

that function in secondary cell wall synthesis (Burton et al.,

2004). Consistent with this are in situ RT–PCR results showing

that HvCSLC transcripts accumulate in all cell types of the root

and coleoptile.

Five barley members of the CAZy glycosyltransferase family

34 (GT34) are on the Barley1 microarray (HvGT1–5). Plant

GT34s include the galactomannan a-(1,6)-galactosyltrans-

ferases and the UDP-Xyl:xyloglucan a-(1,6)-xylosyltransferases

(Cantarel et al., 2008; www.cazy.org/). Correlation analysis

revealed that the expression profiles of HvCSLC3 and HvGT3

were highly correlated (Figure 4 and Supplemental Table 3).

It seems likely that HvGT3 encodes a XyG xylosyltransferase,

as its partial sequence aligns most closely to those of three con-

firmed XyG xylosyltransferases from Arabidopsis: AtXXT1,

AtXXT2, and AtXXT5 (Cavalier and Keegstra, 2006; Cavalier

et al., 2008; Faik et al., 2002; Zabotina et al., 2008). As a recent

study of wheat seedlings found high levels of XyG (23–

39 mol%) in the cell walls of root tips (Leucci et al., 2008)

and our own analysis has confirmed the presence of XyG in

3-day-old barley roots (data not shown), it seems plausible

Figure 8. Barley CSLCs Are Located in the Plasma Membrane.

Transmission electron micrographs showing high-pressure-frozencell suspension-cultured cells of barley probed with gold-labeledHvCSLC2 antibody (A, B). Plasma membrane (pm) indicated byarrows, HvCSLC2 labeling by arrowheads. G, Golgi; mt, mitochon-drion; v, vacuole. Scale bars represent 200 nm.

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to suggest that HvCSLC3 and HvGT3 are involved in XyG bio-

synthesis in barley root tips. Although this conclusion needs to

be confirmed experimentally, as the correlated transcript pro-

files of HvCSLC3 and HvGT1 are not proof that the products of

these genes participate in the same pathway, it is consistent

with the presence of HvCSLC3 and AtCSLC4 in the same CSLC

clade (clade II), as Cocuron et al. (2007) had previously con-

cluded that AtCSLC4 is involved in XyG backbone synthesis.

Among other genes with transcript patterns highly correlated

to HvCSLC3 were two XyG endotransglucosylases/hydrolases

(XET/XTHs) (r = 0.98 and 0.93), which are also likely to be in-

volved in XyG assembly or re-modeling.

Evidence for the other three barley CSLCs being involved in

XyG synthesis is either lacking or equivocal. HvCSLC1, 2, and 4

are all in clade I, which has only one Arabidopsis member

(AtCSLC12). The highest correlation between a clade I barley

CSLC and the barley GT34s was between HvCSLC1 and HvGT1

(r = 0.82). Two other GT34s, HvGT2 and HvGT3, showed

slightly lower correlations to HvCSLC1 (r = 0.71 and 0.80, re-

spectively), and none of these correlations was above the level

assigned as significant (r = 0.89). However, like HvGT3,

HvGT1 and HvGT2 align better to XyG xylosyltransferases from

Arabidopsis than to the galactomannan a-(1,6)-galactosyl-

transferases, which are also in GT34 (Supplemental Figure

3). Furthermore, the HvCSLC1 transcript profile was correlated

to the profile of a XET/XTH gene (r = 0.85). Together, these

data suggest that HvCSLC1 may be involved in XyG backbone

synthesis in tissues other than root tips, such as the coleoptile.

But HvCSLC1 was most highly correlated (r = 0.90) to a gene

related to SHORT HYPOCOTYL 2 (SHY2), that codes for an Ara-

bidopsis protein that promotes cell differentiation by nega-

tively regulating genes involved in auxin redistribution

(Dello Ioio et al., 2008). Further investigation into the function

of HvCSLC1 is therefore required to understand its transcrip-

tional correlation with SHY2. The expression profiles of

HvCSLC2 and HvCSLC4 were not significantly correlated to

those of a HvGT or a XET/XTH (r , 0.5), suggesting that these

genes have functions different from those of HvCSLC3 and pos-

sibly HvCSLC1.

