Gene Expression Profiling Using cDNA Microarray
Analysis of the Sexual Reproduction Stage of the
Unicellular Charophycean Alga Closterium
peracerosum-strigosum-littorale Complex1[W]
Hiroyuki Sekimoto*, Yoichi Tanabe, Yuki Tsuchikane, Hiroshi Shirosaki, Hiroo Fukuda, Taku Demura,
and Motomi Ito
Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University,
Bunkyo-ku, Tokyo 112–8681, Japan (H. Sekimoto); Department of General Systems Studies,
Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153–8902, Japan
(Y. Tanabe, Y. Tsuchikane, H. Shirosaki, M.I.); Plant Science Center, Institute of Physical and Chemical
Research, Yokohama 230–0045, Japan (H.F., T.D.); and Department of Biological Sciences, Graduate
School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113–0033, Japan (H.F.)
The desmid Closterium peracerosum-strigosum-littorale complex, which is the closest unicellular sister to land plants, is the best
characterized of the charophycean green algae with respect to the process of sexual reproduction. To elucidate the molecular
mechanism of intercellular communication during sexual reproduction, we created a normalized cDNA library from mixed
cells of the sexual and the vegetative phases and generated a cDNA microarray. In total, 3,236 expressed sequence tags, which
were classified into 1,615 nonredundant groups, were generated for cDNA microarray construction. Candidate genes for key
factors involved in fertilization, such as those that encode putative receptor-like protein kinase, leucine-rich-repeat receptorlike protein, and sex pheromone homologs, were up-regulated during sexual reproduction and/or by the addition of the
purified sex pheromones, and the expression patterns of these genes were confirmed by quantitative real-time polymerase
chain reaction analysis. This first transcriptome profile of Closterium will provide critical clues as to the mechanism and
evolution of intercellular communication between the egg and sperm cells of land plants.
Fertilization entails an intimate relationship between egg and sperm cells, which interact with each
other at the cellular level. Along with the development
of male and female gametophytes in plants, fertilization has been studied in many plant species (Huang
and Sheridan, 1994; Heslop-Harrison et al., 1999; Faure
et al., 2002). In addition to the pollination process, a
long-lasting period of interaction between the diploid
pistil and the haploid pollen tube is indispensable for
successful fertilization in flowering plants. This interaction phase may be described as the progamic phase,
which starts with the landing of the pollen grain on the
stigmatic surface and ends with syngamy, which is the
1
This work was supported by a grant-in-aid from the 21st
Century Center of Excellence program, Research Center for Integrated Science, of the Ministry of Education, Culture, Sports, Science
and Technology, Japan, to (H. Sekimoto), and from the Japan Society
for the Promotion of Science (to M.I. and H. Sekimoto).
* Corresponding author; e-mail [email protected]; fax 81–3–
5981–3674.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Hiroyuki Sekimoto ([email protected]).
[W]
The online version of this article contains Web-only data.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.106.078048.
fusion of the sexual cells. The mechanism that directs
the pollen tube through the pistil to the female gametophyte has been studied for a long time. The involvement of female tissues in defining a path for the pollen
tube to the female gametophyte has been reported. On
the other hand, the existence of pistil-produced chemotropic substances (i.e. chemoattractive pheromones
for the male gametophyte) has been strongly supported
by several independent studies. Although comprehensive reviews have been published (Higashiyama
et al., 2003; Sanchez et al., 2004; Weterings and Russell,
2004), the intracellular and intercellular events that
occur during fertilization have not been elucidated
fully at the molecular level.
Several studies have reported on the sexual interactions and sexuality of Chlamydomonas reinhardtii,
which is a freshwater green alga of the Chlorophyceae
(Ferris et al., 2002, 2005; Pan et al., 2003). In addition,
expressed sequence tag (EST) and whole-genome
projects are under way (Asamizu et al., 1999, 2000;
Shrager et al., 2003). Using cDNA macroarrays, the
transcriptional program for gametogenesis and the
regulatory networks of gene expression during sexual
differentiation have been analyzed critically (Abe et al.,
2004, 2005).
Charophycean green algae, which are most closely
related to land plants, form a relevant monophyly with
Plant Physiology, May 2006, Vol. 141, pp. 271–279, www.plantphysiol.org Ó 2006 American Society of Plant Biologists
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271
Sekimoto et al.
land plants. Thus, charophyceans and land plants
share many distinctive characteristics with respect to
cellular structures and metabolism and are evolutionarily distant from other major green algae (i.e. the
Chlorophyceae and Ulvophyceae; Graham and Wilcox,
2000; Karol et al., 2001). Charophyceans comprise five
lineages (orders) of freshwater green algae: Charales,
Coleochaetales, Zygnematales, Klebsormidiales, and
Chlorokybales. The desmid Closterium, which belongs
to Zygnematale, is the most successfully studied unicellular charophycean plant in terms of the maintenance of strains and sexual reproduction (Ichimura,
1971; Akatsuka et al., 2003; Fukumoto et al., 2003).
