The Plant Journal 64 Biological implications of the ...€¦ · GH16.html) (Cantarel et al., 2009). In angiosperms, XTHs are typically encoded by large multi-gene families (Okazawa

Biological implications of the occurrence of 32 members ofthe XTH (xyloglucan endotransglucosylase/hydrolase) familyof proteins in the bryophyte Physcomitrella patens

Ryusuke Yokoyama1, Yohei Uwagaki1, Hiroki Sasaki1, Taro Harada1, Yuji Hiwatashi2,3, Mitsuyasu Hasebe2,3,4 and

Kazuhiko Nishitani1,*

1Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University,

Sendai 980-8578, Japan,2National Institute for Basic Biology, Okazaki 444-8585, Japan,3School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan, and4ERATO, Japan Science and Technology Agency

Received 17 April 2010; revised 13 August 2010; accepted 31 August 2010; published online 5 October 2010.*For correspondence (fax +81 22 795 6669; e-mail [email protected]).

SUMMARY

This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and

proteins in bryophytes, based on research using genomic resources that are newly available for the moss

Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in

large multi-gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there

are fewer cell types in P. patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that

there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many

as 32 genes that potentially encode XTH family proteins in the genome of P. patens, constituting a fairly large

multi-gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of

xyloglucan endotransglucosylase activity in this moss indicates that some P. patens XTH proteins exhibit

biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue-

dependent. However, comparison of structural features of families of XTH genes between P. patens and

angiosperms demonstrated the existence of several bryophyte-specific XTH genes with distinct structural and

functional features that are not found in angiosperms. These bryophyte-specific XTH genes might have

evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting

questions about the biological implications of the XTH family of proteins in non-seed plants.

Keywords: xyloglucan endotransglucosylase/hydrolase, Physcomitrella patens, bryophyte, cell wall, gene

family.

INTRODUCTION

The development of specialized cell walls in the evolution of

land plants allowed adaptation to terrestrial environments.

Cell walls in present-day land plants comprise crystalline

cellulose embedded in matrix polymers such as xyloglucans

(Carpita and Gibeaut, 1993). Many enzymes and structural

proteins are required for the construction and remodeling

processes that are involved in producing cell walls (reviewed

by Cosgrove, 2005; Nishitani and Vissenberg, 2006), and

most of these related proteins are encoded by multi-gene

families, with the number of family members and diversity

varying between species (Yokoyama and Nishitani, 2004).

The fundamental diversity among plant species reflected

in cell shape, and, by extension, organ morphology, is

expressed through construction and restructuring of the cell

walls. Therefore, the diversity of plant cell-wall architecture

is expected to be reflected in cell wall-related gene families,

which are potentially representative of the molecular feature

associated with morphological and functional diversity

among various cell types in plants.

The xyloglucan endotransglucosylase/hydrolase (XTH)

family of enzymes was originally identified as capable of

catalyzing molecular grafting reactions among xyloglucan

658 ª 2010 The AuthorsThe Plant Journal ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 64, 658–669 doi: 10.1111/j.1365-313X.2010.04351.x

molecules, and was hypothesized to play a principal role in

the construction and restructuring of xyloglucan crosslinks

in the cellulose/xyloglucan framework (Fry et al., 1992;

Nishitani and Tominaga, 1992). Currently, the XTH class of

enzymes (Rose et al., 2002) is classified as a subfamily of

glycoside hydrolase family 16 (http://www.cazy.org/fam/

GH16.html) (Cantarel et al., 2009). In angiosperms, XTHs

are typically encoded by large multi-gene families (Okazawa

et al., 1993; Nishitani, 1997; Rose et al., 2002). Using avail-

able genome sequence databases, 33, 29 and 41 open

reading frames (ORFs) potentially encoding XTH have been

identified for Arabidopsis thaliana, rice (Oryza sativa) and

poplar (Populus trichocarpa) (Yokoyama and Nishitani,

2000, 2001b; Yokoyama et al., 2004; Geisler-Lee et al.,

2006). Recently, several XTH genes have been found in

non-seed land plants and algae, suggesting that the XTH

family developed in an earlier plant ancestor (Vissenberg

et al., 2003; Van Sandt et al., 2007a,b) via diversification of

ancestral GH16 family genes (Baumann et al., 2007). Identi-

fication of many putative proteins with sequences similar to

known members of the XTH gene family among the

genomic sequences of two basal land plants, Selaginella

moellendorfii and Physcomitrella patens, supports this

hypothesis.

In angiosperms, individual XTH family members exhibit

distinct expression patterns in terms of tissue specificity and

varied responses to hormonal signals (Xu et al., 1996;

Akamatsu et al., 1999; Yokoyama and Nishitani, 2001a,b,

2004; Becnel et al., 2006). The ubiquity of xyloglucans in

various cell types and the cell type-specific expression

profiles of the XTH genes (Nakamura et al., 2003; Vissenberg

et al., 2005; Becnel et al., 2006) imply the involvement of

XTHs in a wide range of physiological processes in vascular

plants. The products of each of the XTH genes appear to play

a specific role in temporal and spatial modulation of various

aspects of wall architecture: seed germination (Chen et al.,

2002), root formation (Vissenberg et al., 2005; Osato et al.,

2006), stem elongation (Catala et al., 1997), flower develop-

ment (Hyodo et al., 2003), fruit ripening (Rose and Bennett,

1999), secondary wall deposition of developing vascular

tissue (Bourquin et al., 2002; Matsui et al., 2005) and reaction

wood formation (Nishikubo et al., 2007). It is worth noting

that most of these developmental processes are character-

istic to vascular plants. If it is supposed that the large XTH

family of genes arose through morphological and functional

diversification of vascular plants, it is logical to predict that

fewer potential orthologs of the XTH family would be found

in non-vascular plants, such as mosses and algae.

Using the complete and fully available genomic sequence

data for the moss P. patens (Hedw.) Bruch & Schimp. subsp.

patens (http://www.jgi.doe.gov/sequencing/why/CSP2005/

physcomitrella.html), we extend the understanding of cell

wall-related gene families to a member of the earliest land

plant lineages (Carey and Cosgrove, 2007; Liepman et al.,

2007; Roberts and Bushoven, 2007) and further developed.

Due to the advantages of using reverse genetics based

on efficient homologous recombination (Schaefer, 2002)

and the abundance of publicly available transcriptome

databases containing more than 100 000 ESTs (Nishiyama

et al., 2003), P. patens offers a valuable opportunity to

investigate the relationship between morphological charac-

teristics and gene family diversification in a wide spectrum

of land plants. In this study, we identified all genes in the

XTH family of P. patens (PpXTH). Histological studies on

XTH proteins and enzyme activity showed that some PpXTH

proteins exhibit biochemical functions similar to those

found in angiosperms. Comparative analyses of PpXTHs

and angiosperm XTHs (Yokoyama and Nishitani, 2001b;

Yokoyama et al., 2004) have identified several bryophyte-

specific XTH genes with distinct protein structures that are

not found in angiosperms, suggesting unique roles in non-

vascular land plants.