An alternative approach to determine whether the barley

CSLCs were involved in XyG biosynthesis was to use

immuno-EM and the LM15 antibody to see whether XyG

was present in the walls of barley cells that accumulated CSLC

transcripts. Coleoptile was used as an example of a tissue in

which both HvCSLC expression and XyG accumulation take

place. To confirm that LM15 specifically detected XyG, coleop-

tile sections were pre-incubated with a variety of (1,4)- and

(1,3)-b-glucose containing polysaccharides/oligosaccharides,

including derivatives of cellulose, callose, and (1,3;1,4)-b-glu-

can. None of these treatments reduced LM15 labeling (Supple-

mental Figure 5). Both primary and secondary cell walls

contained XyG and these cells also accumulated HvCSLC tran-

scripts.

Endosperm was examined because HvCSLC1, 2, and 4 tran-

scripts accumulated to relatively high levels during the initial

stages of wall development (Figure 3B), yet XyG was not

known to be present in endosperm cell walls (Bacic and Stone,

1981; Fincher, 1975). However, immuno-EM with the LM15 an-

tibody detected light labeling in anticlinal and periclinal

walls of developing endosperm cells at 4 DAP (Figure 6 and

Supplemental Figure 6). These walls are known to contain cal-

lose and have been suggested to contain cellulose as well,

based on labeling with gold-conjugated cellobiohydrolase

II (CBH II; Wilson et al., 2006). This conclusion may need to

be revised in light of these findings, as CBH II also binds to

XyG. By 8 DAP, LM15 labeling was still detectable in the sur-

rounding maternal tissues but was no longer evident in en-

dosperm walls, implying that the XyG deposited during

endosperm cellularization was rapidly turned over. XyG is

thus a second example, along with callose, of a polysaccharide

that is deposited and then removed from early endosperm

cell walls. Concomitant with XyG disappearance was a ;10-

fold reduction in the levels of HvCSLC1, 2, and 4 transcripts

(Figure 3B).

Although HvCSLC expression and XyG deposition in devel-

oping endosperm were correlated, experiments with a poly-

clonal antibody to a HvCSLC2 peptide provided evidence

that the CSLCs reside in the PM and are not in the Golgi—an

important observation, given previous evidence that XyG

synthesis occurs in the Golgi (Becker et al., 1995; Delmer,

1999; Gibeaut and Carpita, 1994; Gordon and Maclachlan,

1989). HvCSLC2 is the most abundant CSLC transcript in bar-

ley suspension-cultured cells, although HvCSLC1 and HvCSLC4

transcripts also accumulate to varying degrees (Figure 3A). As

the specificity of the HvCSLC2 antibody to other CSLC isoforms

is unknown, it is possible that the band it detects contains all

three isoforms found in suspension-cultured cells, with

HvCSLC2 probably being the most abundant of these forms

(Figure 7). Thus, one interpretation is that all CSLCs in the ex-

tract preferentially partitioned into the PM-enriched PEG frac-

tion and that the low amount of CSLC in the Golgi-containing

DEX fraction was due to the presence of some PM in this frac-

tion. However, it is equally possible that only the most abun-

dant CSLC isoform (presumably HvCSLC2) partitioned into the

PM fraction, with the DEX fraction containing in other Golgi-

targeted CSLC isoforms. According to this interpretation,

CSLCs are targeted to either the PM or Golgi. Of the two inter-

pretations, only the PM location was supported by immuno-

EM with HvCSLC2 antibody (Figure 8). It therefore appears that

in barley suspension-cultured cells, CSLCs are targeted to the

PM, although we cannot rule out the possibility that some iso-

forms, present in low abundance, are targeted to the Golgi as

well.

However, linkage analysis detected only trace amounts of

XyG in suspension-cultured cell walls (Yulia, 2006) and LM15

labeling of the walls of these cells was very light (Supplemen-

tal Figure 10). HvCSLC2 and the other isoforms present in

these cells consequently do not appear to be involved

in XyG backbone synthesis and may instead play a role in

synthesizing callose or cellulose, as these are the only

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polysaccharides known to be made at the PM. Of the two pol-

ysaccharides, it is most likely that HvCSLC2 is involved in mak-

ing cellulose, as b-(1,4)-glucan synthase activity has already

been shown for another member of the CSLC family (Cocuron

et al., 2007), and callose, a b-(1,3)-glucan, is synthesized by

proteins of the GSL gene family (Brownfield et al., 2007; Li

et al., 2003).

