BC10, a DUF266-containing and Golgi-located type IImembrane protein, is required for cell-wall biosynthesisin rice (Oryza sativa L.)

Yihua Zhou1,†, Shengben Li1,†, Qian Qian2,†, Dali Zeng1, Mu Zhang1, Longbiao Guo2, Xiangling Liu1, Baocai Zhang1,

Lingwei Deng1, Xinfang Liu1, Guanzheng Luo1, Xiujie Wang1 and Jiayang Li1,*

1State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental

Biology, Chinese Academy of Sciences, Beijing 100101, China, and2State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences,

Hangzhou 310006, China

Received 7 June 2008; revised 6 September 2008; accepted 16 September 2008; published online 7 November 2008.

*For correspondence (fax +86 10 64873428; email jyli@genetics.ac.cn).†These authors contributed equally to this work.

Summary

Glycosyltransferases (GTs) are one of the largest enzyme groups required for the synthesis of complex wall

polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have

observable phenotypes, the biological function(s) of most GTs in cell-wall biosynthesis and assembly have

remained elusive. We report here the isolation and in-depth characterization of a brittle rice mutant, brittle

culm 10 (bc10). bc10 plants show pleiotropic phenotypes, including brittleness of the plant body and retarded

growth. The BC10 gene was cloned through a map-based approach, and encodes a Golgi-located type II

membrane protein that contains a domain designated as ‘domain of unknown function 266’ (DUF266) and

represents a multiple gene family in rice. BC10 has low sequence similarity with the domain to a core 2 b-1,6-

N-acetylglucosaminyltransferase (C2GnT), and its in vitro enzymatic activity suggests that it functions as a

glycosyltransferase. Monosaccharide analysis of total and fractioned wall residues revealed that bc10 showed

impaired cellulose biosynthesis. Immunolocalization and isolation of arabinogalactan proteins (AGPs) in the

wild-type and bc10 showed that the level of AGPs in the mutant is significantly affected. BC10 is mainly

expressed in the developing sclerenchyma and vascular bundle cells, and its deficiency causes a reduction in

the levels of cellulose and AGPs, leading to inferior mechanical properties.

Keywords: mechanical strength, glycosyltransferase, cell wall, cellulose, arabinogalactan protein, rice.

Introduction

The plant cell wall is a rigid and dynamic network system

comprising polysaccharides, aromatic substances and gly-

coproteins. Walls are not only central to determining the

mechanical properties of all organs, but also play funda-

mental roles in regulating plant growth and development

and are critical for a wide range of cell functions including

cell adhesion, cell-to-cell communication, and defense

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

et al., 2002; Vogel et al., 2004). The Arabidopsis genome

sequencing project is now complete, and it has been

estimated that, of approximately 27 000 Arabidopsis genes,

approximately 15% are dedicated to the processes of cell-

wall synthesis, modification, assembly and degradation,

and correct regulation of these processes during growth and

development (Carpita et al., 2001). Therefore, the cell wall is

one of the most complicated and the least-understood plant

cell structures. A powerful approach to probe the functions

of individual components of cell walls is through the iden-

tification and characterization of mutants (Reiter et al.,

1997). Mutants defective in mechanical strength, a promi-

nent physical property of the cell wall that depends upon the

structure of various wall polymers and how they interact,

446 ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2009) 57, 446–462 doi: 10.1111/j.1365-313X.2008.03703.x

have been proven to be valuable for identifying genes

involved in the biogenesis and modification of cell walls (Li

et al., 2003b; Tanaka et al., 2003; Taylor et al., 2003; Zhong

et al., 2002). Although much progress has been made in this

area, the molecular mechanisms of how the wall polysac-

charides and glycoproteins are synthesized, deposited and

regulated remain to be elucidated (Cosgrove, 2005; Farrokhi

et al., 2006; Lerouxel et al., 2006; Lytovchenko et al., 2007;

Somerville, 2006; Zhong and Ye, 2007).

Wall polysaccharides and glycoproteins have tremendous

structural complexity, and therefore plants require large

families of glycosyltransferases (GTs) to facilitate their

biosynthesis. Based on sequence similarities and the exis-

tence of certain motifs, hydrophobic clusters and their

catalytic specificity (Rosen et al., 2004; Ross et al., 2001),

GTs are divided into 91 families (http://www.cazy.org/CAZY/).

Among these, both cellulose synthase active subunits

(CESA) (Somerville, 2006) and CESA-like proteins (CSL) are

classified into the GT2 family; the former are responsible for

synthesizing cellulose and the latter are believed to be

responsible for the formation of glycan backbones in the

endoplasmic reticulum (ER) or Golgi apparatus (Burton

et al., 2006; Dhugga et al., 2004). The remaining GTs

distributed in various GT families are usually Golgi-localized

type II integral membrane proteins (Geshi et al., 2004),

which synthesize the backbones of pectin (Bacic, 2006) and

xylan (Faik et al., 2000; Madson et al., 2003; Pena et al.,

2007), or add side chains to polysaccharides (Persson et al.,

2007; Sarria et al., 2001). In addition to polysaccharides,

most wall proteins are also highly glycosylated, and some

have been proposed to interact with the major carbohydrate

components of the wall to form a complex network. Of

these, arabinogalactan proteins (AGPs), located at the outer

leaflets of the plasma membrane or secreted into the cell

wall, are regarded as critical components in maintaining

both the physical and biological functions of walls (Takeda

and Fry, 2004). AGPs often contain >90% carbohydrate,

which is essential for their biological functions (van Hengel

et al., 2001; Wu et al., 2000). Therefore, a large number of

GTs are probably required to synthesize the highly complex

sugar chains of AGPs. However, no enzymes responsible for

the glycosylation of AGPs have yet been identified or

characterized (Gaspar et al., 2001).

In Arabidopsis, more than 400 putative GT genes have

been identified based on their sequences (http://afmb.

cnrs-mrs.fr/CAZY/) (Scheible and Pauly, 2004). However, a

recent survey of the Arabidopsis and rice genomes has

increased that number, and indicates that the proportion of

these enzymes in plant genomes is greater than that in

human or other fully sequenced genomes (Coutinho et al.,

2003). It is likely that each linkage in polysaccharides and

glycoproteins requires the action of a distinct glycosyl-

transferase, leading to the prediction that multi-cellular

organisms contain hundreds of GTs (Henrissat and Davies,

2000). However, little is known of their molecular basis of

reaction specificity, including donor selectivity and acceptor

preference, or their biological functions (Pagny et al., 2003;

Wenderoth and von Schaewen, 2000). Glycosylation in

animal systems most frequently occurs as a post-transla-

tional modification of proteins rather than in polysaccharide

biosynthesis, the exception being hyaluronic acid. Unlike

plants, only about 200 human genes encode proteins related

to the biosynthesis, function and degradation of sugar

chains. For example, core 2 b-1,6-N-acetylglucosaminyl-

transferase (C2GnT), which forms the core 2 branched

O-glycan, is a glycosyltransferase that is involved in bio-

synthesis of mucin-type glycoproteins (O-glycans) (Fukuda,

1996). Generally, GTs identified in animals show low

sequence identity to those in plants, indicating significant

variation between plants and animals.