RESULTS

Localization of XET activity in P. patens

Xyloglucan endotransglucosylase (XET) activity of XTHs

in P. patens was examined by in vivo incorporation of

sulforhodamine-labeled xylogluco-oligosaccharides (XLLG-

SR) into P. patens cultured on BCDAT medium (Nishiyama

et al., 2000). Incorporation activity of XLLG-SR was

observed in protonemata, including both chloronemata and

caulonemata, particularly in the periphery of the apical cells

(Figure 1a,b). The septum of the filaments also showed

prominent incorporation activity (Figure 1c,d). In gameto-

phores, XET activity was observed in buds, axillary hairs

and basal rhizoids (Figure 1m,n,q,r,u,v). To validate the

specificity of this in situ XET activity assay system, moss

tissue incubated with GGGG-SR or unlabeled XLLG

was shown to incorporate little or no fluorescent dye

(Figure 1e–h). Furthermore, incorporation of XLLG-SR into

the tissue was inhibited by addition of 0.5 or 5 mM unla-

beled XLLG in the incubation medium (Figure 1i–l). These

results clearly indicate the high specificity of this enzyme

assay system, and demonstrate the ubiquity and tissue-

dependent presence of XET activity in this bryophyte. This

result is consistent with a recent report by Van Sandt et al.

(2007b), showing the presence of XTH gene activity in

several bryophytes.

Immunolocalization of XTH proteins in P. patens

Based on the demonstrated cell type-dependent distribution

of XET activity in P. patens, we investigated the distribution

profiles of XTH proteins in these cells using a polyclonal

antibody raised against a full-length recombinant azuki bean

(Vigna angularis (Wild.) Ohwi and Ohashi) XTH protein,

VaXTH1 (Yokoyama and Nishitani, 2001a; Nakamura et al.,

2003). Given the high degree of sequence similarity between

XTH family of genes in Physcomitrella patens 659

ª 2010 The AuthorsThe Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669

VaXTH1 and other XTH proteins identified to date, it was

expected that several PpXTHs would be labeled with this

polyclonal antibody. Immunofluorescence signals were

detected on protonemata, and were particularly prominent

at the tip region of apical cells in both chloronemal and

caulonemal filaments (Figure 2a,b). Axillary hairs of ga-

metophores were also strongly labeled with the antibody

(Figure 2i,j). These results are consistent with the results of

in situ localization of XET activity within these cells. By

contrast, no immunofluorescence signal was detected in the

septum of chloronemal and caulonemal filaments. The

apparent discrepancy between immunolocalization of XTH

proteins and in situ XET activity signals in the septum of the

filaments may be due to resistance of these tissues to the

permeabilization that is required for whole-mount immu-

nohistochemical analysis or to reduced sensitivity of the

septum-localized PpXTH(s) to the anti-VaXTH1 antibody.

Recently, Marcus et al. (2008) showed that pectic homoga-

lacturonan masks abundant sets of xyloglucan epitopes in

cell walls. It is therefore likely that XTH proteins, which

co-localize with xyloglucans, are also masked by other

cell-wall components.

P. patens XTH family genes

The presence of XTH family genes in P. patens was deter-

mined by compiling a list of putative ORFs based on

sequences shown to be highly conserved among XTHs

through a search of genomic data newly released by the

Joint Genome Institute (http://genome.jgi-psf.org/Phypa1_1/

Phypa1_1.home.html). ORFs that lacked either the predicted

N- or C-terminal regions (Figure 3) or the conserved struc-

tural features of XTHs identified to date were rejected. The

rejected ORFs were subjected to manual re-interpretation of

coding regions based both on the conserved structural fea-

tures of XTHs and on the sequence data of full-length cDNAs

and ESTs released from PHYSCObase (http://moss.nibb.

ac.jp/). We identified 32 independent ORFs that encode

putative P. patens XTH genes (PpXTH) (Figure 3 and Table

S1). Surprisingly, the number of members of the PpXTH

family identified to date is comparable with the 33 members

in Arabidopsis thaliana (AtXTHs) and the 29 members in rice

(OsXTHs).

The structures of individual PpXTH genes were deter-

mined based on genomic sequences (Figure 3). Of the

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

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

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

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

(q) (r) (s) (t)

(u) (v) (w) (x)

Figure 1. XET activity staining in Physcomitrella

patens.

Epifluorescence images (a, c, e, g, l, k, m, o, q, s,

u, w) and differential interference contrast (DIC)

images (b, d, f, h, j, l, n, p, r, t, v, x) are shown.

Protonemata sub-cultured for 13 days (a–h) or

19 days (i, j) and gametophores (k, l) were used

for experiments. Scale bars = 100 lm.

(a–h) Protonema were incubated with XLLG-SR

(a–d), GGGG-SR (e, f) and without any substrates

(g, h). The inset in (c) shows a magnified

epifluorescence image of the septum or cross

wall.

(i, j, k, l) Protonemata incubated with XLLG-SR in

the presence of 0.5 mM (i, j) or 5 mM (k, l)

unlabeled XLLG.

(m–t) Young gametophores incubated with

XLLG-SR (m, n, q, r) or GGGG-SR (o, p, s, t).

(u–x) Gametophores incubated with XLLG-SR (u,

v) or GGGG-SR (w, x). The inset in (u) shows the

magnified view of axially hairs.

660 Ryusuke Yokoyama et al.


PpXTH genes, 24 were found to have 2–4 intron insertion

sites, and these insertion sites were conserved among the 24

PpXTH genes (Figure 3) and the 33 AtXTH genes (Yokoyama

and Nishitani, 2000, 2001b). However, eight PpXTH genes

lacked introns (Figure 3). This type of single-exon XTH gene

has not previously been found in any of the angiosperms

examined to date.

All of the predicted PpXTH proteins had an overall

structure typical of XTH enzymes, possessing the catalytic

active-site motif ExDxE, which is conserved in glycoside

hydrolase family 16 (Nishitani, 1997; Campbell and Braam,

1999; Henrissat et al., 2001; Johansson et al., 2004;

Baumann et al., 2007). The presence of a signal peptide for

entry into the secretory pathway, and one or more N-linked

glycosylation sites C-terminal to the catalytic site were

predicted by the Wolf PSORT program (http://wolfpsort.

org/) for all PpXTH proteins (Figures 4, 5 and Figure S1).

However, a few amino acid residues in the DEI/LDFEFLG

sequence were found to be replaced in 21 of the 32 PpXTH

genes (Figure 5). In addition, PpXTH30, 31 and 32 lacked the

C-terminal extension region, which is well conserved among

all XTHs identified to date (Figure 4).

Phylogenetic analysis of AtXTH, OsXTH and PpXTH genes

To elucidate the evolutionary relationships of XTH genes, we

generated a phylogenetic tree using full-length A. thaliana,

O. sativa and P. patens XTH protein sequences (Figure 6).

XTH genes in green algae have not been well studied, and

we were unable to specify outgroup genes for rooting.

Although XTH members have historically been classified

into three classes (I, II and III) (Nishitani, 1997), the present

analysis found no apparent divergence between the

previously distinct classes I and II (Yokoyama et al., 2004),

and thus the present classification is into two major groups,

namely group I/II and group III. Twenty six PpXTH genes

(PpXTH3–PpXTH28) were clustered in group I/II, and two

(PpXTH1 and PpXTH2) were members of group III (Figure 6).

We found several monophyletic groups that included only

P. patens proteins, but their relationships to angiosperm

XTH proteins are not specified because of low statistical

support. Baumann et al. (2007) has proposed that group III

actually comprises two distinct clades, designated group IIIA

and group IIIB. They further demonstrated that members of

group IIIA only exhibit hydrolase activity rather than XET

activity. PpXTH1 and PpXTH2 belong to group IIIB, and none

of the P. patens genes identified in this study belong to

group IIIA (Figure 6).

