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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Gene Expression Profiling Using cDNA Microarray Analysis of the Sexual Reproduction Stage of the Unicellular Charophycean Alga Closterium peracerosum-strigosum-littorale Complex^1,[W]

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.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSIONS AND PERSPECTIVES MATERIALS AND METHODS LITERATURE CITED

 
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 receptor-like 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.

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 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 (mt⁺) and mating-type minus (mt^–). 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

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSIONS AND PERSPECTIVES MATERIALS AND METHODS LITERATURE CITED

 

Construction of the cDNA Microarray

The two cDNA libraries (1.6 x 10⁷ and 6.5 x 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 x 10⁶ clones) derived from a mixture of vegetative mt⁺ and mt^– cells was constructed. A normalized cDNA library that contained 1.7 x 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 vector-derived 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 < 1.0e^–5 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, Cy5-labeled cDNA populations from the respective mating types, incubated separately for 8 h in MI medium at a low cell density (mt⁺-L and mt^–-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 (mt⁺-0, mt^–-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 mt⁺-L or mt^–-L cells. Those cDNA spots that were differentially expressed in mt⁺ cells, compared to mt^– 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).

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 (mt⁺-L and mt^–-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 pheromone-responsive 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 ).

View larger version (12K): [in this window] [in a new window]   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.

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. During 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 mt⁺ 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 (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 mt⁺ 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 mt^– 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 mt^– cells. PR-IP inducer has been identified as a sex pheromone that is released from mt^– cells and induces PR-IP production in mt⁺ 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 mt⁺ 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 -tocopherol methyltransferase (ID 216), -tubulin (ID 219), protein kinase (ID 240), epimerase-like protein (ID 397), PREG-like 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.

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 mt^– 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 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 mt⁺ cells but not in mt^– 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 mt⁺ 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) 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 mt⁺ 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 mt^– 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 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--lactamase superfamily (Crowder et al., 1997; Maiti et al., 1997; Zang et al., 2001). A -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).

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 sexual reproduction (Fig. 2 ). The expression levels of CpRLK1, Cp19KSU, and Cp-01 in mt⁺ cells were stimulated by the addition of PR-IP inducer, whereas those of CpRLP1 and Cp-48 in mt^– 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 mt⁺ cells, whereas the accumulation of CpPI mRNA was limited in mt^– 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.

View larger version (18K): [in this window] [in a new window]   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 (mt+-0 h, mt–-0 h). 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 during conjugation is designated as 100.

View larger version (24K): [in this window] [in a new window]   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 mt+ or mt– 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 conjugation-related genes (CpRLK1, Cp19KSU, and Cp-01) in mt+ cells. B, Effect of PR-IP on the expression of two conjugation-related genes (CpRLP1 and Cp-48) in mt– cells. C, Effects of sex pheromones on the expression of the CpGLX2-1 gene: effect of PR-IP inducer on mt+ cells (left) and effect of PR-IP on mt– cells (right).

View larger version (17K): [in this window] [in a new window]   Figure 4. Sex-specific expression of the Cp-41 and CpPI genes under various culture conditions. The cDNAs (mt+-V, mt–-V, mt+-0, mt–-0, mt+-L, and mt–-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.

	CONCLUSIONS AND PERSPECTIVES

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSIONS AND PERSPECTIVES MATERIALS AND METHODS LITERATURE CITED

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

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSIONS AND PERSPECTIVES MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials, RNA Isolation, and Construction of cDNA Libraries

The strains of heterothallic Closterium peracerosum-strigosum-littorale complex (C. pslc) were NIES-67 (mt⁺) and NIES-68 (mt^–), which were obtained from the National Institute for Environmental Studies, Ibaraki, Japan. The respective vegetative cells (mt⁺-V and mt^–-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 x 10⁵ cells/mL) under continuous light for 24 h (high-density preculture; mt⁺-0, mt^–-0). The cells of both mating types (5.4 x 10⁵ each) were mixed in 75 mL fresh MI medium (7.2 x 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 –80°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 (mt⁺-L and mt^–-L; 7.2 x 10³ cells/mL of each) for 8 h without mixing, then harvested and stored. In addition, mt^– cells and mt⁺ cells that had been incubated for 8 h in MI medium (7.2 x 10³ cells/mL) that contained PR-IP and PR-IP inducer at final concentrations of 4.8 x 10^–9 M and 3.0 x 10^–8 M, respectively, were harvested and stored (listed in the tables as PR-IP and inducer).

Poly(A⁺) RNA was isolated using the µMACS 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(A⁺) RNA that was isolated from a mixture of vegetative cells (mt⁺-V and mt^–-V) and cells at various stages of sexual reproduction (mt⁺-0, mt^–-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 (Beckman-Coulter). 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 <1.0e^–5 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 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 mt⁺ or mt^– cells cultured in MI medium at low cell density without mixing (mt⁺-L or mt^–-L), were chosen. We also selected cDNAs that showed a 4-fold difference in expression between the mt⁺ and mt^– 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; returned for revision March 9, 2006; accepted March 11, 2006.

	FOOTNOTES

 
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).

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 (sekimoto{at}fc.jwu.ac.jp).

^[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.

^* Corresponding author; e-mail sekimoto{at}fc.jwu.ac.jp; fax 81–3–5981–3674.

	LITERATURE CITED

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSIONS AND PERSPECTIVES MATERIALS AND METHODS LITERATURE CITED

 
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