The enormous number of sequences available in

databases makes accurate sequence annotation a great

challenge, especially when there are large numbers of

functionally uncharacterized families. Many proteins that

contain domains of unknown function (DUF) have been

predicted in plants (rice and Arabidopsis) and other eukary-

otic genomes (Bateman et al., 2004), but their biological

functions remain to be elucidated. Here, we report the

isolation of a brittle culm mutant (bc10) from rice, cloning of

the gene responsible for the mutant phenotype, and molec-

ular characterization of BC10, which is a DUF266-containing

Golgi-localized glycosyltransferase that perturbs the levels

of AGPs and cellulose, thereby affecting the mechanical

properties of rice plants.

Results

bc10 plants are defective in mechanical strength

Culms and leaves are two major aerial organs of the rice

plant. Sclerenchyma cells and vascular bundles within

these two organs provide mechanical support for body

of the rice plant. To understand the mechanism that regu-

lates the mechanical properties of cereal plants, we sys-

tematically isolated and characterized rice mutants that

show the brittle culm (bc) phenotype (Li et al., 2003b). bc10

was isolated from a japonica cultivar Huang Jin Qin

(Figure 1). The prominent phenotype of bc10 plants is their

decreased mechanical strength in aerial organs, resulting in

brittle culms and leaves that can be broken easily by

bending. To accurately describe the phenotype, we quan-

titatively compared the breaking forces of 140-day-old bc10

and wild-type plants. Figure 1(a,b) shows that the forces

needed to break the bc10 culms and leaves were reduced to

25 and 80%, respectively, compared to the wild-type. The

extension lengths, which reflect the elasticity of plant

tissues, were also significantly decreased in mutant culms

and leaves (Figure 1a,b). In addition, the brittle phenotype

BC10 regulates mechanical strength in rice 447

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is developmentally regulated, and the mutation in BC10

also affects plant growth and development (Figure 1c,d). At

the seedling stage, the mutant plant often has yellowish

leaves and fewer and shorter roots (Figure 1c). At the

mature stage, especially after heading, bc10 aerial organs

become more brittle and show additional morphological

abnormalities, including a significant decrease in plant

height and tiller number (Figure 1d). Therefore, the bc10

mutation causes not only a reduction in mechanical

strength, but also abnormalities in the growth and

development of rice plants.

The bc10 mutation results in altered cell walls in

mechanical tissues

To investigate why the mutant plant has a significant

decrease in mechanical strength, we examined the ana-

tomical features of cells around the vascular bundles and

under the epidermal layer, which is where sclerenchyma

cells differentiate in rice plants. Cross-sections of culms and

leaf sheaths showed that the wall thickness of bc10

mechanical cells is apparently reduced compared with that

of the wild-type (Figure 2a,b). Longitudinal sections of culms

displayed a slight reduction in cell length of the mutant

parenchyma cells (Figure 2c,d), indicating that the

decreased plant height in bc10 plants is probably caused by

a reduction in the cell length of culms. Transmission

electron microscopy analysis demonstrated that the wall

thicknesses of sclerenchyma and fiber cells in bc10 were

reduced (Figure 2e–j). These results suggest that the

reduced mechanical strength in bc10 results from decreased

cell-wall thickness in the secondary walls, which is consis-

tent with the phenotype that brittleness occurs when the

secondary walls are developing.

Map-based cloning of BC10

To understand the molecular mechanism responsible for the

bc10 phenotype, we used a map-based cloning approach to

isolate the BC10 gene. We crossed the mutant with ZF802, a

wild-type polymorphic indica variety, to generate a F2

mapping population. Genetic analysis placed the BC10 locus

between the molecular markers RM7349 and E3528 on

chromosome 5, and was further localized to within a 51 kb

DNA segment covered by two BAC clones, AC084817 and

AC087553 (Figure 3a), using the molecular markers listed in

Table S1.

Annotation analysis of this 51 kb DNA sequence identified

10 putative open reading frames (ORFs). To determine which

one carries the bc10 mutation, we sequenced all of the

putative ORFs and compared them with those of the wild-

type. A 17 bp deletion was found in one putative ORF,

Os05g07790 (Figure 3b), and this was confirmed by com-

paring the corresponding DNA fragment sizes amplified

from bc10 and wild-type plants (Figure 3d). Comparison

between the complementary and genomic DNA sequences

of this ORF revealed that the 17 bp deletion covers the

junction of the second intron and the third exon (Figure 3c),

suggesting that the mutation may disrupt RNA splicing.

Indeed, the 17 bp deletion in the mutant disrupts maturation

(a)

(c) (d)

(b) Figure 1. Phenotypes and physical properties of

wild-type and bc10 mutant plants.

(a) Triple measurements of force and extension

length while the culms of wild-type and bc10

mutant plants were broken.

(b) Triple measurements of force and extension

length while the leaves of wild-type and bc10

mutant plants were broken.

(c) Fourteen-day-old seedlings of bc10 and the

wild-type, showing yellowish leaves and shorter

and fewer roots in bc10. Scale bar = 1.5 cm.

(d) Plants at the mature stage, showing reduced

plant height and tiller number in bc10. Scale

bar = 13 cm.

448 Yihua Zhou et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

of its RNA, leading to an additional 105 bp segment in the

bc10 transcript as demonstrated by sequencing and size

comparison of the cDNA products between bc10 and wild-

type plants (Figure 3c,e). Therefore, bc10 results from the

17 bp deletion, causing a frameshift and producing a

premature translational product (Figure 3c).

To confirm that the deletion was responsible for the

mutant phenotype, plasmids pBC10F containing the entire

ORF and pBC10T carrying a truncated coding region

(Figure 3f) were constructed and introduced into bc10 plants

by Agrobacterium tumefaciens-mediated transformation.

All 18 independent pBC10F transgenic lines completely

rescued the mutant phenotypes (Figure 3g,h), but the 12

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

Figure 2. Anatomical features of sclerenchyma and bundle sheath fiber cells

in the wild-type and bc10 plants.

(a, b) Cross-sections of culms of wild-type (a) and bc10 (b). Scale

bars = 40 lm.

(c, d) Longitudinal sections of culms, showing a slight reduction in the length

of bc10 parenchyma cells (d) compared with those of the wild-type (c). Scale

bars = 40 lm.