Expression profiles of PpXTH genes

The combined sequence and structure analysis indicated

the existence of several bryophyte-specific XTH genes, in

addition to typical angiosperm-type XTH genes. To charac-

terize the detailed expression profiles of these genes, the

coding sequence of b-glucuronidase was inserted in-frame

just before the stop codon of PpXTH16, 21 and 32 by

homologous recombination, enabling histochemical detec-

tion of GUS activity. Protonemata transformed with the

PpXTH32::GUS were strongly stained, whereas those

transformed with PpXTH16::GUS and PpXTH21::GUS were

more weakly stained (Figure 7a–c). The gametophores

showed GUS expression for all three transformants, but with

different intensities and patterns. PpXTH16::GUS was ex-

pressed strongly at the apical portion of young gameto-

phores (Figure 7d,g). GUS expression from the PpXTH21

and PpXTH32 promoters was observed in the base of leaves

and the apical portion of the gametophore (Figure 7e,f,h,i).

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

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

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

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

Figure 2. Immunolocalization of XTH proteins in

P. patens.

Tissues were probed using VaXTH1 antibody (a,

b, i, j) or pre-immune serum (c, d, k, l). Epifluo-

rescence images (a–d, i–l) and differential inter-

ference contrast (DIC) images (e–h, m–p) are

shown. Scale bars = 500 lm.

(a–h) Chloronemal and caulonemal filaments: (b,

d, f, h) are magnified images of (a, c, e, g),

respectively.

(i, j, m, n) Buds.

(k, l, o, p) Gametophores.



In addition, for PpXTH32, GUS expression was clearly

observed in rhizoids (Figure 7f). No signal was detected in

sporangium transformed with PpXTH16::GUS, PpXTH21::

GUS or PpXTH32::GUS (Figure 7j–l).

The expression profile of the PpXTH gene family was also

investigated by comparing the relative abundance of P. pat-

ens ESTs. ESTs for 22 of the PpXTH genes were found in

PHYSCObase (http://moss.nibb.ac.jp/), confirming that they

are transcribed (Figure S2). The divergent occurrence of the

various PpXTH genes in various cDNA libraries also sug-

gests differential expression patterns in terms of organ

specificity and responses to hormonal and environmental

signals (Figure S1).

DISCUSSION

A survey of genomic databases revealed that the moss

P. patens, which is less differentiated than angiosperms in

terms of tissue type, contains a similar number of putative

XTH genes, 32 in total. This finding is somewhat surprising

as circumstantial evidence based on extensive expression

analyses of angiosperm XTH genes suggested that this

family of genes had diversified during the evolution of

angiosperms (Yokoyama and Nishitani, 2001b; Rose et al.,

2002; Becnel et al., 2006; Van Sandt et al., 2007a).

Structural diversity of PpXTH

The exon–intron organization of XTH family genes is well

conserved in AtXTH genes (Yokoyama and Nishitani, 2000,

2001b); the catalytic active-site motif, ExDxE, is located in the

second or third exon. However, the same ExDxE motif is not

always found in the 2nd and 3rd exons in rice, but is located

in the middle of the 1st exon in some genes (Yokoyama

et al., 2004). Therefore, it is interesting that the patterns of

exon–intron organization found in A. thaliana are well con-

served among PpXTH genes (Figure 3). This indicates that

most AtXTH and PpXTH genes retain a common ancestral

gene structure.

However, some PpXTHs possess distinct primary struc-

tures not found in angiosperms. One of the structures typical

of XTH enzymes is the DEI/LDFEFLG motif, which includes

the catalytic active-site residues ExDxE (Campbell and

Braam, 1999; Johansson et al., 2004, Rose et al., 2002).

Although replacement of amino acid residues is found only

in a few of these proteins in angiosperms (Yokoyama and

Nishitani, 2001b; Yokoyama et al., 2004), alterations of

amino acid residues are found within this catalytic region

in 21 of the 32 PpXTHs. Interestingly, the first aspartic acid

residue in the catalytic active site is replaced by a cysteine

residue in seven of these 21 PpXTHs. This residue of the

catalytic active site is oriented into the hydrophobic core of

the protein, and therefore may not significantly affect the

catalytic properties of XTHs (Eklof and Brumer, 2010).

However, given that the mode of enzyme action of XTH

proteins will be directly affected by alteration of amino acid

residues in the vicinity of the catalytic active site, enzymatic

characteristics, including substrate specificity and catalytic

processes, are likely to have diversified and consequently

resulted in different reaction paths.

Three XTHs, PpXTH30, 31 and 32, lack the C-terminal

linker that packs against the conserved core and forms an

additional b-strand and a short a-helix (Figure 4) (Johansson

et al., 2004). Given that the C-terminal extension is required

for the XTH protein to exhibit XET-directed activity, the three

PpXTHs with truncated C-terminal regions may lack this

unique, family-specific enzyme activity. Furthermore, the

PpXTH1

PpXTH2

*PpXTH3

*PpXTH4

*PpXTH5PpXTH6

*PpXTH7*PpXTH8PpXTH9

*PpXTH10*PpXTH11*PpXTH12*PpXTH13

*PpXTH14

PpXTH15

PpXTH16

PpXTH17

*PpXTH18PpXTH19

PpXTH20

[ ]*PpXTH21

*PpXTH27

*PpXTH26

*PpXTH25

*PpXTH24

*PpXTH23

*PpXTH22

*PpXTH28

PpXTH29

PpXTH30

*PpXTH31

*PpXTH32

500 bp

Figure 3. Schematic diagram of the PpXTH gene structures.

The genome organization for PpXTH22 is ambiguous due to uncertainty with

respect to the intron position (potential gene structure in parentheses). White

boxes, exons; lines, introns; gray boxes, putative signal peptide sequence;

black boxes, DEI/LDFEFLG motifs corresponding to the catalytic sites of the

proteins. The dotted lines below the PpXTH gene structures indicate the

portions corresponding to the gene model annotated by the Joint Genome

Institute. Asterisks before gene names indicate the genes whose ESTs have

been deposited in the database http://moss.nibb.ac.jp (Figure S2).



structures of the three PpXTHs are relatively close to that of

AtXTH11, which in turn is classified in an ancestral group of

XTH closer to lichenases (Baumann et al., 2007). This may

imply distinct biochemical activity from that found in

angiosperms.

Accordingly, to obtain insight into the biochemical prop-

erties of bryophyte-specific XTHs, PpXTH32 protein was

recombinantly produced using a Pichia pastoris heterolo-

gous expression system. The purified recombinant

PpXTH32 protein did not exhibit either endotransglucosy-

lase or endohydrolase activity on either xyloglucan, b-1,3/1,4

mixed glucan from barley or lichenan from Cetraria islandica

(Figure S3). These results indicate that PpXTH is not

ordinary XTH or b-1,3/14 glucanase (lichenase), and strongly

suggest that the bryophyte-specific XTH genes encode

proteins whose molecular functions and hence physio-

logical roles are distinct from those encoded by angiosperm

XTH genes.

Functional diversity of PpXTHs

Using XLLG-SR as an acceptor substrate, we demonstrated

prominent tissue-dependent XET activity in the cell walls

of apical cells of protonemata (Figure 1). Immunohisto-

chemical analysis using the polyclonal VaXTH1 antibody

showed localization of XTH proteins at the tips of the

apical cells. In protonemata of P. patens, cell expansion

occurs at the tip of the apical cell (Cove et al., 1997), and

this expansion has recently been shown to occur by tip

growth (Menand et al., 2007). Therefore, XTHs probably

play an important role in the construction and restructur-

ing of xyloglucan cross-links in cell expansion during tip

growth of bryophytes.

Figure 4. Structure-based sequence alignment of PpXTHs, which lack the C-terminal linker, and Xet16A.

