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Keywords:

  • differential gene expression;
  • male germ lineage;
  • preferential fertilization;
  • Plumbago zeylanica;
  • sperm cell;
  • sperm dimorphism

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plumbago zeylanica produces cytoplasmically dimorphic sperm cells that target the egg and central cell during fertilization. In mature pollen, the larger sperm cell contains numerous mitochondria, is associated with the vegetative nucleus (Svn), and fuses preferentially with the central cell, forming endosperm. The other, plastid-enriched sperm cell (Sua) fuses with the egg cell, forming the zygote and embryo. Sperm expressed genes were investigated using ESTs produced from each sperm type; differential expression was validated through suppression subtractive hybridization, custom microarrays, real-time RT-PCR and in situ hybridization. The expression profiles of dimorphic sperm cells reflect a diverse and broad complement of genes, including high proportions of conserved and unknown genes, as well as distinct patterns of expression. A number of genes were highly up-regulated in the male germ line, including some genes that were differentially expressed in either the Sua or the Svn. Differentially up-regulated genes in the egg-targeted Sua showed increased expression in transcription and translation categories, whereas the central cell-targeted Svn displayed expanded expression in the hormone biosynthesis category. Interestingly, the up-regulated genes expressed in the sperm cells appeared to reflect the expected post-fusion profiles of the future embryo and endosperm. As sperm cytoplasm is known to be transmitted during fertilization in this plant, sperm-contributed mRNAs are probably transported during fertilization, which could influence early embryo and endosperm development.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In angiosperms, the male germ lineage is traceable to an asymmetric mitotic division of the post-meiotic microspore, which produces a small lens-shaped generative cell and cleaves off a much larger pollen vegetative cell (Boavida et al., 2005; Ma, 2005). The generative cell undergoes morphogenesis and migration, entering the interior of the vegetative cell and forming a highly polarized spindle-shaped cell. This cell continues to diminish in volume during maturation and divides mitotically to form two small sperm cells (Russell and Strout, 2005). The male gametic lineage is immersed in and thus uniquely dependent on the pollen vegetative cell for nutrition and transportation. Despite the fact that sperm cells and their precursor generative cell appear outwardly dependent, male germ cells are known to have their own enriched and unique molecular repertoire, with both separate and overlapping systems of genetic control (Singh et al., 2008).

Male germ lineage cells transcribe their own separate pools of stable, translatable mRNA (Zhang et al., 1993; Blomstedt et al., 1996), and synthesize proteins unique to the germ lineage (Ueda and Tanaka, 1995a,b). cDNA libraries have been constructed based on generative cells of Lilium longiflorum (lily) (Xu et al., 1998; Okada et al., 2006a) and sperm cells of Oryza sativa (rice) (Gou et al., 2001), Zea mays (maize) (Engel et al., 2003) and Nicotiana tobacco (tobacco) (Xu et al., 2002), revealing some sperm-specific genes (Singh et al., 2008). Transcriptomic examination of Arabidopsis sperm cells (Borges et al., 2008) further supports the existence of unique male lineage products and independent expression.

In Plumbago zeylanica, the two sperm cells are known to be dimorphic, and undergo preferential fertilization based on differences in sperm cell organization, cellular associations and organelle composition. The sperm cell associated with the vegetative nucleus (Svn) is the larger cell and contains many mitochondria (mean 256.18) and few plastids (mean 0.45). The smaller sperm cell (Sua) is not associated with the vegetative nucleus and contains abundant plastids (mean 24.3) and smaller, less numerous mitochondria (mean 39.8) (Russell, 1984). The latter sperm cell fuses with the egg cell in >95% of cases examined, providing evidence of preferentiality during fertilization (Russell, 1985). Such structural differences between the two sperm cells may correlate with differences in gene expression that affect post-fertilization development, as the male cytoplasm in this plant is known to be transmitted into female gametes during double fertilization (Russell, 1980; Weterings and Russell, 2004).