(e)–(h) TEM micrographs of sclerenchyma and fiber cells, showing thinner

walls in bc10 (f, h) compared with the wild-type (e, g). Scale bars = 2 lm.

(i, j) Quantitative comparison of the wall thickness of sclerenchyma (i) and

bundle fiber cells (j) between the wild-type and bc10 (mean of 20 cells � SD).

(a)

(b)

(c)

(d)

(g) (h)

(e) (f)

Figure 3. Map-based cloning and identification of BC10.

(a) The bc10 locus was mapped to a 51 kb region on chromosome 5. White

boxes represent exons and black boxes represent introns.

(b) Partial BC10 DNA sequence corresponding to the second intron (under-

lined). The 17 bp deletion is indicated in bold italics.

(c) Partial transcript sequence of bc10. The fragment sequence resulting from

the unspliced second intron is underlined, and the stop codon is indicated by

an asterisk.

(d, e) The difference between bc10 and the wild-type is shown by the size of

amplified genomic DNA (d) and cDNA (e) fragments.

(f) Complementation constructs. The constructs pBC10F or pBC10T contain

the entire or partial BC10 gene, respectively.

(g, h) Phenotypes of 20- (g) and 70-day-old (h) plants. The bc10 plants are

shown on the left. Scale bars = 1.5 cm (g) or 6 cm (h).

BC10 regulates mechanical strength in rice 449

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pBC10T lines failed to complement the bc10 mutant, and we

therefore conclude that the Os05g07790 is the BC10 gene.

BC10 expression is developmentally regulated

Real-time PCR analysis revealed that BC10 is expressed in all

organs examined, with the highest expression in culms

(Figure 4a). The cellular expression pattern of BC10 was fur-

ther investigated by RNA in situ hybridization, showing that

BC10 is mainly expressed in vascular bundles and the

sub-epidermal regions of leaf sheaths and stems where

sclerenchyma cells are developing (Figure 4b–g). Strong

hybridizationsignalswerealsoobserved in mechanical tissues

of young organs, including young leaf sheaths (Figure 4c),

stems (Figure 4d,e) and tiller buds (Figure 4f), indicating that

BC10 expression is temporally and spatially regulated.

BC10 encodes a DUF266 protein that represents

a large gene family

The BC10 full-length cDNA is 1995 nt long, with an ORF of

1200 nt that encodes a protein of 399 amino acids with a

predicted molecular mass of 45 169 Da. A search using Pfam

(Finn et al., 2006) revealed only one predicted domain,

domain of unknown function 266 (DUF266), in the BC10

protein; this is a plant-specific domain with unknown func-

tion. The bc10 mutation occurs within the DUF266 domain,

resulting in a truncated protein that may disrupt the function

(a)

(b) (c)

(d) (e)

(f) (g)

Figure 4. Expression patterns of BC10 revealed

by real-time PCR and RNA in situ hybridization in

transverse sections of wild-type rice plants.

(a) Expression of BC10 in various organs. The

expression levels are given as a percentage of

that of actin. The results are means � SD of

triplicate assays.

(b)–(g) RNA in situ hybridization in transverse

sections of wild-type plants, showing strong

expression of BC10 in the developing scleren-

chyma and vascular bundle cells. (b) Leaf sheath

with young flower organs. (c) A magnified sec-

tion from (b). (d) Young stem and leaf sheath. (e)

A magnified section of young stem. (f) Stem

section with a tiller bud, showing stronger

signals in young tissues than in older ones. (g)

Background control, hybridized with a sense

probe. V, vascular bundles; Sc, sclerenchyma

cells. Scale bars = 400 lm (b) or 70 lm (c–g).

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ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

of DUF266 (Figure 5a). A BLASTX search for BC10 homologs

identified 18 and 23 DUF266-containing proteins in the Oryza

sativa (japonica) and Arabidopsis thaliana genomes,

respectively. Phylogenetic analysis revealed that these BC10

homologs are clustered into three major clades, implying

that they may have related but distinct functions (Figure 5b).

The closest homolog of BC10 is an Arabidopsis protein,

At5g14550, with 82% sequence identity. RT-PCR amplifica-

tion of total RNAs isolated from various organs of rice

revealed that the BC10-homologous genes tested are

transcriptionally active, with distinct expression patterns

(Figure 5c), suggesting that these DUF266-containing genes

are transcriptionally regulated and may play similar or

partially redundant roles in plant growth and development.

To further confirm whether the DUF266 domain is only

present in plants, we performed profile search for all

proteins in the NCBI nr database (ftp://ftp.ncbi.nih.gov/

blast/db/FASTA/nr.gz). The complete DUF266 domain was

only found in predicted proteins from plant species, such as

Arabidopsis, rice (Oryza sativa), spruce (Picea sitchensis),

poplar (Populus trichocarpa), clover (Medicago truncatula),

grape (Vitis vinifera) and moss (Physcomitrella patens),

indicating that DUF266 is very likely a plant-specific domain.

BC10 is a Golgi-localized type II integral membrane protein

Analysis using TMHMM2.0 (http://www.cbs.dtu.dk/services/

TMHMM-2.0/) showed that BC10 is a type II membrane

(a)

(c)

(b)

Figure 5. Prediction of the primary sequence, phylogenetic tree, and RT-PCR analyses of BC10 homologs.

(a) Deduced amino acid sequence of BC10. Numbers on the left refer to the positions of amino acid residues. The N-terminal cytosolic tail is indicated in bold italics,

the transmembrane domain is shown in white letters on a grey background, and the DUF266 domain is underlined.

(b) Phylogenetic analysis of BC10 homologs from rice and Arabidopsis. The numbers at each node represent the bootstrap support (percentage), and the scale bar is

an indicator of genetic distance based on branch length.

(c) RT-PCR analyses of BC10 homologs, indicating that the BC10 homologs tested are transcribed with various organ patterns.

BC10 regulates mechanical strength in rice 451

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protein that consists of a short cytoplasmic N-terminus

followed by a single transmembrane helix and a long

non-cytoplasmic C-terminus containing the DUF266 domain

(Figure 6a). To determine the membrane topology of BC10,

we generated transgenic rice plants that over-express a

BC10–GFP fusion protein in the bc10 background. The mu-

tant phenotypes were fully rescued in the transgenic plants

(data not shown), indicating that the BC10–GFP fusion pro-

tein functions as a native BC10. Unlike dynamin, a peripheral

membrane protein that may be dissolved by high salt (1 M

NaCl) and alkaline pH (pH 11), the BC10–GFP fusion protein

was partially extracted from the microsomal fraction of

transgenic plants using detergent (Triton X-100) (Figure 6b),

indicating that BC10 is an integral membrane protein. Fur-

thermore, immunoblotting analysis revealed that the BC10–

GFP fusion protein was sensitive to proteinase K treatment

only when microsomes were pre-treated with detergent

(Figure 6c), suggesting that the C-terminus of BC10 is lumi-

nal and that BC10 has type II membrane topology.