Sequences were aligned using Clustal W and generated by ESPript (Gouet et al., 1999). The secondary structure elements indicated above the alignment are those

of Xet16A, whose structure has been experimentally determined (Johansson et al., 2004). Blue frames indicate conserved residues, white letters in red boxes

indicate strict identity, and red letters in white boxes indicate similarity. The predicted a-helices and b-strands are represented by spirals and horizontal arrows,

respectively.



In angiosperms, XTH proteins are exclusively transported

to the cell plate during cytokinesis (Yokoyama and Nishitani,

2001a). Therefore, these XTHs probably play a role in the

integration of newly secreted xyloglucan material into

pre-existing xyloglucan molecules, thereby assembling the

precursors for cellulose/xyloglucan construction (Thomp-

son et al., 1997; Ito and Nishitani, 1999). The prominent

localization of XET activity in the septum of filaments

indicates commitment of some PpXTHs to the construction

of new cell walls during cytokinesis in P. patens.

Seven PpXTHs (PpXTH10, 11, 12, 13, 16, 17 and 18) were

shown to share both the DEI/LDFEFLG motif and the

C-terminal extension region. These two structural features

are strictly conserved in XTH proteins of group I/II and have

been proposed to be essential for XET activity (Baumann

et al., 2007). Thus, it is probable that these seven PpXTHs

exhibit XET activity and are responsible for the in situ XET

activity observed in P. patens tissue in the present study

(Figure 1). PpXTH16, a typical angiosperm-type XTH gene,

was expressed strongly in the apical cells of protonemata

(Figure 7a), implying that the predominant role of PpXTH16

in these cells is as a classical xyloglucan endotranglucosy-

lase. Furthermore, the VaXTH1 antibody, which had been

raised against an XTH of group I/II from Vigna angularis,

bound to proteins in P. patens, supporting this idea.

The phylogenetic tree (Figure 6) indicates that PpXTH

genes in group I/II formed several monophyletic groups that

included only PpXTH genes. This indicates that XTH genes

expanded in the moss and angiosperm lineages in parallel.

EST databases indicate that each PpXTH in group I/II

appears to have a distinct expression profile in terms of

tissue specificity and responses to hormonal signals, sug-

gesting that group I/II XTH genes have diversified to fulfill

distinct physiological roles in P. patens. The biological

implications of these hypothetical roles may provide insight

into the evolutionary implications of xyloglucans in diversi-

fication of cell walls in land plants.

The idea that the PpXTH family has expanded to meet the

specialized needs of bryophytes is also supported by the

presence of unique PpXTHs and the absence of their

orthologs in other angiosperm XTH families. Phylogenetic

analysis also provides strong support for the independent

diversification of several sub-families in the bryophyte

lineage. It has been reported that xyloglucan in bryophytes

differs from typical angiosperm xyloglucan in terms of both

concentration and structure (Popper and Fry, 2003; Pena

et al., 2008). Numerous XTH genes for bryophyte-specific

xyloglucan, originating by gene duplications, may have

resulted in specialized morphological and physiological

evolution in bryophytes. Furthemore, Hrmova et al. (2007)

reported that a barley XTH (HvXET5) can mediate transgly-

cosylation between xyloglucans and other glucans, includ-

ing (1,3/1,4)-b-D-glucans, a reaction that is specific to cereal

plants. Fry et al. (2008) demonstrated that some species of

charophytes and Equisetum contain enzyme activity capable

of transglucosylating (1,3/1,4)-b-D-glucans with xyloglucans.

Thus, we cannot exclude the possibility that mosses, as

DEIDFEFLGL

PpXTH1 YASNGGYYP--YNHDEIDMEFLGVRPGQ-----PYVIQTNPpXTH2 YASNGGSYP--SNHDEIDLEFLGVRPGH-----PYVIQTNPpXTH3 YLSSQG-----HNHDEVDFEFLGNVTG-EP----YVLQTNPpXTH4 YLSSQG-----HDHSEVDFEFLGNKTNTQPGNNEIVLQTNPpXTH5 YTSSDGKK---PYHDEIDIELLGNETS-----SCITMQTNPpXTH6 YTSSDGKM---DDHDEIDIELLGNETS-----KHITMQTNPpXTH7 YMSSQTP----GLHDEMDFEFLGNVTGQ-----PYILQTNPpXTH8 YMSSQTP----GLHDEMDFEFLGNVSGQ-----PYILQTNPpXTH9 YLSSDT-----ARHDEMDFEFLGNVSGQ-----PYILQTNPpXTH10 YLSSAQ-----PNHDELDFEFLGNVSGE-----PYVLQTNPpXTH11 YLSSAQ-----PNHDELDFEFLGNVSGQ-----PYSLQTNPpXTH12 YLSSDQ-----PAHDELDFEFLGNTSGD-----PYILQTNPpXTH13 YLYSPT-----DHHDELDFEFLGNSSGQ-----PYILQTNPpXTH14 YLSSEGGE-----HDEMDFEFLGKGGDQ-----PYILQTNPpXTH15 YLSSEGGQSVRSVHDEMDFEFLGNSSGQ-----PYILQTNPpXTH16 YTSSLSG-----KHDELDFEFLGNQPGK-----LYVLQTNPpXTH17 YTSSLSG-----KHDELDFEFLGNEPGK-----PYVLQTNPpXTH18 YTSSLSG-----KHDELDFEFLGNEAGK-----PYVLQTNPpXTH19 YLQSSTAS-DIDQHDEIDFELLGRISPRD----PYILQTNPpXTH20 YLQSPTAS-DIDQLDEIDFELLGRISPRN----PYILQTNPpXTH21 YMLS-TG----PKYCEFDFEFLGNETGQ-----PFLLHTNPpXTH22 YMTS-PG----PHHCELDFEFLGNQTGE-----PFLLHTNPpXTH23 YMSS-TG----PKHCEFDFEFLGNSSGQ-----PYLLHTNPpXTH24 YMAS-TG----PKHCEFDFEFLGNSSGQ-----PYLLHTNPpXTH25 YMAS-SG----PKHCEFDFEFLGNKPGM-----PYLLHTNPpXTH26 YMSS-QG----DQHYELDMEFLGNTSGQ-----PFLLHTNPpXTH27 YLSS-SG----PEHCELDMEFLGNSTGQ-----PFILHTNPpXTH28 YMMSPQG----DAHCEYDMEFLGNSTGQ-----PYLLHTNPpXTH29 YSKD--------THDEMDFEFLGNVTGE-----PITLQTNPpXTH30 YVSSGEGT---ITQDEIDFEFLGDNKR--------IVQTNPpXTH31 YISSGEGS---TMQDEIDFEFLGDNKR--------IVQTNPpXTH32 YLSSLEGD---KFQDEIDFEFLGKDKT--------IIQTN

Arabidopsis thaliana

Oryza sativa

Physcomitrella patens

(a)

(b)

Figure 5. Conserved sequences of XTH proteins. (a) Sequence alignment of

putative catalytic action-site amino acids residues in XTH proteins from

P. patens. The DEI/LDFEFLG motif is indicated above the alignment. Amino

acid residues that are identical within this motif are indicated by gray shading.

(b) SeqLOGO (http://weblogo.berkeley.edu/) for the conserved portions of the

XTH family. The heights of the letters indicate the frequency of the amino acid

in the XTH sequences of each plant species.



represented by P. patens, have evolved a special class of

XTHs that target polysaccharides other than xyloglucans

that are found specifically in mosses.