Heterothallic strains of Closterium have two morphologically indistinguishable sexes: mating-type plus
(mt1) and mating-type minus (mt2). Sexual reproduction is easily induced when cells of these two sexes are
cultured together in nitrogen-depleted (MI) medium
under light. A possible mechanism for the sexual reproduction of Closterium peracerosum-strigosum-littorale
complex (C. pslc) has been reported (Sekimoto, 2000).
As the first step to understanding the molecular mechanism of C. pslc, 1,190 5#-end ESTs, which included 760
unique sequences, were established from cells in the
sexual reproduction process (Sekimoto et al., 2003).
In this article, we describe the first expression profile
obtained using the charophycean green alga microarray, including 3,064 ESTs (2,047 newly prepared
clones), which will be used to increase our understanding of the molecular mechanism of intercellular communication during the fertilization process in plants.
RESULTS AND DISCUSSION
Construction of the cDNA Microarray
The two cDNA libraries (1.6 3 107 and 6.5 3 106
clones) prepared previously from cells at various
stages of sexual reproduction were used as the primary materials (Sekimoto et al., 2003). In addition, a
cDNA library (4.9 3 106 clones) derived from a mixture of vegetative mt1 and mt2 cells was constructed.
A normalized cDNA library that contained 1.7 3 105
independent clones was established from a mixture of
these three cDNA libraries, with the aim of reducing
redundancy. To analyze both quantitatively and qualitatively genes related to the process of sexual reproduction of C. pslc, 2,304 PCR-amplified cDNA inserts
were isolated from the normalized cDNA library. In
addition to these cDNA inserts, 760 cDNA inserts,
which were derived from previously determined nonredundant EST groups, were prepared (Sekimoto et al.,
2003). These 3,064 cDNAs were spotted twice (left- and
right-hand sides) onto each slide to increase the reliability of the microarray data.
Features of ESTs from Sexual and Vegetative
Closterium Cells
While spotting the cDNA inserts, single-pass sequencing from the 5#-end was performed on the 2,304
uncharacterized cDNAs. After the removal of vectorderived sequences and ambiguous sequences, we
obtained sequence information for 2,046 cDNAs. In addition to the previously obtained 1,190 ESTs (accession
nos. AU294770–AU295959), 3,236 ESTs were established
in all (Supplemental Table I). Clones that showed identity of .98% over stretches of .100 bp were grouped
together, and the ESTs were clustered into 1,615 nonredundant sequences, although the real number of independent genes should be ,1,615 due to nonoverlapping
fragments derived from the same genes.
The EST sequences were compared with the public
nonredundant protein sequence databases using the
BLASTX program, and E , 1.0e25 was set as the level
of significance. Thus, 1,045 of the 1,615 nonredundant
sequences showed similarity to previously registered
genes in the public databases. The source group with
the highest similarity was land plants, including
Arabidopsis (Arabidopsis thaliana).
The search results, which include the names of
the proteins putatively encoded by EST clones as well
as the accession numbers of the ESTs (BW646625–
BW648670), are shown in Supplemental Table I.
cDNA Microarray Analysis of Gene Expression
To monitor gene expression patterns during the
sexual reproduction of C. pslc, Cy5-labeled cDNA
populations were prepared from cells that were incubated in MI medium with the opposite mating type for
2, 8, 24, and 72 h (mixing culture). As controls, Cy5labeled cDNA populations from the respective mating
types, incubated separately for 8 h in MI medium at a
low cell density (mt1-L and mt2-L), were also prepared. These populations were independently hybridized to the microarray. To determine the changes in
gene expression levels during these processes, the
expression levels of the cells just before mixing (mt1-0,
mt2-0) were also examined. After scanning and quantification, normalization was performed for each hybridization (Supplemental Table II).
To identify genes that are specifically expressed
during sexual reproduction, cDNA spots were selected
that showed a 5-fold increase in expression over the
time course of sexual reproduction, as compared to the
mt1-L or mt2-L cells. Those cDNA spots that were
differentially expressed in mt1 cells, compared to mt2
cells, were also chosen as sex-specific genes and vice
versa. To analyze pheromone-responsive genes, cDNA
spots, the expression levels of which were elevated
4-fold by the addition of protoplast-release-inducing
protein (PR-IP) or PR-IP inducer, were selected. In all,
292 cDNA spots were identified. To obtain sequence
information for the selected cDNAs, the 5#-ends of
which showed no sequence similarities, single-pass
sequencing was carried out from the 3#-ends. The
sequences of these 292 cDNAs could be classified into
88 nonredundant gene clusters, and the average values
of the respective gene clusters were used in the final
dataset (Table I; Supplemental Table III).