Most type II membrane proteins are associated with the

ER and/or Golgi apparatus. To ascertain the actual subcel-

lular location of BC10, we expressed the BC10–GFP fusion in

tobacco Bright Yellow 2 (BY2) protoplasts, and found that

the fluorescent signal of BC10–GFP displayed a punctate

pattern (Figure 7a,b), suggesting that BC10 may be targeted

to certain organelles. Co-localization of BC10 with various

organelle-specific markers in BY2 protoplasts demonstrated

that the punctate pattern of BC10–GFP was identical to that

of the red fluorescent protein (RFP)-tagged Golgi marker

sialyltransferase (SialT) (Figure 7e–h); no such match was

found with any other organelle-specific markers, including

the endoplasmic reticulum marker (Figure 7i–l) and the

mitochondrial marker (Figure 7m–p). Therefore, BC10 is a

Golgi-localized type II membrane protein. In addition, we

also showed that a polypeptide consisting of the first 44

amino acid residues at the N-terminus of BC10 is essential

for its subcellular localization and biological function

(Figure S1).

BC10 may function as a glycosyltransferase

As DUF266 is the only domain identified in BC10, it may be

critical for its function. A BLASTP search of BC10 protein

against the NCBI nr database failed to reveal any homolog

with known functions. However, a second iteration of a PSI-

BLAST search identified b-1,6-N-acetylglucosaminyltrans-

ferase (C2GnT), a glycosyltransferase of animals (Van den

Steen et al., 2000), as a homolog, with an E-value less than

1e-25. Moreover, analyses using the Conserved Domain

Architecture Retrieval Tool (CDART) (Geer et al., 2002) and

the FUGUE server showed that the structure similarity

between BC10 and C2GnT is with high certainty

(ZSCORE = 11.72; a ZSCORE > 6 indicates 99% prediction

confidence). The prediction was further supported by a

secondary structure analysis using the PSIPRED Protein

Structure Prediction Program, showing that the top hits (P

value = 1e-09) were also C2GnT (Figure S2a). We therefore

measured the C2GnT enzyme activity of BC10 by expressing

BC10 in Chinese hamster ovary (CHO) cells, which have an

extremely low background of enzyme activity. The enzyme

activity assay with human C2GnT substrates demonstrated

that the expressed BC10 showed a low but significant GT

activity (Table 1), suggesting that BC10 is a glycosyltrans-

ferase. Compared with the positive control, which showed

much higher enzyme activity, the low enzyme activity of

BC10 is probably due to the lack of authentic acceptor or

donor substrates in the assay system. This result was further

strengthened by an in-depth structural comparison between

the published crystal structure of C2GnT (Pak et al., 2006)

and the predicted three-dimensional structure of BC10

(a)

(b)

(c)

Figure 6. BC10 is a type II membrane protein.

(a) BC10 is a type II membrane protein as predicted by the TMHMM2.0

program, showing a transmembrane helix domain between amino acid

residues 20 and 42, a short N-terminal region located on the cytoplasmic side

of the membrane (inside), and a long C-terminal region containing the

DUF266 domain on the non-cytoplasmic side of the membrane (outside).

(b) Supernatant (S) and pellet (P) fractions of the ultracentrifuged microsomal

fractions from BC10–GFP transgenic plants resuspended in the control buffer,

1 M NaCl, alkaline buffer (pH 11) or 1% Triton X-100. Each fraction was probed

with anti-GFP and dynamin antibodies.

(c) Microsomal fractions from BC10–GFP transgenic plants were incubated in

the presence (+) or absence ()) of Triton X-100 and proteinase K and then

subjected to immunoblot analysis with anti-GFP antibodies. LC, loading

control comprising a Coomassie blue-stained gel of the immunoblot.

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ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

(http://robetta.bakerlab.org/ and http://zhang.bioinformatics.

ku.edu/I-TASSER/). As shown in Figure S2(b,c), two of three

structurally conserved amino acid residues (Glu287 and

Lys376) of BC10 are identical to those of C2GnT (Glu320

and Lys401), but the third conserved residue (Lys345) of

BC10 does not match that of C2GnT (Arg378). These three

residues are believed to be pivotal for its substrate binding

and catalytic activity, implying that the substrates or activity

of BC10 might be different from those of C2GnT.

We further carried out a sequence comparison of DUF266-

domain polypeptides between BC10 and its homologs in rice

and Arabidopsis. As shown in Figure S3, two of the three

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

Figure 7. Subcellular localization of BC10–GFP in tobacco BY2 protoplast cells.

(a, b) A BY2 cell expressing BC10–GFP alone (a) and its DIC image (b), showing a punctate signal pattern.

(c, d) A BY2 cell expressing GFP alone (c) and its DIC image (d), showing fluorescent signals in nucleus, membrane and cytoplasm.

(e)–(h) BY2 cells expressing BC10–GFP (e), the Golgi-localized SialT–RFP (f), a merged image (g), and the DIC image (h), showing that the signals of BC10–GFP and

SailT–RFP are superimposable.

(i)–(l) BY2 cells expressing BC10–GFP (i), the endoplasmic reticulum marker ER-mCherry (j), a merged image (k), and the DIC image (l).

(m)–(p) BY2 cells expressing the mitochondria marker GFP–MT (m), BC10–RFP (n), a merged image (o), and the DIC image (p).

Scale bars = 15 lm (a,b,m–p), 10 lm (c–h) or 5 lm (i–l).

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conserved amino acid residues essential for substrate

binding and enzyme activity of C2GnT are unchanged in

all the members. However, the third conserved residue

showed variation in more than half of the members. These

results suggest that this group of proteins may function as

GTs with different donor or acceptor substrates from those

of human C2GnT.

Cellulose contents of total and fractionated cell walls are

decreased in bc10

The cellular and morphological phenotypes suggested that

the cell-wall composition in the mutant plant may also be

altered. We therefore compared the cellulose and lignin

contents between bc10 and wild-type culms. Compared

with the wild-type, the cellulose content of bc10 was

decreased by approximately 30%, but the lignin content

was increased by approximately 20% in bc10 (Table 2).

These results were further confirmed by quantitative

analysis of the neutral monosaccharide and uronic acid

composition in alcohol-insoluble residues of bc10 and

wild-type culms. As shown in Table 2, glucose content was

reduced by approximately 20% in bc10 culms, but the

contents of xylose, arabinose and galactose were slightly

increased, suggesting that the BC10 mutation has an effect

on cell-wall biosynthesis.