To evaluate this hypothesis, further biochemical analysis

of the PpXTH proteins, which have been shown to have

specific structural features in P. patens, together with struc-

tural analysis of P. patens cell walls, will be necessary. A

combination of expression analysis and reverse genetics

may offer the opportunity to explore new aspects of XTH

function, particularly in the context of bryophyte-specific

needs.

EXPERIMENTAL PROCEDURES

Plant material

Physcomitrella patens (Hedw.) Bruch & Schimp. subsp. patens wascultured and transformed as described by Nishiyama et al. (2000).

XET activity assay in P. patens

Sulforhodamine-labeled xyloglucan nonasaccharide (XLLG-SR) andsulforhodamine-labeled cellotetraose (GGGG-SR) were prepared asdescribed by Fry (1997) and Farkas et al. (2005) using HPLC-purifiedxyloglucan nonasaccharide (Tokyo Chemical Industry Co., http://www.tokyokasei.co.jp/) and HPLC-purified cellotetraose (SeikagakuCorporation, http://www.seikagaku.co.jp/english/), respectively.Lissamine rhodamine sulfonyl chloride was purchased from(Molecular Probes Inc., http://ja.invitrogen.com/site/jp/ja/home/brands/Molecular-Probes.html). XLLG-SR and GGGG-SR were puri-fiedbycolumnchromatographyonsilicagelusingmethanol/acetone(1:1v/v)andn-propanol/methanol/water(2:1:1v/v/v)solventsystems.

In situ XET activity was determined as described by Vissenberget al. (2000) using XLLG-SR as the substrate. For this assay, vegeta-tively propagated protonemata and gametophores were cultured onBCDAT medium (Nishiyama et al., 2000). Briefly, intact cells ortissues derived from P. patens culture were incubated in 25 mM MESbuffer solution (pH 5.5) without or with 32.5 mM XLLG-SR or

0.1

OsXTH23OsXTH25

OsXTH24OsXTH27

OsXTH28OsXTH29

AtXTH27AtXTH28

AtXTH29AtXTH30

OsXTH26AtXTH33

PpXTH1PpXTH2

OsXTH19OsXTH20

OsXTH21AtXTH31

AtXTH32OsXTH22

PpXTH30PpXTH31

PpXTH32AtXTH11

PpXTH29AtXTH1

AtXTH2AtXTH3

PpXTH23PpXTH24

PpXTH25PpXTH21

PpXTH22PpXTH26

PpXTH27PpXTH28

PpXTH3PpXTH4PpXTH5

PpXTH6PpXTH19

PpXTH20OsXTH17

AtXTH17AtXTH20

AtXTH18AtXTH19

AtXTH22AtXTH24

AtXTH23AtXTH25AtXTH21

OsXTH10OsXTH11

AtXTH12AtXTH13AtXTH14

AtXTH15AtXTH16

OsXTH12AtXTH26

OsXTH4OsXTH5

OsXTH6OsXTH7OsXTH8

OsXTH9OsXTH18

OsXTH16PpXTH16PpXTH17

PpXTH18AtXTH10OsXTH13

OsXTH14OsXTH15

OsXTH1AtXTH8

OsXTH3AtXTH9

AtXTH4AtXTH5

OsXTH2AtXTH6AtXTH7

PpXTH10PpXTH11

PpXTH12PpXTH13

PpXTH14PpXTH15

PpXTH7PpXTH8

PpXTH9

99

100

96

50

100

66

100

80

8088

100

9397

8865

9651

51

99

8796

58

65

100

8250

54

100

100

956468

7098

54

10082

8687

100100

100

8758

61

10081

6778

56

95

61

66

Group IIIB

Group IIIA

Group I/II

Figure 6. An unrooted neighbor-joining tree for

AtXTH, OsXTH and PpXTH genes.

Bootstrap probabilities >50% are given above the

branches. The horizontal branch length is pro-

portional to the estimated evolutionary distance.

Sub-family assignments (Baumann et al., 2007)

are indicated on the right.



GGGG-SR in the dark for 3 h. In a subset of experiments, unlabeledXLLG at concentrations of 0.5–5 mM was added to the incubationmedium as a competitive inhibitor. Incubation was terminated bywashing plant specimens in ethanol/formic acid/water (15:1:4 v/v/v)for 10 min, followed by an overnight wash in 5% formic acid.Fluorescence signals incorporated into the plant specimens wereobserved under a fluorescence differential interference microscope(Leica MZ APO, http://www.leica.com/) using green (546 � 10 nm)excitation light. Images were recorded using a CAMEDIA C-5050ZOOM camera (Olympus, http://www.olympus-global.com/).

Antibody

Polyclonal VaXTH1 rabbit antibody was raised against therecombinant full-length non-glycosylated azuki bean XTH protein

VaXTH1 (Yokoyama and Nishitani, 2001a; Nakamura et al., 2003).Pre-immune serum was used to quantify non-specific backgroundstaining for the antibody.

Immunocytochemistry

Physcomitrella patens was cultured on BCDAT medium prepared asdescribed by Nishiyama et al. (2000). Immunofluorescence locali-zation was performed on cultured tissues as described by Liepmanet al. (2007). Briefly, tissue was fixed in PBS containing 3.7% form-aldehyde, and washed in PBS. The washed tissue was then pre-incubated in 1% blocking reagent (Invitrogen/Molecular Probes,http://www.invitrogen.com/) for 1 h, followed by incubation withVaXTH1 antibody for 1 h in 1% blocking reagent. After washing inPBS, the tissue was incubated using a tyramide signal amplificationkit (Invitrogen/Molecular Probes) and horseradish peroxidase-con-jugated goat anti-mouse IgG antibody diluted 1:100 in 1% blockingreagent for 1 h at room temperature. Secondary antibody wasdetected by incubation with Alexa Fluor 488 tyramide for 15 minaccording to the manufacturer’s specifications (Invitrogen/Molecu-lar Probes). Samples were observed under a Leica MZ APO binoc-ular microscope equipped with a GFP filter or under a laser scanningconfocal microscope (Olympus Fluoview) equipped with a krypton/argon laser and filter sets suitable for detecting fluorescein. Con-focal images were acquired and processed using Fluoview micro-scope software as described by Yokoyama and Nishitani (2001a).

XTH sequence analyses

Genome assembly release version 1.1 (Phypha 1_1) for P. patens,which was constructed via the Joint Genome Institute annotationpipeline and contains approximately 35 938 gene models availablefor searching at http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html, was used to compile the putative XTH gene of P. pat-ens. We first repeatedly performed BLASTp searches against thegene model set under default conditions using all availablesequences, including 33 AtXTH genes, 29 OsXTH genes and twoknown P. patens XTH sequences (AX172659 and AX172661). If thegene models identified by BLASTp lacked structural features char-acteristic of the XTH family of genes, they were re-examined byalignment of EST sequences and genome sequences. If onlygenomic sequences were available, translated sequences weremanually re-interpreted based on multiple sequence alignments ofpredicted amino acid sequences using the XTH sequences identifiedto date. Intron insertion sites were identified using the NetGene2Server (http://www.cbs.dtu.dk/services/NetGene2/). Protein locali-zation sites were predicted using the Wolf PSORT program (http://wolfpsort.org/).