The CSLC family therefore appears to contain proteins tar-

geted to two distinct sub-cellular locations and participating

in the synthesis of two distinct polysaccharides. Clade II pro-

teins, such as AtCSLC4 and probably HvCSLC3, are targeted to

the Golgi where they might participate in XyG backbone bio-

synthesis (Cocuron et al., 2007; Dunkley et al., 2006). How-

ever, clade I proteins such as HvCSLC2 are targeted to the

PM, where they probably participate in cellulose biosynthe-

sis. From a biochemical perspective, the proposed functional

diversification within the CSLC family is far from implausible

because XyG backbone synthases and cellulose synthases

both make a b-(1,4)-glucan chain. This conclusion is also con-

sistent with the absence of XyG in the walls of charophyte

algae (Popper and Fry, 2003, 2004), suggesting that the

Chara CSLC is not involved in making XyG, but rather partic-

ipates in the synthesis of some other polysaccharide, possibly

cellulose. However, a thorough chemical analysis of the walls

of various charaophycean algae is required to confirm the

absence of XyG from this taxon. An ancestral role for the

CSLCs as cellulose synthases is also in keeping with this family

belonging to an ancient lineage that is evolutionarily dis-

tinct from the CESA lineage to which most other CSL families

belong (Nobles and Brown, 2004; Roberts and Bushoven,

2007).

The cellular locations and functions of some barley isoforms,

specifically HvCSLC1 and HvCSLC4, are at this point uncertain.

They are closely related to each other and phylogenetic anal-

ysis places them in a separate group within clade I (group a in

Figure 2) to HvCSLC2 (group c in Figure 2). HvCSLC4 and

HvCSLC1 most likely share the same function, although there

is ambiguous evidence as to whether this is in cellulose or XyG

synthesis. For instance, although the HvCSLC1 transcript pro-

file shows some correlation to genes involved in XyG synthesis

and deposition in the wall, the HvCSLC4 transcript profile does

not and the list of genes most highly correlated to it provides

few clues towards a likely function (Supplemental Table 3).

However, using the experimental tools described in this paper,

it should be possible to distinguish between these two func-

tions for HvCSLC1 and HvCSLC4.

While this paper was under review, ESTs for a fifth HvCSLC

gene, HvCSLC5, were identified in GenBank (accession num-

bers EX582174, EX582178, EX594308, EX594309, EX590401,

EX590402, EX596001, EX596002). These ESTs are from a

pooled tissue sample library and the HvCSLC5 contig is most

similar to HvCSLC2 and thus highly likely to be the barley CSLC

gene predicted to be missing from clade Ic of Figure 2. The

discovery of this gene does not in any way change the con-

clusions drawn here.

METHODS

Plant Materials, cDNA, and BAC Libraries

Grains of Hordeum vulgare L. cultivars. Schooner and Sloop

were imbibed, sown into soil, and grown in the greenhouse

as previously described (Burton et al., 2004). The barley cv.

Schooner suspension cell culture was initiated from a seed-

derived callus and has been maintained at 23�C in the dark

with shaking (114 rpm) in MS8 basal nutrient salt (ICN Bio-

medical) medium (pH 5.8–6.0) supplemented with 3% w/v

sucrose, 2 mg l�1 2,4-dichlorophenoxyacetic acid (2,4-D)

and 1 mg mL�1 mixed cytokinins (333 lM each of 6-c,c-

dimethylallylaminopurine, 6-benzylaminopurine (BAP), and

kinetin (Sigma-Aldrich). Cells were maintained by sub-

culturing 50 mL of cell suspension to a flask containing

100 mL of fresh medium at weekly intervals. Preparation of

a kZAPII cDNA library from barley suspension-cultured cells

is described in Burton et al. (2004). The barley cv. Morex

BAC library was obtained from the Clemson University

Genomics Institute (CUGI; www.genome.clemson.edu).

Identification of HvCSLC cDNAs and Genes

The barley suspension culture cDNA library was probed with

a DNA fragment amplified from a putative HvCSLC EST (acces-

sion no. BE455720). The barley BAC library was screened with

PCR fragments from the 3’ ends of the HvCSLC1 and HvCSLC2

cDNAs. Hybridizations were performed as described in Burton

et al. (2004).