To understand the detailed changes of polysaccharides in

bc10, we sequentially extracted alcohol-insoluble residues

from walls under conditions that allowed individual frac-

tions enriched in a specific type of wall polymer to be

obtained (see Experimental procedures). The sugar compo-

sition of the five fractions, including cellulose, was then

analyzed by GC–MS. As shown in Table 3, individual

monosaccharides in fractions of pectin extracted using

EGTA-Na2CO3 or digested with endopolygalaturonase plus

pectin methylesterase were not significantly altered in bc10.

Similar results were also obtained for the hemicellulose

fraction, although the contents of some sugars, such as

xylose, showed a slightly increase in bc10 extracts obtained

using 1.0 and 4.0 N KOH. We further investigated the

oligosaccharides of xylan and xyloglucan using MALDI-

TOF by digesting them with xylanase and xyloglucanase,

but found no significant differences between them (Fig-

ure S4). However, the glucose content in the cellulose

fraction was significantly reduced in bc10 (Table 3), consis-

tent with the measured cellulose contents (Table 2). Because

BC10 shows similarity to C2GnT, which adds GlcNAc to

glycan, we determined the N-acetyl-glucosamine content in

the above fractions and found no significant reduction,

suggesting that the substrates of BC10 might be different

from those of C2GnT. Therefore, the mutation in BC10

causes a significant decrease in cellulose content. The

increase in lignin (Table 2) and minor alterations in non-

cellulosic sugars (Tables 2 and 3) probably result from

unidentified compensatory regulations responding to cellu-

lose reduction (Li et al., 2003a; Manfield et al., 2004).

Table 1 Assay for C2GnT enzyme activity of BC10 in CHO cells

Transfectants Enzyme activitya

C2GnT cDNA 9625 � 243BC10 cDNA 101 � 14Denatured crude extracts <10Empty vector <10No transfection <10

aThe enzyme activity (pmol mg)1 protein h)1) is the mean of triplicatemeasurements � SE.

Table 2 Comparison of cell-wall components between wild-type and bc10 culms

Sample Glc Xyl Man Gal Ara Rha Uronic acids Cellulose Lignin

Wild-type 395.0 � 6.5 49.2 � 0.7 7.49 � 0.1 14.1 � 1.0 10.3 � 0.2 4.74 � 0.1 1.38 � 0.09 236.2 � 2.5 144.9 � 0.8bc10 316.0 � 6.3 62.8 � 1.0 7.72 � 0.2 16.6 � 0.6 16.7 � 0.3 5.58 � 0.2 1.51 � 0.07 160.6 � 6.7 177.2 � 5.1

The results are means � SE of five independent assays. Each wall component was calculated as mg g)1 of alcohol-insoluble cell-wall residue.

Table 3 Glycosyl composition of fractionated cell walls fromwild-type and bc10 culms

Cell wallfraction Rha Fuc Ara Xyl Man Glc Gal N-GlcA Total

PectinWild-type 0.8 0.0 4.6 7.7 4.2 110.0 7.1 0.8 135.1bc10 0.5 0.0 4.8 7.9 3.9 111.5 7.3 0.7 136.5

EGTA/Na2CO3

Wild-type 0.1 0.0 1.5 0.8 0.7 1.2 2.2 0.2 6.6bc10 0.1 0.0 1.4 0.7 0.7 0.9 2.3 0.3 6.3

1 N KOHWild-type 0.2 0.2 6.2 24.4 0.3 3.7 1.4 0.1 36.5bc10 0.3 0.1 9.6 36.8 0.3 4.0 2.0 0.2 53.2

4 N KOHWild-type 0.1 0.1 1.0 4.1 0.2 4.1 0.4 0.0 9.9bc10 0.1 0.1 1.8 5.8 0.3 3.4 0.8 0.1 12.4

CelluloseWild-type 318.2 318.2bc10 202.5 202.5

TotalWild-type 1.2 0.2 13.3 37.1 5.4 437.1 11.1 1.1 506.3bc10 1.0 0.2 17.5 51.2 5.1 322.2 12.3 1.3 410.8

The results are means of three independent assays (mg g)1 ofalcohol-insoluble residues). The variance is not shown but is <0.2.

454 Yihua Zhou et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

Formation of AGPs is impaired in bc10

Given that the cellulose content is significantly decreased in

bc10 and BC10 is predicted to function as a type II membrane

glycosyltransferase, we therefore analyzed glycoprotein and

polysaccharide contents using seven monoclonal antibodies

(Appendix S1), which enabled us to compared the carbo-

hydrate epitopes of polysaccharides and glycoproteins

between wild-type and mutant plants. As shown in Figure 8,

immunolabeling of culm sections with these antibodies

indicated an apparent decrease in AGP epitopes recognized

by LM2, LM6 and JIM13 in the bc10 plants. AGPs recognized

by LM2 (Figure 8a–d) and JIM13 (Figure 8e–h) were found in

all cells including vascular bundles, sclerenchyma and

parenchyma cells in the wild-type, but were significantly

decreased in mutant plants (Figure 8a–h and Table S2). A

difference in signal intensity when using LM6 was only de-

tected in the vascular bundles and sclerenchyma cells (Fig-

ure 8i–l and Table S2). In contrast, JIM7 equally labeled the

pectin in wild-type and mutant plants (Figure 8m,n and Ta-

ble S2). Staining with b-glucosyl Yariv reagent (bGlcY), a

chemical that is widely used to localize and quantify AGPs,

also revealed a reduction in AGPs in hand-cut stem sections

of the bc10 mutant plant, as indicated by a substantial

diminution in the reddish-brown color (Figure 8o, p), but no

difference was found using b-mancosyl Yariv reagent

(bManY), an inactive analog of bGlcY (data not shown). To

investigate the distribution of AGPs in cell walls, immuno-

gold analysis was performed, and revealed that the bc10

sclerenchyma and parenchyma cell walls were less labeled

by LM2 and JIM13 (Figure 9a–h and Table S2), but a less

labeled pattern was only observed in sclerenchyma cell

walls in mutants probed with LM6 (Figure 9i,j and Table S2),

results that are consistent with those observed in the

immunofluorescent analyses. These results indicate that the

deficiency in BC10 affects AGP abundance in cell walls.

To confirm the immunological analyses, we compared the

abundance and composition of AGPs between the mutant

and wild-type plants by measuring AGP contents and their

monosaccharide composition. As shown in Table 4, the

content of AGPs in bc10 was decreased by 70% compared

with that in the wild-type. The reduced AGP content results

in a significant reduction in total sugars, especially

arabinose and galactose, the two major sugars of AGPs

(Table 4).