Sequence alignment and phylogenetic tree

Alignments of full-length amino acid sequences comprising 32PpXTHs, 33 AtXTHs and 29 OsXTHs were generated using MAFFT

version 6.611b (Katoh et al., 2005).After manual revision of the alignment and removal of all gaps, 91

amino acids were used to calculate evolutionary distances using theJTT model (Jones et al., 1992) and to construct a neighbor-joiningtree (Saitou and Nei, 1987). Statistical support for internal branchesby bootstrap analyses was calculated for 1000 replications using thePROTDIST, NEIGHBOR, SEQBOOT and CONSENSE programs in thePHYLIP version 3.69 software package (J. Felsenstein, Department ofGenetics, University of Washington, Seattle, WA).

Plasmid construction

The 5¢ DNA fragments of PpXTH16, 21 and 32 genomic DNA fromthe intron or 5¢ flanking region to just before the stop codon were

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

Figure 7. Histochemical detection of GUS activity in transgenic P. patens

expressing PpXTH16::GUS, PpXTH21::GUS and PpXTH32::GUS.

(a, d, g, i) GUS activity in PpXTH16::GUS-transformed plants.

(b, e, h, k) GUS activity in PpXTH21::GUS-transformed plants.

(c, f, i, l) GUS activity in PpXTH32::GUS-transformed plants.

(a–c) Representative protonemata cultured for 7 days on standard medium.

(d–f) Representative gametophyte cultured for 3 weeks.

(g–i) Representative gametophyte cultured for 5 weeks.

(j–l) Representative 4-month-old gametophyte. Insets show sporangia.

Scale bars = 100 lm (a–c) and 1 mm (d–l).



amplified from genomic DNA of P. patens using the followingforward and reverse PCR primer sets: PpXTH16, 5¢-CGGGG-TACCGGTAACGGTGCTGAGTTGAAA-3¢ and 5¢-CCCATCGATGTTC-TCAAGAACCTTCAACCC-3¢; PpXTH21, 5¢-CGGGGTACCGACTGAA-GGCACAGAAACTT-3¢ and 5¢-CCCAAGCTTCAGGGTGTTGTGA-GCGC-3¢; PpXTH32, 5¢-CGGGGTACCCACTCAGATCGGCTTCT-AGCT-3¢ and 5¢-CCCAAGCTTCTGATCAGGGAGATGAGGAAC-3¢.The underlined nucleotides indicate restriction sites that were usedin subsequent plasmid construction. The amplified fragment wasinserted into the XhoI–HindIII, KpnI–ClaI or KpnI–HindIII sites of thepTN85 plasmid (accession number AB267707), which contains thecoding sequence of uidA, the nopaline synthase polyadenylationsignal and an NPTII cassette (Nishiyama et al., 2000), thereby cre-ating an in-frame fusion of the PpXTH and uidA genes. Fragmentscontaining the 3¢ region of PpXTH genes were produced by PCRusing the following forward and reverse PCR primer sets: PpXTH16,5¢-CGCGGATCCGCCTGATTTAACTGGCAATT-3¢ and 5¢-GATGCGGCCGCGCGCAATGACATAAAATGA-3¢; PpXTH21, 5¢-CGCGGATCCTAGAGACCGTTGCAGTCACC-3¢ and 5¢-CGCGAGCTCGGCTGTGTTGCTGTTCATACT-3¢; AtXTH32, 5¢-CGCGGATCCTCAACAACACCC-GATTGTG-3¢ and 5¢-CGCGAGCTCCTCTGTGCAAATTATCAATGT-3¢.Each of these primers contained one restriction site, which isunderlined. The amplified fragment was inserted into the BamHI–NotI or BamHI–SacI site of the plasmid containing the 5¢ DNA frag-ment. The resulting plasmid was linearized using KpnI and SacI orNotI to generate the PpXTH–GUS fusion constructs. Polyethyleneglycol-mediated transformation was performed as previouslydescribed (Nishiyama et al., 2000). Of 80 independent stable trans-formants, approximately 20 lines were identified by PCR ascontaining the GUS fusion construct at the PpXTH locus. UsingSouthern analyses, five lines containing a single insertion in thePpXTH locus were selected.

Transgenic P. patens plants were incubated in X-gluc solution(0.5 mM 5-bromo-4-chloro-3-indolyl-b-glucuronide, 50 mM sodiumphosphate, pH 7.0) at 37�C for 2–24 h. Fixation was performed in 5%v/v formalin for 10 min, followed by incubation for 10 min in 5% v/vacetic acid. Pigments in the tissues were removed by serialincubation in 30%, 50%, 70% and 100% ethanol.

Analysis of the distribution of ESTs in P. patens

The relative abundances of PpXTH transcripts in the EST databaseswere estimated based on the total number of clones identified fromeither 5¢ or 3¢ sequences in the libraries (Nishiyama et al., 2003) andother P. patens clones deposited in public databases. The followinglibraries were used: untreated protonema and gametophore library(9944 5¢ ESTs and 9352 3¢ ESTs), auxin-treated library (16 733 5¢ESTs and 16 763 3¢ ESTs), cytokinin-treated library (16 450 5¢ ESTsand 15 000 3¢ ESTs), library for protoplasts during the first celldivision (10 535 5¢ ESTs and 10 975 3¢ ESTs), and library for sporo-phytes before meiosis with surrounding archegonia (8514 5¢ ESTsand 8241 3¢ ESTs).

Production and purification of recombinant PpXTH32

protein

The cDNA of the PpXTH32 was amplified using forward and reverseprimers 5¢-CCGCTCGAGAAAAGAATGCAGTGCATGGCC-3¢ and5¢-TGCTCTAGATTACTGATCAGGGAGATGAGG-3¢, respectively,and cloned into the pBluescript II SK+ vector (Invitrogen, http://www.invitrogen.com/site/us/en/home.html). The XhoI/XbaI frag-ment was excised from the pBluescript clone, and cloned into thepPICZaA vector in-frame with the a factor secretion signal sequence(Invitrogen). The resulting pPICZaA-PpXTH32 expression vectorwas used to transform Pichia pastoris strain X-33, and recombinant

PpXTH32 protein was generated according to the manufacturer’sprotocol for Pichia expression kit, followed by purification bysuccessive anion-exchange chromatography (Hitrap Q FF andResource Q) and gel-permeation chromatography (Superdex 7510/300GL) with HPLC (AKTA purifier; GE Healthcare, http://www.gehealthcare.com).

Enzyme assay

For measurement of hydrolytic activity, the enzyme reaction mix-ture consisted of 62 ng recombinant PpXTH32, 10 lg nasturtium(Tropaeolum majus L.) xyloglucan or approximately 30 lg lichenanderived from Cetraria islandica, in 50 ll of 25 mM sodium acetatebuffer at pH 5, 6 or 7. As a control experiment, denatured PpXTH32was added. After incubation at 30�C for 3 h, the molecular weightdistribution profile of the polysaccharide substrate was estimatedby size-exclusion chromatography on columns of TSK-GELG3000PWXL and TSK-GEL G5000PWXL (Tosoh Corporation, http://www.tosoh.com/) connected in series using an HPLC system(Dionex D500, http://www.dionex.com/en-us/index.html) equippedwith a pulsed amperometric detector as described by Nishitani andTominaga (1992).

For measurement of XET activity, the reaction mixture contained62 ng recombinant PpXTH32, 10 lg nasturtium xyloglucan and0.1 mM 2-pyridylamino xyloglucan oligomer in 50 ll of 25 mM

sodium acetate buffer at pH 5.5. After incubation at 30�C for 3 h,the reaction mixture was separated by size-exclusion chromato-graphy on the same columns using the Dionex D500 HPLC equippedwith both a pulsed amperometric detector and a fluorescencedetector (Nishitani and Tominaga, 1992).

