全 文 ：Received 19 May 2003 Accepted 7 Jul. 2003
Supported by the National Natural Science Foundation of China (30170090).
* Author for correspondence. Tel: +86 (0)10 62789384; Fax: +86 (0)10 62788604; E-mail: .
http://www.chineseplantscience.com
.Rapid Communication.
A Novel Endo-1,4-b-Glucanase Gene (LlpCel1) Is Exclusively Expressed
in Pollen and Pollen Tubes of Lilium longiflorum
ZHOU Wei1, Hiroyuki TAKEDA2, LIU Xi-Zhen1, Naoki NAKAGAWA2,
Naoki SAKURAI2, HUANG Jian1, LI Yi-Qin1*
(1. Protein Science Laboratory of the Ministry of Education, School of Life Sciences and Engineering,
Tsinghua University, Beijing 100084, China;
2．Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan)
Abstract: A full-length cDNA (LlpCel1) encoding an endo-1,4-b-glucanase (EGase) was isolated from
pollen tubes of Lilium longiflorum Thunb. by RT-PCR and RACE. The deduced protein, which is predicted to
be a compact globular protein, is of 490 amino acids, including a putative 21 amino acid-signal peptide.
LlpCel1 shares high level of homology (about 50%) with plant secreted EGases, which bear apparently
neither trans-membrane domain, nor cellulose-binding domain (CBD). LlpCel1 belongs to glycosyl hydrolase
family 9. LlpCel1 transcripts were detected in pollen grains, germinating pollen and elongating pollen tubes
with a similar level, whereas LlpCel1 transcripts were not detectable in all other lily tissues examined. The
specific expression pattern suggests that LlpCel1 is confined to lily pollen germination and pollen tube
elongation.
Key words: endo-1,4-b-glucanase (EGase, cellulase); Lilium longiflorum ; pollen germination; pollen
tube growth
Pollen germination and pollen tube elongation are cru-
cial processes of sexual reproduction in higher plants. Pol-
len tube, a representative tip-growing cell, grows at prodi-
gious rate, and travels a long distance within the female
tissues to deliver the sperm cells to the ovary. The regula-
tion mechanism of pollen tube growth has been documented
to link to complex factors, including cell wall rheological
properties. Cytological studies revealed that cell wall of
pollen tubes exhibit a distinctive structure, an outer pecto-
cellulosic layer and an inner callosic layer. The cell wall of
pollen tube tip is regarded as the weakest and the only
extensible portion, because of the lacking of callose. In
pollen tube wall distal from the tip, parallel 1,4-b-glucan
chains form crystalline cellulose microfibrils (CMF).
However, the presence of cellulose and its texture in the
wall of the pollen tube apex is still disputed (Li et al., 1997;
Taylor and Hepler, 1997; Derksen et al., 1999; Lord, 2000;
Hepler et al., 2001).
Pollen germination and pollen tube growth are attrib-
uted to alterations and modifications of the composition
and structure of cell wall polysaccharides, which are cata-
lyzed by appropriate enzymes. Besides the more valued
pectin-related enzymes, we reported that exo-b-glucanases
hydrolyzed both 1,3- and 1,4-glucosyl linkages in
hemicellulosic polysaccharides isolated from the cell wall
of Lilium longiflorum pollen tubes, and played a role in the
regulation of pollen tube growth (Kotake et al., 2000; Li et
al., 2001). Based on this finding, we hold on our interests at
the turnover of cellulosic compounds in pollen and pollen
tube walls. There are considerable reports demonstrating
the function of plant endo-1,4-b-glucanase (EGase, EC 3.2.
1.4, cellulase) on modifying cell wall architecture during
plant developmental processes, such as fruit ripening, leaf
abscission and cell expansion and elongation (Cosgrove,
1999; 2000; Hayashi, 2000; Darley et al., 2001; Levy et al.,
2002; Mølhøj et al., 2002). However, knowledge on EGase
acting on cellulose in pollen tube wall is very limited and
indirect. The existence of cellulose in pollen tube walls has
long been argued based on the ultrastructural observation,
including the more accurate localization by the use of
CellobiohydrolaseⅠprobe. Some reports showed no cel-
lulose depositing at the pollen tube tip wall (Ferguson et
al., 1998; Derksen et al., 1999). While Roggen and Stanley
(1969) described that addition of exogenous cellulase to
the germination medium induced significant increment of
pear pollen tube length. In addition, recombinant bacterial
EGase could promote pollen tube elongation in vitro
(Shoseyov and Dekel-Reichenbach, 1993). The discrepancy
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 142-147
ZHOU Wei et al.: A Novel Endo-1,4-b-Glucanase Gene Is Exclusively Expressed in Pollen and Pollen Tubes 143
prompted us to find an access via gene analysis to search
for new evidence. It would be greatly significant, if the endo-
beta-1,4-glucanase gene could be isolated from pollen
tubes, and the gene expression is evidenced to
be associated with pollen germination and pollen tube
growth.