Discussion

The rapid increase in the availability of genomic resources

and improvement in molecular techniques have led to

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

Figure 8. Immunohistochemical localization of AGP in culm sections of wild-type and bc10 plants.

(a)–(l) Immunolocalization using LM2 (a–d), JIM13 (e–h) or LM6 (i–l). The bc10 plant shows weak fluorescent signals in all cells including the vascular bundle,

sclerenchyma (b, f) and parenchyma (d,h) cells visualized with antibodies LM2 and JIM13. However, immunostaining with antibody LM6 showed weaker signals in

vascular bundles and sclerenchyma cells (j), but not in parenchyma cells (l).

(m, n) Immunolocalization of pectin with JIM7, showing no detectable differences between bc10 (n) and the wild-type (m).

(o, p) Staining of hand-cut stem sections with bGlcY, showing a substantially stronger signal in the wild-type (o) than in bc10 (p).

Scale bars = 70 lm (a, b, e, f, i, j), 25 lm (c, d, g, h, k, l), 36 lm (m, n) or 160 lm (o, p).

BC10 regulates mechanical strength in rice 455

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

numerous GTs being identified through bioinformatics and

an increasing number being predicted to play roles in cell-

wall metabolism. However, only a small number have been

functionally characterized (Somerville et al., 2004). In this

study, we demonstrate that a loss-of-function mutation of

BC10, a DUF266-containing Golgi-localized protein that

represents a large family of proteins in higher plants and

may function as a glycosyltransferase, disrupts the levels of

cellulose and arabinogalactan proteins in cell walls. These

findings are important for understanding cell-wall biosyn-

thesis in monocotyledonous plants.

BC10 encodes a Golgi-localized DUF266-containing protein

belonging to a superfamily

The plant Golgi apparatus is a critical cytoplasmic organelle

involved in cell-wall polysaccharide and glycoprotein bio-

synthesis and modification (Andreeva et al., 1998). Based

upon bioinformatic analysis, microscopic co-localization

with organ-specific markers and biochemical experiment,

we conclude that BC10 encodes a 399 amino acid long Golgi-

localized type II membrane protein. Identification of the first

44 amino acid residues of BC10 comprising the N-terminal

cytosolic tail (CT) and transmembrane (TM) domains as the

Golgi targeting signal (Figure S1), and characterization of

the mutant’s inferior mechanical properties strongly suggest

that BC10 functions in cell-wall biosynthesis.

Proteins associated with cell-wall biosynthesis and mod-

ification are generally believed to be clustered into families

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

Figure 9. Immunogold labeling of sclerenchyma and parenchyma cell walls of wild-type and bc10 plants.

(a)–(d) Labeling with the LM2 monoclonal antibody, showing that the bc10 plant has fewer particles in sclerenchyma (b) and parenchyma (d) cell walls.

(e)–(h) Labeling with the JIM13 monoclonal antibody, showing that the bc10 plant has fewer particles in sclerenchyma (f) and parenchyma (h) cell walls.

(i, j) Labeling with the LM6 monoclonal antibody, showing that the bc10 plant has fewer particles only in sclerenchyma (j) cell walls.

(k, l) Labeling with the JIM7 monoclonal antibody, showing equal labeling in wild-type (k) and mutant (l) cell walls.

Scale bars = 500 nm (a–j) or 1 lm (k, l).

Table 4 Comparison of the abundance of AGPs and sugar compo-sition in bc10 and wild-type plants

Samples Wild-type bc10

AGPs 69.8 � 2.6 20.1 � 1.1Total sugar 40.3 � 2.3 17.9 � 1.0Gal 24.0 � 2.3 5.7 � 0.3Ara 14.5 � 1.1 5.5 � 0.3

The AGP content was measured and calculated as lg g)1 fresh weightof tissues. The results are means � SE of three independent assays.

456 Yihua Zhou et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

(Sarria et al., 2001; Scheible and Pauly, 2004). For example,

the Arabidopsis genome contains a total of nine sequences

with high homology to AtFUT1 (Sarria et al., 2001) and eight

genes with significant similarity to GalT (Edwards et al.,

1999, 2004; Keegstra and Raikhel, 2001). BC10 contains a

DUF266 domain that does not display homology with any

domain of known function. Intensive genomic surveys in the

NCBI and pFam databases indicated that this family of

proteins is plant-specific and ubiquitous in higher plants,

with 18 homologs in rice, 23 in Arabidopsis, and some in

poplar, spruce, grape, etc.

BC10 functions as a glycosyltransferase

As the first identified mutant in the family, bc10 provides an

intriguing opportunity to unravel functions of the BC10

family genetically and biologically. Bioinformatic studies

involving CDART analysis and the prediction of protein

secondary and crystal structures revealed that BC10 shows

some similarities to the animal glycosyltransferase C2GnT,

consistent with a previous prediction that At5g11730 and

At3g21310, two BC10 homologs in Arabidopsis, may func-

tion as GTs related to PF02485 (C2GnT) based on pFam

bioinformatic analysis (Egelund et al., 2004). BC10 shares

two of the three essential conserved amino acid residues

and three of nine conserved Cys residues that are important

for binding substrates and maintaining an active confor-

mation of C2GnT (Pak et al., 2006; Yang et al., 2003). On the

other hand, sequence variations between BC10 and C2GnT

may also reflect their activity differences. This speculation is

substantiated by the low but significant GT enzyme activity

of BC10 in the in vitro assay system. Further studies are

required to understand the biochemical nature of BC10,

especially donor selectivity and acceptor preference, which

has proven to be a real challenge for most GTs in higher

plants (Farrokhi et al., 2006; Keegstra and Raikhel, 2001;

Zhang, 2003).

GTs involved in cell-wall biosynthesis have been grouped

into more than 70 families (http://www.cazy.org) based on

their sequence similarities or sometimes on the existence of

certain motifs, hydrophobic clusters and catalytic specifici-

ties (Rosen et al., 2004). All the BC10 homologs in the current

databases, which share common features including a length

of 340–420 amino acid residues, the presence of a DUF266

domain and prediction as type II membrane proteins, and

should thus be grouped into a separate sub-family or family

of GTs. However, which GT family does BC10 and its

homologs belong to? Based on several lines of experimental

and bioinformatic evidence, we categorize BC10 and its

homologs into family GT14 (http://www.cazy.org). First,

bioinformatic studies, especially the structural comparison

between BC10 and C2GnT using the CDART and PSIPRED

programs, have shown that BC10 is homologous to C2GnT,

which is a GT14 member. Second, the BC10 protein has low

GT enzymatic activity using a C2GnT assay system. Third,

some BC10 homologs, such as At5g11730 and At3g21310,

show a close association with GT14 (Egelund et al., 2004).