ACKNOWLEDGEMENTS

We thank Ms. Kei Saito for her excellent technical assistance. Thiswork was supported by Grants-in-Aid for Scientific Research onPriority Areas number 19039003 to K.N. and by Grants-in-Aid forScientific Research (B) number 19370014 to K.N. and (C) number19370014 to R.Y. from the Ministry of Education, Culture, Sports,Science and Technology of Japan.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Alignment of the amino acid sequences of 32 PpXTHproteins constructed using the Clustal W program.Figure S2. Frequency of ESTs from tissue-specific full-length cDNAlibraries.Figure S3. Size-exclusion chromatograms of reaction products forPpXTH32 protein and polysaccharide.Table S1. XTH family genes in Physcomitrella patens.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

REFERENCES

Akamatsu, T., Hanzawa, Y., Ohtake, Y., Takahashi, T., Nishitani, K. and

Komeda, Y. (1999) Expression of endoxyloglucan transferase genes in

acaulis mutants of Arabidopsis. Plant Physiol. 121, 715–721.

Baumann, M.J., Eklof, J.M., Michel, G., Kallas, A.M., Teeri, T.T., Czjzek, M. and

Brumer, H. III (2007) Structural evidence for the evolution of xyloglucanase

activity from xyloglucan endo-transglycosylases: biological implications

for cell wall metabolism. Plant Cell, 19, 1947–1963.



Becnel, J., Natarajan, M., Kipp, A. and Braam, J. (2006) Developmental

expression patterns of Arabidopsis XTH genes reported by transgenes and

genevestigator. Plant Mol. Biol. 61, 451–467.

Bourquin, V., Nishikubo, N., Abe, H., Brumer, H., Denman, S., Eklund, M.,

Christiernin, M., Teeri, T.T., Sundberg, B. and Mellerowicz, E.J. (2002)

Xyloglucan endotransglycosylases have a function during the formation of

secondary cell walls of vascular tissues. Plant Cell, 14, 3073–3088.

Campbell, P. and Braam, J. (1999) In vitro activities of four xyloglucan endo-

transglycosylases from Arabidopsis. Plant J. 18, 371–382.

Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V.

and Henrissat, B. (2009) The Carbohydrate-Active EnZymes database

(CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37,

233–238.

Carey, R.E. and Cosgrove, D.J. (2007) Portrait of the expansin superfamily in

Physcomitrella patens: comparisons with angiosperm expansins. Ann. Bot.

95, 1131–1141.

Carpita, N.C. and Gibeaut, D.M. (1993) Structural models of primary cell walls

in flowering plants: consistency of molecular structure with the physical

properties of the walls during growth. Plant J. 3, 1–30.

Catala, C., Rose, J.K.C. and Bennett, A.B. (1997) Auxin-regulation and spatial

localization of an endo-1,4-b-D-glucanase and a xyloglucan endotransgly-

cosylase in expanding tomato hypocotyls. Plant J. 12, 417–426.

Chen, F., Nonogaki, H. and Bradford, K.J. (2002) A gibberellin-regulated

xyloglucan endotransglycosylase gene is expressed in the endosperm cap

during tomato seed germination. J. Exp. Bot. 53, 215–223.

Cosgrove, D.J. (2005) Growth of the plant cell wall. Nat. Rev. Mol. Cell Biol. 6,

850–861.

Cove, D.J., Knight, C.D. and Lamparter, T. (1997) Mosses as model systems.

Trends Plant Sci. 2, 99–105.

Eklof, J.M. and Brumer, H. (2010) The XTH gene family: an update on enzyme

structure, function, and phylogeny in xyloglucan remodeling. Plant Phys-

iol. 153, 456–466.

Farkas, V., Ait-Mohand, F. and Stratilova, E. (2005) Sensitive detection of

transglycosylating activity of xyloglucan endotransglycosylase/hydrolase

(XTH) after isoelectric focusing in polyacrylamide gels. Plant Physiol.

Biochem. 43, 431–435.

Fry, S.C. (1997) Novel ‘dot-blot’ assays for glycosyltransferases and glyco-

sylhydrolases: optimization for xyloglucan endotransglycosylase (XET)

activity. Plant J. 11, 1141–1150.

Fry, S.C., Smith, R.C., Renwick, K.F., Martin, D.J., Hodge, S.K. and Matthews,

K.J. (1992) Xyloglucan endotransglycosylase, a new wall-loosening

enzyme activity from plants. Biochem. J. 282, 821–828.

Fry, S.C., Mohler, K.E., Nesselrode, B.H.W.A. and Frankova, L. (2008) Mixed-

linkage b-glucan:xyloglucan endotransglucosylase, a novel wall-remodel-

ling enzyme from Equisetum (horsetails) and charophytic algae. Plant J. 55,

240–252.

Geisler-Lee, J., Geisler, M., Coutinho, P.M. et al. (2006) Poplar carbohydrate-

active enzymes. Gene identification and expression analyses. Plant Phys-

iol. 140, 946–962.

Gouet, P., Courcelle, E., Stuart, D.I. and Metoz, F. (1999) ESPript: analysis of

multiple sequence alignments in PostScript. Bioinformatics, 15, 305–308.

Henrissat, B., Coutinho, P.M. and Davies, G.J. (2001) A census for carbohy-

drate-active enzymes in the genome of Arabidopsis thaliana. Plant Mol.

Biol. 47, 55–72.

Hrmova, M., Farkas, V., Lahnstein, J. and Fincher, G.B. (2007) A barley xylo-

glucan xyloglucosyl transferase covalently links xyloglucan, cellulosic

substrates, and (1,3,1,4)-b-D-glucans. J. Biol. Chem. 282, 12951–12962.

Hyodo, H., Yamakawa, S., Takeda, Y., Tsuduki, M., Yokota, A., Nishitani, K.

and Kouchi, T. (2003) Active gene expression of a xyloglucan endotrans-

glucosylase/hydrolase gene, XTH9, in inflorescence apices is related to cell

elongation in Arabidopsis thaliana. Plant Mol. Biol. 54, 473–482.

Ito, H. and Nishitani, K. (1999) Visualization of EXGT-mediated molecular

grafting activity by means of a fluorescently labeled xyloglucan oligomer.

Plant Cell Physiol. 40, 1172–1176.

Johansson, P., Brumer, H. III, Baumann, M.J., Kallas, A.M., Henriksson, H.,

Denman, S.E., Teeri, T.T. and Jones, T.A. (2004) Crystal structures of a

poplar xyloglucan endotransglycosylase reveal details of transglycosyla-

tion acceptor binding. Plant Cell, 16, 874–886.

Jones, D.T., Taylor, W.R. and Thornton, J.M. (1992) The rapid generation of

mutation data matrices from protein sequences. Comput. Appl. Biosci. 8,

275–282.

Katoh, K., Kuma, K., Toh, H. and Miyata, T. (2005) MAFFT version 5:

improvement in accuracy of multiple sequence alignment. Nucleic Acids

Res. 33, 511–518.

Liepman, A.H., Nairn, C.J., Willats, W.G.T., Sørensen, I., Roberts, A.W. and

Keegstra, K. (2007) Functional genomic analysis supports conservation of

function among cellulose synthase-like A gene family members and sug-

gests diverse roles of mannans in plants. Plant Physiol. 143, 1881–1893.

Marcus, S.E., Verhertbruggen, Y., Herve, C., Ordaz-Ortiz, J.J., Frakas, V.,

Pedersen, H.L., Willats, W.G. and Knox, J.P. (2008) Pectic homogalacturo-

nan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC

Plant Biol. 8, 60.