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Microarray Analyses in Closterium
Table I. Representative conjugation-related, sex pheromone-responsive, or sex-specific genes identified by cDNA microarray analysis
Cluster IDa
76
97
1,456
136
609
1,283
12
414
285
1
104
247
Description
19-kD subunit
of PR-IP
Aquaporin-like
protein
Glyoxalase II
LRR transmembrane
protein
Luminal BiP 2
Luminal BiP
No significant
homology
No significant
homology
No significant
homology
PR-IP inducer
Receptor-like
protein kinase
Tonoplast membrane
integral protein
a
Gene
Name
Mix/mt1-Lb
2h
d
8h
d
Mix/mt2-Lc
d
24 h
d
72 h
d
2h
8 hd
24 hd
72 hd
mt1-L/
mt2-Le
Inducer/
mt1-Lf
PR-IP/
mt2-Lg
mt1-L/
mt1-Vh
mt2-L/
mt2-Vi
Groupj
Cp19KSU
5.46 10.63 10.33 15.72 4.34
8.45
8.21 12.49
0.81
17.64
1.83
0.50
0.93 C-D
CpAQP1
1.27
0.69
0.41 6.19
2.74
3.36
1.98
4.58
1.31
1.32
0.38
0.25 A
CpGLX2-1
2.75
5.88 14.00
CpRLP1
10.85 18.52 19.87
4.38 1.00
9.01 4.21
2.14
7.29
5.09
7.73
1.59
3.68
0.34
0.43
6.13
2.03
4.42
11.36
0.09
0.62
0.50 C-D-E
1.45 C-E
CpBiP1
CpBiP2
Cp-01
3.02
2.78
1.79
0.96 3.30
3.04
1.96
1.05
6.84
5.29
3.84
1.90 4.93
3.81
2.77
1.37
1.91 13.48 25.32 26.46 3.82 26.96 50.64 52.93
1.01
0.66
2.00
1.53
1.71
30.09
5.28
5.90
1.46
1.62
1.35
1.65
1.22 C-E
1.87 C-E
0.78 C-D
Cp-41
0.26
0.31
0.52
0.60 1.92
2.31
3.85
4.46
7.69
0.53
1.42
0.80
0.72 A
Cp-48
2.30
8.60 10.97
4.33 1.25
4.69
5.98
2.36
0.55
1.80
10.22
1.11
0.82 C-E
33.95 36.00 29.50 19.45 0.64
0.67
0.55
0.36
2.60
5.04
8.43
8.44 5.27 10.22 17.08 17.11
0.02
2.00
1.95
6.44
1.37
1.74
0.61
1.25
17.52 B
0.71 C-D
5.54
1.16
1.50
0.44
0.19 A
CpPI
CpRLK1
CpAQP2
1.18
0.56
0.58
0.58
0.39 6.85
3.35
3.35
2.25
b
c
IDs for 1,615 nonredundant sequences after clustering.
Expression ratios of cells in mating to mt1 cells, at low cell density in MI medium.
Expression ratios
d
e
of cells in mating to mt2 cells, at low cell density in MI medium.
Incubation time for induction of mating (conjugation).
Expression ratios of mt1 cells to mt2
f
cells, at low cell density in MI medium.
Expression ratios of PR-IP inducer-treated mt1 cells to pheromone-free mt1 cells, at low cell density in MI
g
h
Expression ratios of PR-IP-treated mt2 cells to pheromone-free mt2 cells, at low cell density in MI medium.
Expression ratios of mt1 cells incubated in
medium.
i
j
MI medium to vegetative mt1 cells.
Expression ratios of mt2 cells incubated in MI medium to vegetative mt2 cells.
Groups classified with respect to similar
expression patterns during conjugation, sex specificity, and responses to sex pheromone treatment.
Clustering of Sexual Reproduction-Related Genes with
Respect to Expression Patterns
The 88 gene clusters were subdivided into groups
with respect to similar expression patterns during
conjugation, sex specificity, and responses to sex pheromone treatment. We classified 13 and 25 gene clusters
in which gene expression was observed preferentially
in one of the sexes (mt1-L and mt2-L) as groups A and
B, respectively. Of the remaining 50 gene clusters, 44
clusters whose expression was elevated during conjugation were classified into group C. In addition, 25 and
16 gene clusters that were expressed in response to
PR-IP inducer and PR-IP were classified into groups D
and E, respectively. Because most of the pheromoneresponsive genes showed elevated expression levels
during conjugation, these genes were placed into
subgroups C-D, C-E, D-E, and C-D-E (Fig. 1).
ing sexual reproduction, condensation of the cytoplasm and release of protoplasts from the cell wall are
observed (Sekimoto et al., 1990). For these processes,
the content of water and/or some solutes in cells must
undergo change. These proteins may play a role in
these processes. In higher plants, aquaporins comprise
four different groups of highly divergent proteins,
being present in the tonoplast, in the plasma membrane, and possibly in other internal membranes.