In this study, a novel cDNA of EGase was cloned from
lily pollen tubes. The sequence of the putative protein was
characterized, and the patterns of gene expression in pol-
len grains, germinating pollen and elongating pollen tubes,
as well as in various lily tissues were analyzed.
1 Materials and Methods
1.1 Plant materials
Fresh anthers were detached from flowers of lily (Lilium
longiflorum Thunb.) and dehydrated in a desiccator
overnight. Pollen was collected and stored at –80 ℃ until
use (Li et al., 1983). Leaves, stems, petals, pistils, ovaries
and filaments were removed from lily plants, rinsed thor-
oughly in cold double-distilled water, immediately frozen in
liquid nitrogen and stored at –80 ℃.
1.2 Pollen culture
Lily pollen was cultured in liquid medium containing
1.29 mmol/L Ca(NO3)2, 0.99 mmol/L KNO3, 0.16 mmol/L
H3BO3 and 0.29 mol/L sucrose (Dickinson, 1968) at 25 ℃.
Germinated pollen with tubes (hereafter called pollen tubes)
were collected and washed as described before (Kotake et
al., 2000).
1.3 Isolation of total RNA
The total RNAs of de-hydrated/hydrated pollen grains
and pollen tubes collected following the time course of
incubation (1-5 h) were isolated, respectively, with guani-
dine-HCl method (Logemann et al., 1987). The total RNAs
were also extracted from lily tissues described above with
SDS-phenol method, respectively (Ausubel et al., 1987).
1.4 Isolation of EGase cDNA
Full-length EGase cDNA was generated from total RNA
of lily pollen tubes by RT-PCR and RACE. The 1st strand
cDNA of mRNA was obtained with SUPERSCRIPT Ⅱ
RNase H-Reverse Transcriptase (Invitrogen, Carlsbad, CA).
RT-PCR was performed using two degenerated primers de-
signed according to conserved amino acid domains of
known plant EGase sequences. A 500-bp amplified frag-
ment was obtained, recovered from the 1.5% agarose gel
with QIAEXⅡgel extraction kit (QIAGEN, Valencia, CA),
cloned into the vector pGEM-T (Invitrogen, Carlsbad, CA),
and sequenced. 3-RACE was conducted with oligo (dT) -
primer and a specific 20-mer primer designed according to
DNA sequence of the 500-bp fragment. A 1 000-bp
fragment was obtained and sequenced. 5-RACE was ac-
complished with RACE Core Set (TaKaRa, Tokyo) accord-
ing to the manufacturer’s protocol. The top 400-bp ampli-
fied fragment was recovered and sequenced. The full-length
cDNA was further convinced by PCR with KOD DNA poly-
merase (TOYOBO, Osaka) and a pair of primers designed
according to the 5-end and the 3-end sequences,
respectively. A 1 600 bp cDNA was attained and sequenced.
1.5 Sequence analysis
The amino acid sequence deduced from full-length
cDNA was subjected to homology analysis with BLAST
program (http://www.ncbi.nlm.nif.org), and was aligned with
other plant EGases using ClustalW program (http://www.
ddbj.nig.ac.jp/). The signal peptide was predicted with
SignalP program (http://www.cbs.dtu.dk/services/SignalP/).
The amino acid sequence was also subjected to molecular
weight and isometric point analysis with Compute pI/Mw
tool (http://www.expasy.org/tools/pi_tool.html). Prediction
of protein globularity was conducted using GLOBE pro-
gram (http://cubic.bioc.columbia.edu/predictprotein). Po-
tential glycosyl hydrolase active sites and N-linked
glycosylation sites were predicted using PROSITE motif
research program (Hofmann et al., 1999).
1.6 Northern hybridization
RNA samples (20-30 mg) were loaded on a 1.2% agar-
ose gel containing formaldehyde, and transferred to
HybondTM-N membrane (Amersham Pharmacia Biotech,
USA) after electrophoresis. Northern hybridization was con-
ducted with the labeled 250 bp probe, which came from the
fragment located at the 3 untranslated region (UTR) of the
cDNA by using Gene Images Random-Prime Labeling and
Detection System (Amersham Pharmacia Biotech., USA).