Matsui, A., Yokoyama, R., Seki, M., Ito, T., Shinozaki, K., Takahashi, T.,

Komeda, Y. and Nishitani, K. (2005) AtXTH27 plays an essential role in cell

wall modification during the development of tracheary elements. Plant J.

42, 525–534.

Menand, B., Calder, G. and Dolan, L. (2007) Both chloronemal and caulonemal

cells expand by tip growth in the moss Physcomitrella patens. J. Exp. Bot.

58, 1843–1849.

Nakamura, T., Yokoyama, R., Tomita, E. and Nishitani, K. (2003) Two azuki

bean XTH genes, VaXTH1 and VaXTH2, with similar tissue-specific

expression profiles, are differently regulated by auxin. Plant Cell Physiol.

44, 16–24.

Nishikubo, N., Awano, T., Banasiak, A. et al. (2007) Xyloglucan endo-trans-

glycosylase (XET) functions in gelatinous layers of tension wood fibers in

poplar – a glimpse into the mechanism of the balancing act of trees. Plant

Cell Physiol. 48, 843–855.

Nishitani, K. (1997) The role of endoxyloglucan transferase in the organization

of plant cell walls. Int. Rev. Cytol. 173, 157–206.

Nishitani, K. and Tominaga, R. (1992) Endoxyloglucan transferase, a novel

class of glycosyltransferase that catalyzes transfer of a segment of xylo-

glucan molecule to another xyloglucan molecule. J. Biol. Chem. 267,

21058–21064.

Nishitani, K. and Vissenberg, K. (2006) Roles of the XTH protein family in

the expanbng cell. In The Expanding Cell. Plant Cell Monographs, Vol. 5

(Verbelen, J.-P. and Vissenberg, K., eds). Berlin/Heidelberg: Springer-

Verlag, pp. 89–116.

Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M. and Hasebe, M. (2000)

Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by

shuttle mutagenesis. DNA Res. 7, 9–17.

Nishiyama, T., Fujita, T., Shin-I, T. et al. (2003) Comparative genomics of

Physcomitrella patens gametophytic transcriptome and Arabidopsis thali-

ana: implication for land plant evolution. Proc. Natl Acad. Sci. USA, 100,

8007–8012.

Okazawa, K., Sato, Y., Nakagawa, T., Asada, K., Kato, I., Tomita, E. and

Nishitani, K. (1993) Molecular cloning and cDNA sequencing of endoxy-

loglucan transferase, a novel class of glycosyltransferase that mediates

molecular grafting between matrix polysaccharides in plant cell walls.

J. Biol. Chem. 268, 25364–25368.

Osato, Y., Yokoyama, R. and Nishitani, K. (2006) A principal role for AtXTH18

in Arabidopsis thaliana root growth: a functional analysis using RNAi

plants. J. Plant. Res. 119, 153–162.

Pena, M.J., Darvill, A.G., Eberhard, S., York, W.S. and O’Neill, M.A. (2008)

Moss and liverwort xyloglucans contain galacturonic acid and are struc-

turally distinct from the xyloglucans synthesized by hornworts and vas-

cular plants. Glycobiology, 18, 891–904.

Popper, Z.A. and Fry, S.C. (2003) Primary cell wall composition of bryophytes

and charophytes. Ann. Bot. 91, 1–12.

Roberts, A.W. and Bushoven, J.T. (2007) The cellulose synthase (CESA) gene

superfamily of the moss Physcomitrella patens. Plant Mol. Biol. 63, 207–

219.

Rose, J.K.C. and Bennett, A.B. (1999) Co-operative disassembly of the cellu-

lose–xyloglucan network of plant cell walls: parallels between cell expan-

sion and fruit ripening. Trends Plant Sci. 4, 176–183.

Rose, J.K.C., Braam, J., Fry, S.C. and Nishitani, K. (2002) The XTH family of

enzymes involved in xyloglucan endotransglucosylation and endohydro-

lysis: current perspectives and a new unifying nomenclature. Plant Cell

Physiol. 43, 1421–1435.

Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

Schaefer, D.G. (2002) A new moss genetics: targeted mutagenesis in Physc-

omitrella patens. Annu. Rev. Plant Biol. 53, 477–501.



Thompson, J.E., Smith, R.C. and Fry, S.C. (1997) Xyloglucan undergoes

interpolymeric transglycosylation during binding to the plant cell wall

in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation

in caesium trifluoroacetate. Biochem. J. 327, 699–708.

Van Sandt, V.S.T., Guisez, Y., Verbelen, J.-P. and Vissenberg, K. (2007a)

Xyloglucan endotransglycosylase/hydrolase (XTH) is encoded by a multi-

gene family in the primitive vascular land plant Selaginella kraussiana.

Plant Biol. 9, 142–146.

Van Sandt, V.S.T., Stieperaere, H., Guisez, Y., Verbelen, J.-P. and Vissenberg,

K. (2007b) XET activity is found near sites of growth and cell elongation in

bryophytes and some green algae: new insights into the evolution of pri-

mary cell wall elongation. Ann. Bot. 99, 39–51.

Vissenberg, K., Martinez-Vilchez, I.M., Verbelen, J.-P., Miller, J.G. and Fry,

S.C. (2000) In vivo colocalization of xyloglucan endotransglycosylase

activity and its donor substrate in the elongation zone of Arabidoposis

roots. Plant Cell, 12, 1229–1238.

Vissenberg, K., Van Sandt, V.S.T., Fry, S.C. and Verbelen, J.-P. (2003) Xylo-

glucan endotransglucosylase action is high in the root elongation zone and

in the trichoblasts of all vascular plants from Selaginella to Zea mays.

J. Exp. Bot. 54, 335–344.

Vissenberg, K., Oyama, M., Osato, Y., Yokoyama, R., Verbelen, J.-P. and

Nishitani, K. (2005) Differential expression of AtXTH17, AtXTH18, AtXTH19

and AtXTH20 genes in Arabidopsis roots. Physiological roles in specifica-

tion in cell wall construction. Plant Cell Physiol. 46, 192–200.

Xu, W., Campbell, P., Vargheese, A.K. and Braam, J. (1996) The Arabidopsis

XET-related gene family: environmental and hormonal regulation of

expression. Plant J. 9, 879–889.

Yokoyama, R. and Nishitani, K. (2000) Functional diversity of xyloglucan-

related proteins and its implications in the cell wall dynamics in plants.

Plant Biol. 2, 598–604.

Yokoyama, R. and Nishitani, K. (2001a) Endoxyloglucan transferase is local-

ized both in the cell plate and in the secretary pathway destined for the

apoplast in tobacco cells. Plant Cell Physiol. 42, 292–300.

Yokoyama, R. and Nishitani, K. (2001b) A comprehensive expression analysis

of all members of a gene family encoding cell-wall enzymes allowed us to

predict cis-regulatory regions involved in cell-wall construction in specific

organs of Arabidopsis. Plant Cell Physiol. 42, 1025–1033.

Yokoyama, R. and Nishitani, K. (2004) Genomic basis for cell-wall diversity in

plants: a comparative approach to gene families in rice and Arabidopsis.

Plant Cell Physiol. 45, 1111–1121.

Yokoyama, R., Rose, J.K.C. and Nishitani, K. (2004) A surprising diversity and

abundance of xyloglucan endotransglucosylase/hydrolases in rice. Classi-

fication and expression analysis. Plant Physiol. 134, 1088–1099.



Documents

The Plant Journal 64 Biological implications of the ...€¦ · GH16.html) (Cantarel et al., 2009). In angiosperms, XTHs are typically encoded by large multi-gene families (Okazawa