Given that CpAQP1 and CpAQP2 comprised a monophyletic clade and were separate from genes derived
from other land plant lineages (data not shown), their
actual roles in mt1 cells as well as their cellular
localizations remain unclear.
Genes with significant levels of similarity to carbonate
dehydratase (IDs 106 and 1,043), carbonate anhydrase
Genes Expressed Specifically in One of the Mating Types
Among the 13 gene clusters in group A, eight
showed similarities with known proteins. Two of these
clusters (cluster IDs 97 and 247) showed similarities
with aquaporins, which represent an ancient family of
channel proteins that transport water and certain
neutral solutes across biological membranes (Chaumont
et al., 2001), and these clusters were termed CpAQP1
and CpAQP2, respectively. The deduced amino acid
sequences of CpAQP1 and CpAQP2 showed 93.1%
identity with each other and contained Asn-Pro-Ala
motifs, which were conserved within the family. Dur-
Figure 1. Overlapping of conjugation-related and pheromone-responsive
genes. The numbers of genes with elevated levels of expression during
conjugation (C), after treatment with PR-IP inducer (D), or after treatment
with PR-IP (E) are indicated.
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Sekimoto et al.
(ID 245) and glyceraldehyde-3-P dehydrogenase (ID 201)
were also identified in group A. These genes are known
to have roles in cellular metabolism, although specific
roles in mt1 cells remain to be analyzed. Cluster 1,441
showed similarity with the heparanase-like protein from
Rattus norvegicus. The deduced proteins from Closterium
and Rattus contain the tandem repeat sequences
(P/S)AD(N/S)DNTVAV(V/I) and D(S/H)D(V/D)
SSGPVDS, which are not significantly similar to each
other. Because the apparent sequence similarity may
be attributed to the repeated structures, a functional
relationship cannot be deduced.
Among the genes that were expressed specifically in
mt2 cells (group B), the expression levels of the genes
that encode PR-IP inducer (ID 1) and the homolog (ID
179) were found to be most critically restricted in the
mt2 cells. PR-IP inducer has been identified as a sex
pheromone that is released from mt2 cells and induces
PR-IP production in mt1 cells during the sexual reproduction of C. pslc. Recently, recombinant PR-IP
inducer produced by yeast (Saccharomyces cerevisiae)
cells has been shown to induce sexual cell division in
mt1 cells in addition to its PR-IP-inducing activity
(Tsuchikane et al., 2005). Production of the recombinant protein is under way to facilitate evaluation of the
biological functions of the PR-IP inducer-like protein.
In addition, seven gene clusters showed significant
similarities to known proteins, such as g-tocopherol
methyltransferase (ID 216), a-tubulin (ID 219), protein
kinase (ID 240), epimerase-like protein (ID 397), PREGlike protein (ID 1,261), glycosyltransferase family protein (ID 1,279), and ubiquitin-specific protease 2 (ID
1,374). These sex-regulated genes from groups A and B
are useful in the identification of differences between
the sexes and in the elucidation of the sex determination mechanisms of C. pslc.
classified into subgroup C-D. In this group, gene cluster
104 showed significant similarity to the receptor-like
protein kinase and was named CpRLK1. The conserved regions of the kinase domain were found in
the deduced amino acid sequence, which suggests that
the gene is functional, although the full-length cDNA
sequence is not currently available. In plants, many
genes encode receptor-like protein kinases (Shiu and
Bleecker, 2001), although the functions of most of these
genes are unknown. In addition, information regarding their ligands is rather limited (Kachroo et al., 2001;
Takayama et al., 2001; Wang et al., 2001; Matsubayashi
et al., 2002; Scheer and Ryan, 2002). In the case of the
CpRLK1 protein, the ligand is unknown, although
PR-IP inducer is a candidate. A receptor for PR-IP
inducer should be expressed in mt1 cells but not in mt2
cells. It is reasonable to assume that the expression
of the gene that encodes this type of receptor is activated after acquisition of the ligand itself during
sexual reproduction. To facilitate characterization of
the gene product, cloning of the full-length cDNA is
under way.
Gene clusters that encode the 19- and 42-kD subunits of PR-IP (IDs 76 and 3), which is a sex pheromone
produced in mt1 cells, were classified into subgroup
C-D; in addition, our results corroborate a previous
report that showed the gene expression patterns
(Sekimoto et al., 1994). Furthermore, genes that encode
homologs of the 19- and 42-kD subunits (IDs 9 and 239)
Genes Expressed during Sexual Reproduction But Not
in Response to Pheromones
Among the remaining 50 gene clusters, 11 showed
high expression levels during sexual reproduction but
were not remarkably responsive to the pheromone
treatment. Five genes showed similarities to known
proteins, which included Gly dehydrogenase (ID
1,226), the 22-kD protein of PSII (ID 223), hypothetical
proteins (ID 988), putative pectin acetylesterase (ID
988), and low-CO2-inducible proteins (LCIB and/or
LCIC) from C. reinhardtii (IDs 952 and 1,205). These are
the first genes implicated in the sexual reproduction of
Closterium; furthermore, they will be useful in characterizing the metabolic changes that occur during the
process of reproduction.