Sequence Analysis and Bioinformatics

DNA sequencing was done at a commercial sequencing facility

(Australia Genomic Research Facility, Australia) and the chro-

matograms analyzed using Sequencher� 3.0 (Gene Codes Cor-

poration, Inc., Michigan, USA).

Arabidopsis, rice, and barley CSLC sequences were used in

iterative searches of public databases, including the now dis-

continued Stanford Cell Wall website, NCBI (www.ncbi.nlm.

nih.gov/), HarvEST (http://harvest.ucr.edu/), GrainGenes

(http://wheat.pw.usda.gov/GG2/index.shtml), BarleyGene In-

dex (http://compbio.dfci.harvard.edu/tgi/plant.html), and Bar-

leyBase (www.barleybase.org) to obtain full-length cDNAs and

identify other members of this gene family in barley. Sequen-

ces were assembled into contigs using either Sequencer� 3.0

(Gene Codes) or ContigExpress, a module of Vector NTI Ad-

vance 9.1.0 (Invitrogen). DNA and protein alignments were

performed using CLUSTALW2 (www.ebi.ac.uk/) or CLUSTALX

v 2.0.9 (Larkin et al., 2007).

CSLC sequences from other species were downloaded

from TAIR (At, www.arabidopsis.org/), TIGR Gene Indices (Os,

http://compbio.dfci.harvard.edu/tgi/plant.html),MIPS(Sb,http://

mips.gsf.de/proj/plant/jsf/sorghum/index.jsp), JGI (Pt, www.jgi.

doe.gov/poplar/; Cg, Pp, Sm, http://genome.jgi-psf.org/), TIGR

Plant Transcript Assemblies (Cg, Mt, Sl, Vv, http://plantta.

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tigr.org/, http://genome.jgi-psf.org/), and NCBI (Zm, www.ncbi.

nlm.nih.gov/).

Phylogenetic trees were constructed from sequence align-

ments using the distance algorithm of Paup 4.0b10 (Swofford,

2000) using the PaupUp v1.0.3.1 graphical interface (Calendini

and Martin, 2005). Default distance settings were used with

the Neighbour Joining clustering option. Trees were bootstrap-

ped with 1000 replicates to assess the robustness of each node.

MEGA4.0 was used to view and edit the resulting trees (Tamura

et al., 2007). Sequence identities and similarities were calculated

using MatGat 2.02 using default settings (Campanella et al.,

2003). Transmembrane domains and protein topologies were

predicted using WoLF PSORT (http://wolfpsort.org/; Horton

et al., 2007).

The Affymetrix Barley1 22K GeneChip reference dataset for

Experiment BB3: ‘Transcription patterns during barley devel-

opment’ (Druka et al., 2006) was downloaded from PLEXdb

(Wise et al., 2007; www.plexdb.org/). The downloaded text file

contained the robust multi-array average (RMA) treatment

means for all probe sets. The RMA treatment includes back-

ground adjustment, normalization, and log2-transformation

of perfect match values calculated from triplicate hybridiza-

tions. These data were imported into Excel 2007, row-to-

column transformed, and the correlation of CSLC and GT

probe sets calculated using the CORREL function.

Quantitative PCR

QPCR of HvCSLC gene expression used a previously described

collection of barley cDNAs (Burton et al., 2004), except for the

suspension cell culture cDNA, which was prepared from the

suspension culture cell line described above 1 week after sub-

culture. QPCR amplification was performed in a total reaction

volume of 20 ll using the method described by Burton et al.

(2008). Relative expression levels were normalized by geomet-

ric averaging of the internal control genes calculated using

geNorm (Vandesompele et al., 2002). Barley genes for glycer-

aldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin,

a-tubulin and elongation factor 1a (EF1a) were used as the in-

ternal controls. Supplemental Table 5 lists the primers used to

amplify the four CSLC genes. Primers for the control genes are

listed in Doblin et al. (2009).

In Situ PCR

In situ PCR was performed on coleoptile and root tissues

harvested 3 d after germination using the method of Koltai

and Bird (2000) with the modifications listed in Doblin et al.

(2009). Synthesis of cDNA was carried out using Thermoscript

RT (Invitrogen, USA) and one of the gene-specific primers

listed in Supplemental Table 5. A typical PCR profile was as

follows: initial denaturation period of 96�C for 2 min, 40 cycles

of 94�C for 30 s, 30 s at an annealing temperature chosen

based on the primer pair being used, 72�C for 2 min. The prod-

ucts of all primer combinations were analyzed by agarose gel

electrophoresis and sequenced to ensure that they gave only

the expected product (data not shown).