However, when BC10, C2GnT and some randomly selected

members from individual GT families were analyzed for their

phylogenetic relationship, BC10 did not cluster in the same

clade as C2GnT and other GT14 proteins (data not shown),

suggesting the possibility that BC10 and its homologs might

form a new GT family. Elucidation of its biochemical nature

will enable this question to be answered.

BC10 regulates mechanical properties by perturbing the

levels of cellulose and AGPs

Based on its subcellular localization and biochemical anal-

ysis, BC10 very probably functions in a manner that leads to

perturbation of the biosynthesis of glycoprotein and/or non-

cellulosic polysaccharides. Therefore, the defect in cellulose

biosynthesis in bc10 revealed by the quantitative analysis of

cell-wall fractions may not be the primary effect that results

in the mutant phenotypes because cellulose is formed at the

plasma membrane involving CESAs in the catalytic subunits

(Delmer, 1999). However, deficiency in formation of glyco-

proteins or the polysaccharide matrix often causes aberrant

cellulose synthesis. For example, an Arabidopsis cyt1

mutant that is defective in mannose-1-phosphate guanylyl-

transferase involved in N-linked glycosylation was found to

have a reduced level of cellulose (Lukowitz et al., 2001).

Xylan synthesis mutants also exhibit reduced secondary

wall deposition and hence reduced cellulose (Brown et al.,

2005, 2007). A likely explanation might be that proteins such

as BC10 play a glycosylation role, modifying CESA subunits

and/or other component(s) of the cellulose synthase

machinery, because CESAs have been predicted to have

several putative N-linked glycosylation sites and mutants of

Arabidopsis with defects in the processing of N-linked gly-

cans are deficient in cellulose synthesis (Gillmor et al., 2002).

Another possibility is that BC10 might play a role in modi-

fying plasma membrane-associated proteins such as

KORRIGAN, COBRA or KOBITO, which have been demon-

strated to be important for cellulose biosynthesis in the

cellulose-deficient mutants kor (His et al., 2001; Molhoj

et al., 2001), cobra (Schindelman et al. 2001; Roudier et al.,

2005) and bc1 (Li et al., 2003b). N-glycosylation of KOR and

COB has been demonstrated to be required for the enzy-

matic activity, which in turn affect cellulose synthesis.

Therefore, the perturbation of cellulose levels in bc10

probably results from malfunction in glycosylation of CESAs

or related proteins.

Monosaccharide analysis of total and fractioned wall

residues revealed no significant alteration in pectin and

hemicellulose in bc10. Therefore, the decrease in the cellu-

lose content in bc10 may result from deficient biosynthesis

of some wall glycoproteins, for example AGPs. It has been

BC10 regulates mechanical strength in rice 457

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

predicted that AGPs may be secreted to the cell surface in

parallel with cellulose synthesis, and that their carbohydrate

chains bind to cellulose, directly or indirectly regulating the

cellulose synthesis and mechanical properties of cell walls

(Casero et al., 1998; Ito et al., 2005; Loopstra et al., 2000).

Indeed, AGP-like glycoproteins show a tight physical asso-

ciation with flax fiber cellulose (Girault et al., 2000). The

presence of AGP carbohydrate epitopes (Dolan and Roberts,

1995; Stacey et al., 1995) and backbone peptides (Gao and

Showalter, 1999, 2000) in the secondary wall suggest their

crucial roles in the secondary cell-wall thickening. Because

AGPs are often composed of a short protein backbone and

branched carbohydrate chains (Nothnagel, 1997; Seifert and

Roberts, 2007), glycosylation of AGPs is a complicated

process. Therefore, it is plausible that BC10 is responsible

for adding a key moiety to carbohydrate portion of AGPs that

is required for complete synthesis of AGPs. The dramatic

decrease in AGP carbohydrate epitopes in the secondary

walls of bc10 may explain its brittle phenotype, as the lower

level of AGPs in bc10 might directly or indirectly result in a

significant reduction in the cellulose content, which in turn

affects the formation of the secondary cell walls and thus

leads to the brittle phenotype.

Experimental procedures

Plant materials

The bc10 mutant was isolated from a natural population of thejaponica cultivar Huang Jin Qin. A F2 mapping population wasgenerated from a cross between bc10 and Zhefu802 (ZF802), apolymorphic indica cultivar.

Measurements of physical properties

The breaking force and extensibility (measured as extension length)of rice culms and leaves were measured using a digital force/lengthtester (5848 microtester, Istron, http://www.instron.com).

Microscopy

Culms and leaves were fixed in 2.5% v/v glutaraldehyde. Sampleswere post-fixed in 2% w/v OsO4 for 2 h, dehydrated through anethanol gradient, and infiltrated and embedded in butyl methylmethacrylate. Sections 3 lm thick were cut, stained with toluidineblue, and viewed under a microscope. For transmission electronmicroscopy, the samples were embedded using a Suprr kit forelectron microscope (Sigma, http://www.sigmaaldrich.com/), sec-tioned (80 nm ultra-thin) using an Ultracut E ultramicrotome (Leica,http://www.leica.com), and viewed under a Hitachi (http://www.hitachi-hta.com) H7500 transmission electron microscope.

Mapping of BC10

BC10 was mapped and cloned using 870 F2 mutant plants and themolecular markers listed in Table S1. For complementation anal-ysis, an 8.69 kb genomic DNA fragment containing the entire

BC10 coding region, a 1.98 kb upstream sequence and a 2.3 kbdownstream sequence were inserted into the binary vectorpCAMBIA 1300 to generate the transformation plasmid pBC10F. Acontrol plasmid, pBC10T, containing a 3¢ truncated BC10 gene(4.5 kb) was also constructed. The binary plasmids were intro-duced into Agrobacterium tumefaciens strain EHA105 by electro-poration and transformed into bc10 as described previously (Hieiet al., 1994).

Bioinformatics analysis of the BC10 gene

Domain prediction for BC10 was performed using Pfam onlineprediction software (http://pfam.sanger.ac.uk/). Systematic identifi-cation of DUF266-containing proteins in the NCBI nr databases wasperformed using a TimeLogic implemented profile search enginewith default parameters through a CodeQuest work station (http://www.timelogic.com). Sequences with the identified DUF266domain were selected as DUF266-containing proteins. A phyloge-netic tree of Arabidopsis and rice DUF266-containing proteins wasgenerated using MAGE4 (Tamura et al., 2007), with 1000 bootstrapreplications. A PSI-BLAST search for BC10 homologs was per-formed using the NCBI BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A structure homology search for BC10 was carried outusing the FUGUE server (http://www-cryst.bioc.cam.ac.uk/fugue/).