PR-IP Inducer-Responsive Genes Specifically
Expressed in mt2 Cells
Twenty-five gene clusters responded to the addition
of PR-IP inducer. Of these, 20 gene clusters were
further up-regulated during sexual reproduction and
Figure 2. Expression patterns of six representative conjugation-related
genes from C. pslc, as analyzed by real-time PCR. cDNAs were
prepared from mating-induced cultures at the indicated times (2, 4,
8, 24, and 72 h). cDNAs were also prepared from the respective mating
types just before mating induction (mt1-0 h, mt2-0 h). The relative realtime PCR values are shown as the means from two independent
experiments, with normalization to the expression of 18S rRNA. The
expression level of each gene from an individual cDNA is indicated as
the relative abundance; the maximal value for the expression of each
gene during conjugation is designated as 100.
274
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Microarray Analyses in Closterium
were also found to be involved, which suggests that
the gene products also play roles in the progress of
sexual reproduction, presumably as pheromones of
unknown function.
One of the remaining genes showed significant
similarity with phytochrome A repressor proteins
(ID 1,454). Indeed, light is indispensable for the sexual
reproduction of Closterium, although the light requirement for mating activation has been suggested
as being primarily dependent on photosynthesis
(Ueno and Sasaki, 1978; Kato et al., 1983; Sekimoto
and Fujii, 1992). Some photosensing molecules and a
regulatory mechanism should be involved in the regulation of the progression of sexual reproduction
(Sekimoto and Fujii, 1992). The products of two genes
showed high levels of sequence similarity to the glutathione S-transferase of Nostoc punctiforme (ID 502)
and an unknown protein of Arabidopsis (ID 819). The
remaining 12 genes did not show any significant
similarities to known proteins.
PR-IP-Responsive Genes Expressed Specifically
in mt1 Cells
In subgroup C-E, 11 gene clusters were found.
Among these, we identified cDNA clone 136, the
product of which showed high-level similarity to
Leu-rich repeats (LRR) containing transmembrane
protein kinase. Several LRRs and a transmembrane
domain were found at the 5#- and 3#-ends, respectively, whereas conserved regions for the kinase
domain could not be found at the 3#-end (data not
shown). Therefore, this cDNA clone was named
CpRLP1 (receptor-like protein-1). The primary function of the LRR motifs is to provide a versatile structural framework for the formation of protein-protein
interactions (Kobe and Kajava, 2001). Because CpRLP1
was expressed specifically in mt2 cells and its expression was stimulated by the addition of PR-IP, the
primary function of the CpRLP1 protein appears to be
related to the acquisition of PR-IP. In vivo binding
assays have demonstrated the presence of a receptor
for the 19-kD subunit of PR-IP and its appearance
during sexual differentiation (Sekimoto et al., 1993).
However, molecular biological information for this
molecule is lacking. As in the case of the CLV2 protein
of Arabidopsis (Jeong et al., 1999), the CpRLP1 protein
may form a heterodimer with another protein, such as
a receptor-like protein kinase, to transduce the extracellular signal of PR-IP to the intracellular compartment.
Two genes (IDs 609 and 1,283) of subgroup C-E
encoded homologs of luminal binding proteins (BiP)
and were named CpBiP1 and CpBiP2, respectively. BiP
Figure 3. Effects of sex pheromones on the expression of six representative conjugation-related genes
of Closterium. The cDNAs were prepared from cultures that were treated with sex pheromones at the
indicated final concentrations. The relative real-time
PCR values are shown as the means from two independent experiments, with normalization to the expression of 18S rRNA. The expression level of each
gene from an individual cDNA is indicated as the
relative abundance; the mean signal values detected
for mt1 or mt2 cells incubated in sex pheromone-free
medium are assigned the value of 1.0. A, Effect of
PR-IP inducer on the expression of three conjugationrelated genes (CpRLK1, Cp19KSU, and Cp-01) in mt1
cells. B, Effect of PR-IP on the expression of two
conjugation-related genes (CpRLP1 and Cp-48) in
mt2 cells. C, Effects of sex pheromones on the
expression of the CpGLX2-1 gene: effect of PR-IP
inducer on mt1 cells (left) and effect of PR-IP on mt2
cells (right).