2 Results and Discussion
2.1 Full-length cDNA isolated from lily pollen tubes en-
codes a novel EGase
Analysis of the full-length cDNA sequence revealed a
single open reading frame (ORF) of 1 470 bp, a 15 bp 5 -
UTR and a 99 bp 3 -UTR. The deduced protein consists of
490 amino acids, including a 21 amino acid-signal peptide
at the N-terminal region, and has a predicted molecular mass
of 53 kD and a pI of 5.67 (Fig.1). Structural prediction re-
vealed that it is a compact globular protein, without any
apparent trans-membrane domain. There are two predicted
glycosyl hydrolase active sites, and six N-glycosylation
sites (Fig.1). BLAST result turned out it encodes a typical
EGase (cellulase), which belongs to glycosyl hydrolase fam-
ily 9 (Henrissat et al., 1989). The cDNA clone was then
denominated as LlpCel1 (Lilium longiflorum pollen/pollen
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004144
tube cellulase 1), and its sequence has been deposited in
GenBank under the accession number of AY205300
(LlpCel1).
2.2 LlpCel1 shows similarity to secreted EGases
Figure 2 demonstrates the phylogenetic tree produced
by comparing predicted amino acid sequences of LlpCel1
to published plant EGases. The tree is divided into two
main branches: secreted EGases and membrane-bound
EGases. The secreted EGases contain two subgroups. Most
known EGases classified into GroupⅠhave no cellulose-
binding domain (CBD). In GroupⅡ the genes underlined
were reported to have putative CBDs (Catala and Bennett,
1998; Trainotti et al., 1999; Goellner et al., 2001). LlpCel1
shares very high level of homology with secreted EGases,
e.g. 57.4% with orange Celb1, and around 50% with the
others. Whereas, it shares as less as 30% identity with
membrane-bound EGases. The location of LlpCel1 in the
phylogenetic tree may draw out that LlpCel1 is a secreted
EGase bearing no CBD. This is well in accordance with the
result of gene analysis that LlpCel1 does not show any
apparent trans-membrane domain. Moreover, LlpCel1
shares high similarity to tomato Cel7 and pea EGL1 (Fig.2),
which were revealed to have close relationship to expan-
sive growth of the cells (Wu et al., 1996; Catala et al., 1997).
Most identified plant EGases belong to secreted type
and function on the degradation of cellulose in the cell
wall. Besides secreted EGases a few membrane-bound
EGases were also found in plants and presumed to edit
cellulose synthesis in addition to hydro lys i s , e . g .
Arabidopsis KOR mutants showed an abnormal
Fig.1. Optimal alignment of the deduced amino acid sequences of lily LlpCel1, orange Celb1, tomato Cel4 and Arabidopsis Cel1. Gray
shading depicts identical amino acids among the sequences. The vertical arrow designates the putative signal peptide cleavage site of the
protein. Horizontal arrows designate two degenerated primers corresponding to the conserved regions used to amplify LlpCel1. Two
open boxes indicate the putative glycosyl hydrolase active sites. N-linked glycosylation sites are shown by bold overlines.
ZHOU Wei et al.: A Novel Endo-1,4-b-Glucanase Gene Is Exclusively Expressed in Pollen and Pollen Tubes 145
phenotype of cell expansion which was due to the forma-
tion and assembly of cellulose microfibrils (Brummell et al.,
1997; Nicol et al., 1998; Williamson et al., 2002). The phylo-
genetic evaluation leads us to propose that LlpCel1 would
act, as a secreted EGase, upon the degradation of cellulose
in the wall of pollen and pollen tubes.
2.3 LlpCel1 is expressed exclusively in pollen and pol-
len tubes
The different expression patterns of LlpCel1 in lily pol-
len grains, pollen tubes and other various tissues were in-
vestigated by Northern hybridization. In Fig.3, comparable
amounts of total RNAs were loaded in each lane of the gel,
which was confirmed by the density of rRNA bands on the
HybondTM-N membrane stained with methylene blue.
LlpCel1 transcripts were detected in dehydrated pollen
grains (Pg) and pollen tubes (Pt). However, no signal for
LlpCel1 transcript could be seen in tissues including leaves,
stems, petals, pistils, ovaries and filaments (Fig.3A). It
indicates that LlpCel1 specifically expresses in pollen and
pollen tubes. In appropriate medium lily pollen begins to
germinate after 1.0-1.5 h incubation. Cultured for 2 h, most
pollen germinated and pollen tubes grew healthily. During
3-5 h incubation the growth of pollen tubes was continu-
ously active. Figure 3B demonstrates that the level of
LlpCel1 transcripts remains apparently stable in dehydrated
pollen grains (Pg), re-hydrated pollen (0 h), germinating
pollen (1 h) and elongating pollen tubes (2-5 h).