HvCSLC Antibody Production

The region of the HvCSLC2 cDNA coding for amino acids

411–466 was amplified by PCR and cloned into the expression

vector pProEX HTa (Invitrogen). The resultant plasmid

(pEVCSLC) was transformed into Escherichia coli BL21(DE3)

cells. Expression was induced in a 500-ml culture by addition

of isopropyl-b-D-thiogalactoside (IPTG) to 1 mM and the pep-

tide enriched using Ni-NTA affinity chromatography (Qiagen)

as described by the manufacturer. After assessment of Ni-NTA-

eluate fractions by SDS–PAGE, fractions containing expressed

peptide were pooled and dialyzed. Antibodies were raised

by intramuscular injection of 500 lg expressed protein with

Freund’s complete adjuvant (Sigma) into New Zealand white

rabbits (Monash University, Melbourne, Australia). Booster

injections were given 21, 49, and 77 d after the initial injection

with the same amount of protein but with Freund’s incom-

plete adjuvant (Sigma) and the rabbits were exsanguinated

94 d after the initial immunization. Affinity purification was

conducted using the method of Brownfield et al. (2007).

Two-Phase Partitioning

Barley suspension culture cells (;20 g) were homogenized

with a glass-to-glass grinder (Tenbroek, Pyrex, USA) in 80 mL

homogenization buffer (50 mM potassium phosphate buffer

(pH 7.5), 20 mM KCl, 0.5 M sucrose, 10 mM DTT and 400 ll

plant protease inhibitor cocktail (Sigma). Disrupted cells were

filtered through Miracloth (Calbiochem) and centrifuged at

10 000 g, 4�C for 10 min and the pellet discarded. The super-

natant, labeled the homogenate (HM) fraction, was centri-

fuged at 125 000 g for 1 h at 4�C and the resultant pellet

was labeled the mixed membrane (MM) fraction.

A PM-enriched fraction was prepared using the two-phase

partitioning method (Larsson et al., 1987). The MM pellet was

re-suspended in a solution composed of 6.5% (w/v) polyethyl-

ene glycol (PEG) 3350 (Sigma), 6.5% (w/v) dextran T500 (DEX)

(Pharmacosmos A/S, Holbaek, Denmark), 3 mM KCl, 0.25 M su-

crose and 5 mM potassium phosphate buffer (pH 7.5) and pro-

cessed according to Natera et al. (2008). The third PEG phase

was diluted two-fold with a buffer composed of 20 mM HEPES,

20 mM KCl, and 0.2 M sucrose and pelleted at 125 000 g, 4�Cfor 45 min. The pellet was re-suspended in the same buffer and

labeled the PM fraction.

The purity of the PM fraction was assessed by Western blots

with antisera to known marker proteins and by TEM (data not

shown; Dwivany, 2003; Yulia, 2006).

Western Blotting

Homogeneous 12% polyacrylamide gels were prepared

and Western blotting performed using standard methods

(Sambrook et al., 1989) with an OSMONIC Nitropure 22 lm ni-

trocellulose membrane (GE Osmonics). After blocking, the

membrane was incubated in the appropriate dilution of pri-

mary antibody in PBS (see below), followed by incubation in

a 1:10 000 dilution of a goat anti-rabbit IgG antibody

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conjugated to either alkaline phosphatise (Sigma) or horserad-

ish peroxidase (Pierce). Detection was performed using Super-

Signal West Pico chemiluminescent substrate (Pierce). The

protein content of all fractions was determined by the BCA pro-

tein assay (Pierce).

The plasma membrane marker Arabidopsis H+-ATPase AHA3

(P-type) antibody was generously provided by Dr Ramon Ser-

rano (Universidad Politecnica de Valencia-CSIC, Valencia,

Spain) and was used at 1:2000 dilution. The Golgi marker

Pisum sativum anti-reversibly glycosylated protein 1 (RGP1,

also known as UDP-arabinopyranose mutase; Konishi et al.,

2007) antibody was kindly provided by Dr Kanwarpal Dhugga,

Pioneer Hi-Bred International Inc., Des Moines, IA, USA, and

was used at a 1:100 000 dilution. Naser Farrokhi (ACPFG, Uni-

versity of Adelaide, Australia) kindly provided the IgG-purified

HvGlyT4 Golgi marker antibody generated towards a bacteri-

ally expressed barley GT47 family glycosyltransferase (Supple-

mental Figures 7 and 8) that was used at a 1:1000 dilution. The

affinity-purified HvCSLC2 antibody, generated as described

above, was used at a 1:1000 dilution.