Plant Physiol. Vol. 141, 2006
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Sekimoto et al.
is a member of the Hsp70 family and acts as a molecular chaperone that aids in the folding and assembly of
proteins routed through the endoplasmic reticulum
(Denecke et al., 1991). Although BiP is present constitutively during normal growth, rapid induction occurs
under conditions that cause the accumulation of abnormal proteins in the lumen of the endoplasmic
reticulum, and BiP accomplishes its function in the
lumen (Wrobel et al., 1997). During sexual reproduction, cells have to change from the vegetative to the
sexual state, with consequent de novo protein synthesis and protein degradation. The CpBiP proteins may
promote protein folding and assembly and may dissolve protein aggregates that are formed during these
processes.
Among the other eight genes of subgroup C-E, one
gene (ID 285) did not show any sequence homology to
known proteins. At present, the roles of these genes in
sexual interaction remain unclear.
Genes Expressed in Response to Both Pheromones
Two genes were up-regulated not only during sexual reproduction but also in response to both pheromones (subgroup C-D-E). One of these genes (ID
1,484) showed weak sequence similarity to the hypothetical protein of Dictyostelium discoideum, whereas
the other (ID 1,456) showed a high level of similarity to
putative glyoxalase II (AtGLX2-3; U74610). The latter
gene was named CpGLX2-1. Glyoxalase II is part of the
glutathione-dependent glyoxalase detoxification system and is thought to be involved in cell proliferation.
It belongs to the metallo-b-lactamase superfamily
(Crowder et al., 1997; Maiti et al., 1997; Zang et al.,
2001). A b-lactamase-like domain and a metal-binding
domain (T-H-X-H-X-D-H) were conserved in the deduced CpGLX2-1 protein. In Arabidopsis, the presence
of at least four glyoxalase II isozymes has been
reported (Maiti et al., 1997). AtGLX2-3 has been suggested as encoding a mitochondrial isozyme, although biochemical and physiological characterizations
have not been reported. Analysis of the deduced
CpGLX2-1 protein using PSORT (Nakai and Kanehisa,
1992) predicted that, unlike the AtGLX2-3 protein,
CpGLX2-1 localizes to microbodies (accuracy rate of
0.64) or to the cytoplasm (accuracy rate of 0.45).
sexual reproduction (Fig. 2). The expression levels of
CpRLK1, Cp19KSU, and Cp-01 in mt1 cells were stimulated by the addition of PR-IP inducer, whereas those
of CpRLP1 and Cp-48 in mt2 cells were also stimulated
by the addition of PR-IP (Fig. 3, A and B). In the case of
CpGLX2-1, the stimulatory effects of both sex pheromones in opponent cells were also confirmed (Fig. 3C).
In the case of two sex-specific genes (Cp-41 and the
PR-IP inducer gene CpPI), the expression of Cp-41 was
restricted in mt1 cells, whereas the accumulation of
CpPI mRNA was limited in mt2 cells, especially at low
cell density in MI medium (Fig. 4). These results are
concordant with the expression patterns obtained
from the microarray analyses and indicate that the
microarray data presented here are highly reliable.
CONCLUSIONS AND PERSPECTIVES
In this study, we generated an additional 2,046 ESTs.
The resulting 1,615 nonredundant ESTs, which include
the 760 previously characterized nonredundant
clones, are available for downstream experiments
and in silico analyses. Subsequently, the cDNA microarrays, on which 3,064 ESTs were spotted, were constructed to reveal comprehensive gene expression
profiles during the sexual reproduction process (Supplemental Table II). According to the expression patterns, the genes spotted on the glasses could be
classified into several groups. Thirty-eight matingtype-specific genes and 50 conjugation-related and/or
pheromone-responsive genes were discovered, none
of which have previously been deposited in the public
Real-Time PCR Analysis Clustering of Sexual
Reproduction-Related Genes with Respect to
Expression Patterns
To confirm the expression patterns obtained in
our microarray analyses and to check the reliability
of the data, quantitative real-time PCR was performed
using TaqMan probes for eight representative genes
(Supplemental Table IV). The expression levels of six
conjugation-related genes (CpRLP1, CpRLK1, the gene
encoding the 19-kD subunit of PR-IP [Cp19KSU],
CpGLX2-1, and genes showing no homology [tentatively named Cp-01 and Cp-48]) were elevated during
Figure 4. Sex-specific expression of the Cp-41 and CpPI genes under
various culture conditions. The cDNAs (mt1-V, mt2-V, mt1-0, mt2-0,
mt1-L, and mt2-L) were prepared from the respective cultures noted in
the figure. The various culture conditions are described in ‘‘Materials and
Methods.’’ The relative real-time PCR values are shown as the means
from two independent experiments, with normalization to the expression of 18S rRNA. The expression level of each gene from an individual
cDNA is indicated as the relative abundance; the maximal value for the
expression of each gene among cultures is designated as 100.
