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The Plant Journal (2010) 64, 658–669
doi: 10.1111/j.1365-313X.2010.04351.x
Biological implications of the occurrence of 32 members of
the XTH (xyloglucan endotransglucosylase/hydrolase) family
of proteins in the bryophyte Physcomitrella patens
Ryusuke Yokoyama1, Yohei Uwagaki1, Hiroki Sasaki1, Taro Harada1, Yuji Hiwatashi2,3, Mitsuyasu Hasebe2,3,4 and
Kazuhiko Nishitani1,*
1
Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University,
Sendai 980-8578, Japan,
2
National Institute for Basic Biology, Okazaki 444-8585, Japan,
3
School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan, and
4
ERATO, 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 tissuedependent. 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).
658
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
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd
XTH family of genes in Physcomitrella patens 659
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 available 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). Identification 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 development (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 characteristic 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 characteristics 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 bryophytespecific XTH genes with distinct protein structures that are
not found in angiosperms, suggesting unique roles in nonvascular 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 (XLLGSR) 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 gametophores, 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 unlabeled 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 tissuedependent 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
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
660 Ryusuke Yokoyama et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
(q)
(r)
(s)
(t)
(u)
(v)
(w)
(x)
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 gametophores 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 immunohistochemical analysis or to reduced sensitivity of the
septum-localized PpXTH(s) to the anti-VaXTH1 antibody.
Recently, Marcus et al. (2008) showed that pectic homogalacturonan 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.
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.
P. patens XTH family genes
The presence of XTH family genes in P. patens was determined 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 structural 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 features 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 determined based on genomic sequences (Figure 3). Of the
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
XTH family of genes in Physcomitrella patens 661
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). Epifluorescence images (a–d, i–l) and differential interference 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.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
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 characterize 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 detection 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 expressed strongly at the apical portion of young gametophores (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 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
662 Ryusuke Yokoyama et al.
specificity and responses to hormonal and environmental
signals (Figure S1).
PpXTH1
PpXTH2
*PpXTH3
DISCUSSION
*PpXTH4
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).
*PpXTH5
PpXTH6
*PpXTH7
*PpXTH8
PpXTH9
*PpXTH10
*PpXTH11
*PpXTH12
*PpXTH13
*PpXTH14
PpXTH15
Structural diversity of PpXTH
PpXTH16
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 conserved 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 structures 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 (Eklöf 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
PpXTH17
*PpXTH18
PpXTH19
PpXTH20
*PpXTH21
*PpXTH22
[ ]
*PpXTH23
*PpXTH24
*PpXTH25
*PpXTH26
*PpXTH27
*PpXTH28
PpXTH29
500 bp
PpXTH30
*PpXTH31
*PpXTH32
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).
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. patens 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 suggests differential expression patterns in terms of organ
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
XTH family of genes in Physcomitrella patens 663
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.
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 properties of bryophyte-specific XTHs, PpXTH32 protein was
recombinantly produced using a Pichia pastoris heterologous expression system. The purified recombinant
PpXTH32 protein did not exhibit either endotransglucosylase 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). Immunohistochemical 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 restructuring of xyloglucan cross-links in cell expansion during tip
growth of bryophytes.