Immuno-Electron Microscopy (Immuno-EM)

Coleoptile, developing grain, and suspension-cultured cells

(4 d after sub-culture) of barley were fixed and processed

for immuno-EM using the method described in Wilson

et al. (2006). A high-pressure freezing method described in

Brownfield et al. (2008) was also used to prepare the barley

suspension-cultured cells. Thin sections were incubated in a

1:50 dilution of LM15 rat monoclonal antibody (PlantProbes)

in PBS, pH 7.4, containing 1% w/v BSA (+/– competing polysac-

charides) for 1 h at room temperature and then overnight at

4�C. Grids were washed in PBS and then incubated in a 1:20

dilution of goat anti-rat secondary antibody conjugated to

18-nm gold particles (Jackson ImmunoResearch). Sections

then were washed, post-stained, and viewed by TEM as de-

scribed in Burton et al. (2006). Polysaccharide/oligosaccharide

solutions (1 mg mL�1) used in competitive antibody incuba-

tions were carboxymethyl cellulose, laminarin and cellohex-

aose (Sigma), crystalline microcellulose (Merck), barley flour

(1,3;1,4)-b-D-glucan and tamarind seed XyG (Megazyme), and

N. plumbaginifolia-extracted XyG (Sims and Bacic, 1995; Sims

et al., 1996). For sub-cellular location studies, the affinity-

purified HvCSLC2 antibody was used at a 1:10 dilution with

the goat anti-rabbit secondary antibody conjugated to

18-nm gold particles (Jackson ImmunoResearch) used at

a 1:20 dilution.

Accession Numbers

Sequence data from this article can be found in the EMBL/

GenBank data libraries under accession numbers GQ386981

to GQ386984. Accession numbers for other CSLCs are as follows:

Arabidopsis thaliana (TAIR gene id. At3g28180, At4g31590,

At3g07330, At2g24630, At4g07960; Richmond and Somerville,

2000), Oryza sativa (TIGR gene id. Os01g56130, Os09g25900,

Os08g15420, Os05g43530, Os03g56060, Os07g03260; Hazen

et al., 2002), Sorghum bicolor (SbSb02g002090.1,

Sb01g006820.1, Sb09g025260.1, Sb03g035660.1,

Sb07g007890.1; Paterson et al., 2009), Populus trichocarpa

(Poptr1_1:763645, Poptr1_1:578365, Poptr1_1:816437, Poptr1_

1:818429, Poptr1_1:830588; Suzuki et al., 2006), Vitis vinifera

(CAN83466, CAN78456, CAN82135, CAN82493, AM430199),

Medicago truncatula (AC171266), Solanum lycopersicum

(AP009283), Tropaeolum majus (nasturtium) (Cocuron et al.,

2007), Zea mays (van Erp and Walton, 2009), Physcomitrella pat-

ens (DQ898284, DQ898285, DQ898286, EF608235; Roberts and

Bushoven, 2007), Chara globularis (AY995817), Selaginella

moellendorffii (scaffold_92|650930, scaffold_26|411112, scaf-

fold_57|824521, scaffold_0|6575806).

SUPPLEMENTARY DATA

Supplementary Data are available at Molecular Plant Online.

FUNDING

We gratefully acknowledge the Grains Research and Development

Corporation for funding. F.M.D. was funded by a QUE Project Schol-

arship from the Indonesian Government and D.Y. by an Australian

Development Scholarship from the Australian government.

ACKNOWLEDGMENTS

We thank Dr Andrew Harvey (ACPFG, University of Adelaide) for his

assistance with designing primers for QPCR, Dr Filomena Pettolino

(School of Botany, University of Melbourne) for assistance with the

analysis of barley suspension-cultured cell walls, and Ms Cherie

Walsh (School of Botany, University of Melbourne) for her assis-

tance with immuno-EM. No conflict of interest was declared.

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