It is reported that the function of EGases is related to
the molecular structure of the enzymes. Bacterial and fun-
gal cellulases with one or more CBDs linked to the catalytic
domain can efficiently hydrolyze crystalline cellulose (Levy
et al., 2002). Most plant EGases shown in groupⅠ(Fig.2),
lacking of CBDs, have little activity towards the crystalline
cellulose, but act on non-crystallized cellulose such as car-
boxymethyl-cellulose (CMC) and hemicellulose in the pri-
mary cell wall (Hayashi, 2000; Levy et al., 2002). LlpCel1
lacks of CBD and thus probably acts on amorphous non-
fibrous cellulose in the cell wall. Pollen and pollen tubes
exhibit distinct wall structure in comparison with the cell
wall of other tissue cells. While the pollen are germinating
the outer layer of the pollen tube wall which appears to be
an extension of intine of the pollen grain. The intine shows
the characteristic three-layered structure. The sign of the
tube emergence at the aperture is the weakening of the
cellulosic inner layer of the intine (Heslop-Harrison, 1987).
Fig.2. Phylogenetic comparison of plant EGases based on the
deduced amino acid sequences. Genes falling into the upper branch
are secreted and soluble EGases. Others belong to membrane-
anchored type EGase in the lower branch. The three genes in
Group Ⅱ underlined are included as members of plant EGases
with putative cellulose-binding domains. Sequences used with
accession numbers in parentheses are: Arabidopsis Cel1 (X98544),
KOR (U37702); barley Cel1 (AB040769); orange CEL-a1
(AF000135), CEL-b1 (AF000136); pea EGL1 (L41046); pepper
CCel3 (X97189); poplar PopCel1 (AB049199), PopCel2
(AB025796); tobacco Cel2 (AF362948), Cel7 (AF362947), Cel8
(AF362949); tomato Cel2 (U13055), Cel3 (U78526), Cel4
(U20590), Cel5 (AF077339), Cel7 (Y11268), Cel8 (AF098292);
strawberry faEg3 (AJ006349).
Fig.3. Expression of LlpCel1 transcripts in lily pollen, pollen
tubes and other tissues. A. Northern blotting analysis for LlpCel1
in pollen and different tissues. Fi, filament; Le, leaves; Ov, ovary;
Pe, petal; Pg, pollen grain; Pi, pistil; Pt, pollen tube; St, stem. B.
Northern blotting analysis for LlpCel1 in pollen and pollen tubes
at different incubation time (0-5 h). In bottom panels in both A
and B, methylene blue-stained blot shows the quality and amount
of RNA transferred on the nylon membrane, based on the inten-
sity of the ribosome RNAs. Fragment from 3-URF was labeled
and used as probe for hybridization.
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004146
The fragile tip wall of the growing pollen tubes is unique
and thought to be pecto-cellulosic (Li et al., 1997; Taylor
and Hepler, 1997). Ferguson et al. (1998) used enzyme-gold
affinity labelling to localize cellulose in lily pollen tubes
and found no signal in the tip wall. The enzyme they used
is Cellobiohydrolase Ⅰ (CBHI; EC 3.2.1.91), which is iso-
lated from Trichoderma reesei, composed of a CBD and
targets crystallized cellulose. Our lily LlpCel1 (EC 3.2.1.4) is
without CBD and belongs to the secreted EGase group.
The target of LlpCel1 is most likely the amorphous non-
fibrous cellulose located in intine and the highly extensive
tip wall of the pollen tubes. The distinctive structural fea-
tures of pollen and pollen tube walls, thus, lead to inter-
preting the exclusive expression of LlpCel1 in pollen and
pollen tubes.
There are a large number of genes active in the male
gametophyte, especially when compared with the vegeta-
tive tissue (Hamilton and Mascarenhas, 1997). Pollen-spe-
cific genes have been characterized as “early” and “late”
genes according to the expression patterns. The late genes
probably encode for proteins with functions associated
with pollen maturation and/or germination and pollen tube
growth (Mascarenhas, 1990). LlpCel1 expressed in de-hy-
drated pollen grains, re-hydrated pollen and pollen tubes
can be regarded as a “late” gene. In some plant species,
RNAs needed for germination are available in the mature
pollen and the encoding proteins are synthesized at the
onset of pollen germination and during tube elongation
(Mascarenhas, 1993; Fernando et al., 2001). According to
the expression patterns we might propose that LlpCel1
mRNA has already been synthesized and stored in mature
pollen. LlpCel1 may be associated with cell wall loosening
by attacking the non-crystallized b-1,4-glucan components,
and contributes to lily pollen germination and pollen tube
elongation.
For better understanding the role of LlpCel1, further
study to confirm that the product of LlpCel1 expressed in
vitro has EGase activity and to identify its endogenous
substrate should be addressed.
Acknowledgements: We thank Dr. Toshihisa KOTAKE
in Hiroshima University in Japan, for kindly providing the
degenerate primers.
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