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
664 Ryusuke Yokoyama et al.
(a)
PpXTH1
PpXTH2
PpXTH3
PpXTH4
PpXTH5
PpXTH6
PpXTH7
PpXTH8
PpXTH9
PpXTH10
PpXTH11
PpXTH12
PpXTH13
PpXTH14
PpXTH15
PpXTH16
PpXTH17
PpXTH18
PpXTH19
PpXTH20
PpXTH21
PpXTH22
PpXTH23
PpXTH24
PpXTH25
PpXTH26
PpXTH27
PpXTH28
PpXTH29
PpXTH30
PpXTH31
PpXTH32
DEIDFEFLG
L
YASNGGYYP--YNHDEIDMEFLGVRPGQ-----PYVIQTN
YASNGGSYP--SNHDEIDLEFLGVRPGH-----PYVIQTN
YLSSQG-----HNHDEVDFEFLGNVTG-EP----YVLQTN
YLSSQG-----HDHSEVDFEFLGNKTNTQPGNNEIVLQTN
YTSSDGKK---PYHDEIDIELLGNETS-----SCITMQTN
YTSSDGKM---DDHDEIDIELLGNETS-----KHITMQTN
YMSSQTP----GLHDEMDFEFLGNVTGQ-----PYILQTN
YMSSQTP----GLHDEMDFEFLGNVSGQ-----PYILQTN
YLSSDT-----ARHDEMDFEFLGNVSGQ-----PYILQTN
YLSSAQ-----PNHDELDFEFLGNVSGE-----PYVLQTN
YLSSAQ-----PNHDELDFEFLGNVSGQ-----PYSLQTN
YLSSDQ-----PAHDELDFEFLGNTSGD-----PYILQTN
YLYSPT-----DHHDELDFEFLGNSSGQ-----PYILQTN
YLSSEGGE-----HDEMDFEFLGKGGDQ-----PYILQTN
YLSSEGGQSVRSVHDEMDFEFLGNSSGQ-----PYILQTN
YTSSLSG-----KHDELDFEFLGNQPGK-----LYVLQTN
YTSSLSG-----KHDELDFEFLGNEPGK-----PYVLQTN
YTSSLSG-----KHDELDFEFLGNEAGK-----PYVLQTN
YLQSSTAS-DIDQHDEIDFELLGRISPRD----PYILQTN
YLQSPTAS-DIDQLDEIDFELLGRISPRN----PYILQTN
YMLS-TG----PKYCEFDFEFLGNETGQ-----PFLLHTN
YMTS-PG----PHHCELDFEFLGNQTGE-----PFLLHTN
YMSS-TG----PKHCEFDFEFLGNSSGQ-----PYLLHTN
YMAS-TG----PKHCEFDFEFLGNSSGQ-----PYLLHTN
YMAS-SG----PKHCEFDFEFLGNKPGM-----PYLLHTN
YMSS-QG----DQHYELDMEFLGNTSGQ-----PFLLHTN
YLSS-SG----PEHCELDMEFLGNSTGQ-----PFILHTN
YMMSPQG----DAHCEYDMEFLGNSTGQ-----PYLLHTN
YSKD--------THDEMDFEFLGNVTGE-----PITLQTN
YVSSGEGT---ITQDEIDFEFLGDNKR--------IVQTN
YISSGEGS---TMQDEIDFEFLGDNKR--------IVQTN
YLSSLEGD---KFQDEIDFEFLGKDKT--------IIQTN
(b)
Arabidopsis thaliana
Oryza sativa
Physcomitrella patens
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.
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 (Thompson 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 endotranglucosylase. 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, suggesting 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 diversification 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 transglycosylation between xyloglucans and other glucans, including (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
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
XTH family of genes in Physcomitrella patens 665
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 proportional to the estimated evolutionary distance.
Sub-family assignments (Baumann et al., 2007)
are indicated on the right.
61
0.1
56
78
67
81
100
66
58
100
61
OsXTH23
OsXTH25
OsXTH24
OsXTH27
OsXTH28
OsXTH29
AtXTH27
AtXTH28
AtXTH29
95
AtXTH30
AtXTH33
PpXTH1
PpXTH2
87
OsXTH19
OsXTH20
OsXTH21
AtXTH31
AtXTH32
OsXTH22
100
PpXTH29
AtXTH1
AtXTH2
AtXTH3
PpXTH23
95
64
PpXTH24
68
PpXTH25
PpXTH21
98
PpXTH22
70
PpXTH26
54
PpXTH27
PpXTH28
PpXTH3
PpXTH4
PpXTH5
100
PpXTH6
100
PpXTH19
PpXTH20
OsXTH17
58 AtXTH17
AtXTH20
100
AtXTH18
50
AtXTH19
65
82
AtXTH22
AtXTH24
54
AtXTH23
AtXTH25
AtXTH21
OsXTH10
OsXTH11
87 AtXTH12
96
AtXTH13
AtXTH14
99
AtXTH15
AtXTH16
OsXTH12
51
AtXTH26
88
OsXTH4
65
OsXTH5
OsXTH6
96
OsXTH7
51
OsXTH8
OsXTH9
OsXTH18
OsXTH16
82
PpXTH16
100
PpXTH17
PpXTH18
AtXTH10
93
OsXTH13
97
OsXTH14
OsXTH15
OsXTH1
100
AtXTH8
80
OsXTH3
AtXTH9
AtXTH4
80
88
AtXTH5
OsXTH2
AtXTH6
100
AtXTH7
PpXTH10
50
PpXTH11
100
PpXTH12
PpXTH13
PpXTH14
PpXTH15
87
66
99
100
96
PpXTH7
PpXTH8
PpXTH9
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 structural 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 was
cultured and transformed as described by Nishiyama et al. (2000).