Gene expression analysis

Total RNA was extracted from rice plants as described previously(Wadsworth et al., 1988). Complementary DNA (cDNA) was syn-thesized from total RNA using a reverse transcriptional kit fromPromega (http://www.promega.com/). The RT-PCR primers foramplification of BC10, its homologs and actin are given in Table S3,and the reactions were performed on quantitative PCR system7900HT from Applied Biosystems (http://www.appliedbiosystems.com/).

RNA in situ hybridization was performed as described byKao et al. (2002). The 3¢ end of BC10 was sub-cloned into thepGEM-T vector (Promega) and used as a template to generateRNA probes. Hybridization was performed on wax-embeddedtransverse sections (10 lm) using probe labeled with digoxin(Roche, http://www.roche.com).

Subcellular localization

To determine its exact subcellular location, BC10 cDNA was fusedin-frame with either EGFP or mRFP and ligated into the PUC19vector. The expression constructs were co-transfected into tobaccoBY2 protoplasts with mRFP/mCherry- or EGFP-tagged markers forvarious organelles. These markers include the Golgi-localized sial-yltransferase (Lee et al., 2002), and ER and mitochondria localiza-tion constructs containing the ER and mitochondria retentionsequences, respectively (Nelson et al., 2007). The transformedprotoplasts were examined using a confocal microscope (LSM 510META, Zeiss, http://www.zeiss.com/).

C2GnT enzyme activity assay

Human C2GnT and BC10 cDNA clones were inserted into the pEGFP-N1 vector (Clontech, http://www.clontech.com/). The plasmid DNAswere transiently transfected into Chinese hamster ovary (CHO) cellsusing a modified lipofection procedure. The cells were harvested,washed with PBS buffer, and suspended in lysis buffer (10 mM TrisCl,

458 Yihua Zhou et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 446–462

pH 8.0, 0.1 mM EDTA, 5 mM DTT, 0.9% NaCl, 1% Triton X-100). Aftergently shaking at 4�C for at least 2 h, the cell lysate was centrifuged at800 g at 4�C for 5 min. The supernatant was transferred, divided intoaliquots (250 ll each), and stored at )80�C until use. Protein con-centration was determined using a Bio-Rad protein assay kit (http://www.bio-rad.com/) with bovine serum albumin (BSA) as the stan-dard. C2GnT activities were determined as described previously(Bierhuizen and Fukuda, 1992). Briefly, a total of 50 ll reaction mix-ture containing 50 mM HEPES-NaOH, pH 7.0, 1 mM UDP-[glucosa-mine-U-14C] GlcNAc (3.7 kBq, Amersham Pharmacia Biotech, http://www5.amershambiosciences.com/), 1 mM Galb1 fi 3GalNAca1 fip-nitrophenyl (Sigma), 0.1 M GlcNAc, 5 mM DTT and 250 lg lysatewas incubated at 37�C for 2–3 h and the reaction was stopped byadding 5 ml deionized water. The reaction products were purifiedusing a C18 Sep-Pak column (Waters, http://www.waters.com),washed with 20 ml of water, and eluted with methanol. Finally, theproducts were measured using a liquid scintillation counter (1450MicroBeta TriLux; Perkin-Elmer, http://www.perkinelmer.com).

Compositional analysis of neutral glycosyl residues

of whole cell walls

Alcohol-insoluble residues of culms were prepared as described byLi et al. (2003b), and the cellulose content was determined as pre-viously described (Tanaka et al., 2003). A phenol/sulfuric acid assaywas used to quantify hexoses attributed to cellulose content.Sugars, alditol acetates and lignin were analyzed as describedpreviously (Hoebler et al., 1989; Kirk and Obst, 1988).

Fractionation of walls for glycosyl composition analysis and

measurement of uronic acids

Ground material from the second culm internode was fractioned asdescribed by Persson et al. (2007) and Zhong et al. (2005). Uronicacid content was determined as described by Filisetti-Cozzi andCarpita (1991). For details, see Appendix S1.

Chemical and immunological analyses of AGPs

For immunolocalization analyses, see Appendix S1. For histo-chemical staining, fresh hand-cut sections of stems of wild-type andbc10 plants were incubated in bGlcY (Yariv et al., 1962) [2 mg ml)1

(b-D-glucosyl)3 in 0.1 M NaCl] or the control reagent bManY[2 mg ml)1 (b-D-mannosyl)3 in 0.1 M NaCl] for 1 h. The sectionswere then visualized using a fluorescent microscope (Leica).

AGPs were isolated from the aerial organs of wild-type and bc10plants as described previously (Kreuger and van Holst, 1993). Theconcentration of AGPs was determined by the single radial diffusionmethod developed by van Holst and Clarke (1985). Monosaccharidecomposition was determined by GC–MS analysis of alditol acetatesderived from sugars in the hydrolysates of AGPs (van Hengel andRoberts, 2002).

Acknowledgements

We thank Antony Bacic (The School of Botany, University of Mel-bourne, Melbourne) and Vincent L. Chiang (Department of Forestry,North Carolina State University, Raleigh) for critical comments onthe manuscript, Taihua Zhang for measurement of breaking force ofrice plants, Dongqiao Shi for confocal microscope examination,Minoru Fukuda (Cancer Research Center, Burnham Institute, LaJolla, CA) for providing the C2GnT cDNA clone, Inhwan Hwang(Division of Molecular and Life Sciences, Pohang University of

Science and Technology, Pohang, Korea) for providing the Golgimarker gene SailT–RFP, and Xianming Pan (Department of Biolo-gical Sciences and Biotechnology, Tsinghua University) and Yun He(Institute of Biophysics, Chinese Academy of Sciences, Beijing) forprotein structure prediction. Development and distribution ofantibodies JIM7 and JIM13 were supported in part by NationalScience Foundation grants DBI-0421683 and RCN-0090281. Thiswork was supported by grants from the National Natural ScienceFoundation of China (30221002, 30470112 and 30330040).

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Subcellular localization of GFP-tagged BC10 andBC10DN44 in onion epidermal and rice root cells.Figure S2. Secondary and three-dimensional structures of the BC10protein.Figure S3. Alignment of BC10 homologs in rice and Arabidopsis.Figure S4. Comparison of MALDI-TOF mass spectra of b-endoxy-lanase- and xyloglucanase-digested products from 1 and 4 N KOHfractions of wild-type and bc10 culms.Table S1. Molecular markers developed for map-based cloning ofBC10.Table S2. Quantification of fluorescent intensity and gold particlesdetected in immunostaining.Table S3. Primers for RT-PCR analysis of BC10 homolog in rice.Appendix S1. Additional experimental procedures and referencesPlease note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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