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Microarray Analyses in Closterium
databases, with the exception of the previously reported
sex pheromone genes, which encode two PR-IP subunits and the PR-IP inducer. In addition, although
many of the deduced amino acid sequences of the ESTs
on the array have no similarity to known proteins, a
number of interesting ESTs, such as those related to
intercellular communication, receptor-like protein kinase (CpRLK1), and LRR-containing receptor-like protein (CpRLP1), were unearthed by data mining. In
Closterium, intercellular communication between two
mating types via pheromonal substances is essential
for successful conjugation (Tsuchikane et al., 2003,
2005). To date, direct information on the relationships
between sex pheromones and these molecules has
been lacking. Cloning of the full-length cDNAs for
these receptor candidates and testing of the binding
activities between the extracellular domains and sex
pheromones will shed light on the molecular mechanisms of intercellular communication during the sexual reproduction of Closterium.
In higher plants, the fertilization process occurs in
the ovules and, thus, it is not easy to analyze the
cellular responses of gametes in vivo. In addition, it is
difficult to isolate sufficient sperm and eggs from the
plants for in vitro molecular investigations of fertilization. However, it is relatively easy to induce sexual
reproduction in C. pslc, and some of the sexually
related genes in C. pslc showed similarities to previously deposited genes from land plants, including
hypothetical genes and/or genes whose functions
were unknown (Supplemental Table III). It would be
important to investigate whether such homologous
genes are also up-regulated in sexual cells. If any show
a promising expression pattern, phenotypic analyses
by reverse genetics should be applied. Because
charophycean green algae are the closest relatives to
land plants, the expression profiles generated from
this array and some genes detected by these analyses
should help reveal the molecular mechanisms of gametogenesis and intercellular communication during
fertilization in land plants.
The Closterium microarray resource is a potentially
useful and beneficial tool for the exploration of genes
that are regulated in response to environmental
changes, as well as sex-related phenomena. We can
easily monitor gene expression changes caused by
environmental modifications without influences from
other tissues and organs, because Closterium is a
unicellular plant. In addition, Closterium has an experimental advantage that results from its evolutionary position on ‘‘the tree of life.’’ This unicellular plant
is most closely related to land plants, and its cellular
features and metabolism are more similar to those of
land plants than those of the green yeast Chlamydomonas. Recently, we isolated the MADS-box gene
(CpMADS1) from C. pslc and implicated it in sexual
differentiation (Tanabe et al., 2005). Loss-of-function
experiments and this microarray technique are indispensable for studies concerned with unveiling the
impact on critical phenomena in plants of the genetic
network that is under the regulation of the MADS-box
protein and other key regulators. The phenotypic
profiles prospectively obtained from transformation
experiments with Closterium cells, as well as the
transcriptome resources, will provide insights into
intercellular communication and the evolution of
land plants.
MATERIALS AND METHODS
Plant Materials, RNA Isolation, and Construction of
cDNA Libraries
The strains of heterothallic Closterium peracerosum-strigosum-littorale complex (C. pslc) were NIES-67 (mt1) and NIES-68 (mt2), which were obtained from
the National Institute for Environmental Studies, Ibaraki, Japan. The respective
vegetative cells (mt1-V and mt2-V) were obtained from cultures grown in
nitrogen-supplemented medium (C medium; http://www.nies.go.jp/biology/
mcc/home.htm), as described previously (Sekimoto et al., 1990).
The sexual reproduction of C. pslc was induced as follows. Vegetatively
growing cells of the two mating types were harvested, washed three times
with MI medium (Ichimura, 1971), and incubated separately in MI medium
(3.0 3 105 cells/mL) under continuous light for 24 h (high-density preculture;
mt1-0, mt2-0). The cells of both mating types (5.4 3 105 each) were mixed in
75 mL fresh MI medium (7.2 3 103 cells/mL) in 300-mL Erlenmeyer flasks
and incubated under light (low cell density culture). At various time intervals
(2, 8, 24, and 72 h), the cells were harvested, frozen in liquid nitrogen, and
stored at 280°C (mix-2 h, mix-8 h, mix-24 h, and mix-72 h, respectively). As
controls, cells of the respective mating types were incubated in MI medium at
a low cell density (mt1-L and mt2-L; 7.2 3 103 cells/mL of each) for 8 h
without mixing, then harvested and stored. In addition, mt2 cells and mt1
cells that had been incubated for 8 h in MI medium (7.2 3 103 cells/mL) that
contained PR-IP and PR-IP inducer at final concentrations of 4.8 3 1029 M and
3.0 3 1028 M, respectively, were harvested and stored (listed in the tables as
PR-IP and inducer).
Poly(A1) RNA was isolated using the mMACS mRNA isolation kit
(Miltenyi Biotec) or the PolyATtract mRNA isolation system (Promega)
following treatment with TRIzol reagent (Invitrogen) in accordance with the
manufacturer’s instructions.