100
Group IIIB
OsXTH26
Group IIIA
PpXTH30
PpXTH31
PpXTH32
AtXTH11
86
Group I/II
XET activity assay in P. patens
Sulforhodamine-labeled xyloglucan nonasaccharide (XLLG-SR) and
sulforhodamine-labeled cellotetraose (GGGG-SR) were prepared as
described by Fry (1997) and Farkaš et al. (2005) using HPLC-purified
xyloglucan nonasaccharide (Tokyo Chemical Industry Co., http://
www.tokyokasei.co.jp/) and HPLC-purified cellotetraose (Seikagaku
Corporation, 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 purified by column chromatography on silica gel using methanol/acetone
(1:1v/v)andn-propanol/methanol/water(2:1:1v/v/v) solventsystems.
In situ XET activity was determined as described by Vissenberg
et al. (2000) using XLLG-SR as the substrate. For this assay, vegetatively propagated protonemata and gametophores were cultured on
BCDAT medium (Nishiyama et al., 2000). Briefly, intact cells or
tissues derived from P. patens culture were incubated in 25 mM MES
buffer solution (pH 5.5) without or with 32.5 mM XLLG-SR or
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
666 Ryusuke Yokoyama et al.
(a)
(b)
(c)
VaXTH1 (Yokoyama and Nishitani, 2001a; Nakamura et al., 2003).
Pre-immune serum was used to quantify non-specific background
staining for the antibody.
Immunocytochemistry
(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).
Physcomitrella patens was cultured on BCDAT medium prepared as
described by Nishiyama et al. (2000). Immunofluorescence localization was performed on cultured tissues as described by Liepman
et al. (2007). Briefly, tissue was fixed in PBS containing 3.7% formaldehyde, and washed in PBS. The washed tissue was then preincubated in 1% blocking reagent (Invitrogen/Molecular Probes,
http://www.invitrogen.com/) for 1 h, followed by incubation with
VaXTH1 antibody for 1 h in 1% blocking reagent. After washing in
PBS, the tissue was incubated using a tyramide signal amplification
kit (Invitrogen/Molecular Probes) and horseradish peroxidase-conjugated goat anti-mouse IgG antibody diluted 1:100 in 1% blocking
reagent for 1 h at room temperature. Secondary antibody was
detected by incubation with Alexa Fluor 488 tyramide for 15 min
according to the manufacturer’s specifications (Invitrogen/Molecular Probes). Samples were observed under a Leica MZ APO binocular microscope equipped with a GFP filter or under a laser scanning
confocal microscope (Olympus Fluoview) equipped with a krypton/
argon laser and filter sets suitable for detecting fluorescein. Confocal images were acquired and processed using Fluoview microscope 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 annotation
pipeline and contains approximately 35 938 gene models available
for searching at http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.
home.html, was used to compile the putative XTH gene of P. patens. We first repeatedly performed BLASTp searches against the
gene model set under default conditions using all available
sequences, including 33 AtXTH genes, 29 OsXTH genes and two
known P. patens XTH sequences (AX172659 and AX172661). If the
gene models identified by BLASTp lacked structural features characteristic of the XTH family of genes, they were re-examined by
alignment of EST sequences and genome sequences. If only
genomic sequences were available, translated sequences were
manually re-interpreted based on multiple sequence alignments of
predicted amino acid sequences using the XTH sequences identified
to date. Intron insertion sites were identified using the NetGene2
Server (http://www.cbs.dtu.dk/services/NetGene2/). Protein localization sites were predicted using the Wolf PSORT program (http://
wolfpsort.org/).
Sequence alignment and phylogenetic tree
GGGG-SR in the dark for 3 h. In a subset of experiments, unlabeled
XLLG at concentrations of 0.5–5 mM was added to the incubation
medium as a competitive inhibitor. Incubation was terminated by
washing 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 were
observed 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-5050
ZOOM camera (Olympus, http://www.olympus-global.com/).
Alignments of full-length amino acid sequences comprising 32
PpXTHs, 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 the
JTT model (Jones et al., 1992) and to construct a neighbor-joining
tree (Saitou and Nei, 1987). Statistical support for internal branches
by bootstrap analyses was calculated for 1000 replications using the
PROTDIST, NEIGHBOR, SEQBOOT and CONSENSE programs in the
PHYLIP version 3.69 software package (J. Felsenstein, Department of
Genetics, University of Washington, Seattle, WA).
Antibody
Plasmid construction
Polyclonal VaXTH1 rabbit antibody was raised against the
recombinant full-length non-glycosylated azuki bean XTH protein
The 5¢ DNA fragments of PpXTH16, 21 and 32 genomic DNA from
the intron or 5¢ flanking region to just before the stop codon were
ª 2010 The Authors
The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669
XTH family of genes in Physcomitrella patens 667
PpXTH32 protein was generated according to the manufacturer’s
protocol for Pichia expression kit, followed by purification by
successive anion-exchange chromatography (Hitrap Q FF and
Resource Q) and gel-permeation chromatography (Superdex 75
10/300GL) with HPLC (AKTA purifier; GE Healthcare, http://
www.gehealthcare.com).
amplified from genomic DNA of P. patens using the following
forward and reverse PCR primer sets: PpXTH16, 5¢-CGGGGTACCGGTAACGGTGCTGAGTTGAAA-3¢ and 5¢-CCCATCGATGTTCTCAAGAACCTTCAACCC-3¢; PpXTH21, 5¢-CGGGGTACCGACTGAAGGCACAGAAACTT-3¢ and 5¢-CCCAAGCTTCAGGGTGTTGTGAGCGC-3¢; PpXTH32, 5¢-CGGGGTACCCACTCAGATCGGCTTCTAGCT-3¢ and 5¢-CCCAAGCTTCTGATCAGGGAGATGAGGAAC-3¢.
The underlined nucleotides indicate restriction sites that were used
in subsequent plasmid construction. The amplified fragment was
inserted into the XhoI–HindIII, KpnI–ClaI or KpnI–HindIII sites of the
pTN85 plasmid (accession number AB267707), which contains the
coding sequence of uidA, the nopaline synthase polyadenylation
signal and an NPTII cassette (Nishiyama et al., 2000), thereby creating an in-frame fusion of the PpXTH and uidA genes. Fragments
containing the 3¢ region of PpXTH genes were produced by PCR
using the following forward and reverse PCR primer sets: PpXTH16,
5¢-CGCGGATCCGCCTGATTTAACTGGCAATT-3¢ and 5¢-GATGCG
GCCGCGCGCAATGACATAAAATGA-3¢; PpXTH21, 5¢-CGCGGATCC
TAGAGACCGTTGCAGTCACC-3¢ and 5¢-CGCGAGCTCGGCTGTGTT
GCTGTTCATACT-3¢; AtXTH32, 5¢-CGCGGATCCTCAACAACACCCGATTGTG-3¢ and 5¢-CGCGAGCTCCTCTGTGCAAATTATCAATGT-3¢.