The cDNA library was prepared from poly(A1) RNA that was isolated
from a mixture of vegetative cells (mt1-V and mt2-V) and cells at various
stages of sexual reproduction (mt1-0, mt2-0, mix-2 h, mix-8 h, mix-24 h, and
mix-72 h) using a cDNA library synthesis kit (SuperScript plasmid system;
Invitrogen), according to the manufacturer’s instructions. This primary cDNA
library and two previously prepared cDNA libraries (Sekimoto et al., 2003)
derived from cells in the various stages of sexual reproduction were mixed.
Normalization was performed as described previously (Sekimoto et al., 2003)
and used to derive EST sequence information.
Sequence Analysis
The EST sequences were determined using the multicapillary automated
DNA sequencers RISA-384 (Shimadzu Corporation) and CEQ 2000XL (BeckmanCoulter). Both vector-derived and ambiguous sequences were removed from
the collected EST sequences by computer-aided analyses. Each sequence was
then subjected to similarity searching against the National Center for Biotechnology Information (NCBI) nonredundant protein database using the
BLASTX algorithm. Sequence similarities were considered significant when
the expected value was ,1.0e25 at the amino acid sequence level. EST
redundancy was checked using an alignment program (BLAST) with a dataset
of itself (Altschul et al., 1990). Clones that showed .98% identity for stretches
of .100 bp were grouped together. The identified sequences have been
deposited in the DNA Data Bank of Japan (accession nos. BW646625–
BW648670).
Preparation and Analysis of the cDNA Microarray
The inserts of the cDNA clones were amplified by PCR using T7 and SP6
universal primers that were complementary to the vector sequences flanking
Plant Physiol. Vol. 141, 2006
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Sekimoto et al.
both sides of the cDNA insert. The PCR products were purified using 96-well
MultiScreen PCR plates (Millipore). Each purified cDNA insert was mixed
with reagent D (Amersham Biosciences), and each cDNA clone was spotted in
duplicate on aluminum-coated and DMSO-optimized glass slides using the
Array Spotter Generation III (Amersham Biosciences). Labeling, hybridization, and scanning were performed as described previously (Endo et al., 2002).
The fluorescence intensity for each spot was captured and quantified using
Microarray Suite (Scanalitics). Because each cDNA clone was spotted in
duplicate (right- and left-hand sides), normalization was performed against
the respective sides of the same glass slide, as described below. The 50th
percentiles of all values obtained from the respective sides were used as the
synthetic positive controls for each cDNA, and the value for each cDNA was
divided by these synthetic positive controls. These results were then filtered to
eliminate those cDNAs with ratios between the right and left position of ,1/3
or .3, and those cDNAs with values before normalization of ,50 in all
hybridizations. The average value of the duplicated spots was used as the
normalized value for each cDNA for each hybridization. After normalization,
cDNAs with .5-fold increase in expression during conjugation, as compared
with mt1 or mt2 cells cultured in MI medium at low cell density without
mixing (mt1-L or mt2-L), were chosen. We also selected cDNAs that showed a
4-fold difference in expression between the mt1 and mt2 cells or a 4-fold
increase in expression following the addition of purified sex pheromones
(PR-IP and PR-IP inducer). The reproducibility of the expression analysis
results was confirmed in two biologically independent experiments.
Real-Time PCR
Real-time PCR analyses were performed with the ABI PRISM 7000 (Applied Biosystems), according to the manufacturer’s instructions. Primers and
probes for the target sequences were designed using the Primer Express
software (Applied Biosystems). The cDNA was synthesized from total RNA
using the TaKaRa RNA LA PCR kit (AMV) version 1.1 (TaKaRa Bio) and
random primers, according to the manufacturer’s instructions. The primers
and probes for each gene are shown in Supplemental Table IV. PCR was
performed for 43 cycles at 95°C for 15 s and at 60°C for 1 min. The signals were
detected in the ABI Prism 7000 as fluorescent emissions generated by the
dissociation of fluorescent chemicals from the TaqMan probes. The transcript
levels were determined by the slope of the curve generated by amplification of
serially diluted plasmids that carried the respective genes. For the cycle in
which all the signals were amplified exponentially, the signals were converted
into numerical values of 18S rRNA using the predeveloped TaqMan assay
reagent for eukaryotic 18S rRNA (Applied Biosystems) to normalize all
signals.
Statistical analysis was performed according to standard procedures, as
indicated. The reproducibility of the expression analysis results was confirmed in two independent experiments and SEs are shown.
ACKNOWLEDGMENTS
We thank Ms. Ayumi Ihara and Ms. Sayaka Mikami for technical assistance.
Received January 28, 2006; revised March 9, 2006; accepted March 11, 2006;
published March 24, 2006.
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