Each of these primers contained one restriction site, which is
underlined. The amplified fragment was inserted into the BamHI–
NotI or BamHI–SacI site of the plasmid containing the 5¢ DNA fragment. The resulting plasmid was linearized using KpnI and SacI or
NotI to generate the PpXTH–GUS fusion constructs. Polyethylene
glycol-mediated transformation was performed as previously
described (Nishiyama et al., 2000). Of 80 independent stable transformants, approximately 20 lines were identified by PCR as
containing the GUS fusion construct at the PpXTH locus. Using
Southern analyses, five lines containing a single insertion in the
PpXTH 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 sodium
phosphate, pH 7.0) at 37C for 2–24 h. Fixation was performed in 5%
v/v formalin for 10 min, followed by incubation for 10 min in 5% v/v
acetic acid. Pigments in the tissues were removed by serial
incubation in 30%, 50%, 70% and 100% ethanol.
For measurement of hydrolytic activity, the enzyme reaction mixture consisted of 62 ng recombinant PpXTH32, 10 lg nasturtium
(Tropaeolum majus L.) xyloglucan or approximately 30 lg lichenan
derived from Cetraria islandica, in 50 ll of 25 mM sodium acetate
buffer at pH 5, 6 or 7. As a control experiment, denatured PpXTH32
was added. After incubation at 30C for 3 h, the molecular weight
distribution profile of the polysaccharide substrate was estimated
by size-exclusion chromatography on columns of TSK-GEL
G3000PWXL 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) equipped
with a pulsed amperometric detector as described by Nishitani and
Tominaga (1992).
For measurement of XET activity, the reaction mixture contained
62 ng recombinant PpXTH32, 10 lg nasturtium xyloglucan and
0.1 mM 2-pyridylamino xyloglucan oligomer in 50 ll of 25 mM
sodium acetate buffer at pH 5.5. After incubation at 30C for 3 h,
the reaction mixture was separated by size-exclusion chromatography on the same columns using the Dionex D500 HPLC equipped
with both a pulsed amperometric detector and a fluorescence
detector (Nishitani and Tominaga, 1992).
Analysis of the distribution of ESTs in P. patens
SUPPORTING INFORMATION
The relative abundances of PpXTH transcripts in the EST databases
were estimated based on the total number of clones identified from
either 5¢ or 3¢ sequences in the libraries (Nishiyama et al., 2003) and
other P. patens clones deposited in public databases. The following
libraries 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¢ ESTs
and 15 000 3¢ ESTs), library for protoplasts during the first cell
division (10 535 5¢ ESTs and 10 975 3¢ ESTs), and library for sporophytes before meiosis with surrounding archegonia (8514 5¢ ESTs
and 8241 3¢ ESTs).
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Alignment of the amino acid sequences of 32 PpXTH
proteins constructed using the Clustal W program.
Figure S2. Frequency of ESTs from tissue-specific full-length cDNA
libraries.
Figure S3. Size-exclusion chromatograms of reaction products for
PpXTH32 protein and polysaccharide.
Table S1. XTH family genes in Physcomitrella patens.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
Production and purification of recombinant PpXTH32
protein
The cDNA of the PpXTH32 was amplified using forward and reverse
primers 5¢-CCGCTCGAGAAAAGAATGCAGTGCATGGCC-3¢ and
5¢-TGCTCTAGATTACTGATCAGGGAGATGAGG-3¢,
respectively,
and cloned into the pBluescript II SK+ vector (Invitrogen, http://
www.invitrogen.com/site/us/en/home.html). The XhoI/XbaI fragment was excised from the pBluescript clone, and cloned into the
pPICZaA vector in-frame with the a factor secretion signal sequence
(Invitrogen). The resulting pPICZaA-PpXTH32 expression vector
was used to transform Pichia pastoris strain X-33, and recombinant
Enzyme assay
ACKNOWLEDGEMENTS
We thank Ms. Kei Saito for her excellent technical assistance. This
work was supported by Grants-in-Aid for Scientific Research on
Priority Areas number 19039003 to K.N. and by Grants-in-Aid for
Scientific Research (B) number 19370014 to K.N. and (C) number
19370014 to R.Y. from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
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The Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 658–669