The embryo and endosperm follow strongly divergent developmental pathways after double fertilization, which underscores the importance of gene expression in female gametes preceding double fertilization (Le et al., 2005; Sprunck et al., 2005), especially in the light of post-fertilization paternal silencing in flowering plants (Grossniklaus et al., 1998; Vielle-Calzada et al., 2000). Little consideration, however, has been given to male contributions during early embryogenesis that may occur during plasmogamy. As a prelude to studying the role of sperm cell contributions in embryo and endosperm development, in this study we examine the transcriptional profile of sperm cells of Plumbago zeylanica, a plant with dimorphic sperm cells that displays preferential fertilization, including distinct patterns of gene expression in each type of sperm cell.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation of high-quality sperm cell populations

Sperm cells are readily released from anthesis-stage pollen into isolation buffer (Figure 1a), and may be separated by size, shape and contents (Figure 1b,c,e). Micropipette-collected cells yielded populations of extremely high purity with high viability based on fluorescein diacetate accumulation (Figure 1d,f).

image

Figure 1.  Isolation of sperm cells from anthesis-stage pollen of Plumbago zeylanica. (a) Anthesis-stage pollen 5 min after scattering on sperm cell isolation buffer. (b) Released sperm cells. The larger Svn is associated with the vegetative nucleus, whereas the smaller Sua is not associated with the vegetative nucleus. Arrows indicate the released sperm cells, and these are magnified in the inset. (c, e) Populations of isolated Sua and Svn, respectively. (d, f) Fluorochromatic reaction demonstrates viable and intact isolated Sua and Svn, respectively. Images are from bright-field microscopy (a, b, c, e) and epifluorescence microscopy (d, f). Scale bars = 50 μm (a) and 20 μm (b–f).

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Sperm-cell ESTs from representative cDNA libraries

Populations of at least 12 000 sperm cells of each morphotype (Sua and Svn) were used to construct two separate size-fractionated (>400 bp) cDNA libraries. After 24 and 26 amplification cycles in the Sua and the Svn, respectively, the resulting cDNA had a size distribution of 0.1–6.0 kb, with highest abundance between 0.5 and 3.0 kb in both types of sperm cell (Figure S1a,b). PCR-amplified cDNA was ligated into λTriplEx2 vector and packaged, yielding unamplified libraries of 2.1 × 107 and 3.2 × 107 pfu ml−1 for the Svn and the Sua, respectively. Libraries were of high quality: approximately 90% of the clones had inserts, and >95% of the inserts >500 bp. The insert size of examined cDNA clones in the Sua cDNA library ranged from 0.6 to 2.5 kb, centered around 1.5 kb (Figure S1c), and from 0.4 to 1.5 kb in the Svn cDNA library, centered around 1.0 kb (Figure S1d).

EST sequencing was performed from both 5′ and 3′ ends for 615 Sua and 593 Svn randomly picked clones. A total of 826 Sua ESTs of high quality were obtained and assembled to 426 non-redundant clusters containing 207 contigs and 219 singletons (Table S1). A total of 606 Svn ESTs of high quality were assembled to 419 non-redundant clusters containing 84 contigs and 335 singletons (Table S2). ESTs from one clone were assigned to one cluster. Only 25.4% of the 426 Sua EST clusters and 26.5% of the 419 Svn EST clusters could be assigned to putative functions (Tables S1, S2 and S3). Most EST clusters (58.9% of Sua and 62.8% of Svn) showed no significant homology with National Center for Biotechnology Information (NCBI) databases. The remaining sequence clusters (15.7% of Sua and 10.7% of Svn) displayed similarities but could not be assigned functions.

Among annotationally classified ESTs, the Sua is enriched in transcription-related genes (13.9% versus 8.1%) and the Svn is enriched in protein fate genes (18.9% versus 13.9%). Interestingly, sperm morphotypes frequently had similar percentages among categories of classified genes (Figure S2a,b and Table S3), although these were often represented by different genes. Of recovered ESTs, 17.63% of these clusters were common to both sperm cells, with 40.24% found only in Svn ESTs and 42.13% only in Sua ESTs. Among the most abundant ESTs in both the Sua and the Svn were ubiquitin ligase, ubiquitin-conjugating enzyme, heat shock protein, high mobility group proteins, various histones and phosphatases (Table 1).

Table 1.   Most abundant transcripts in the Sua and the Svn
Putative identityaAGIbe-valuecClonesdPutative function
  1. aBased on sequence similarity to the genes in the non-redundant Genbank protein database.

  2. bThe AGI codes of the most similar Arabidopsis genes to the Sua and Svn EST sequences.

  3. cThe best e-value from a BLASTX search for corresponding Sua and Svn EST sequences.

  4. dNumber of sequenced Sua and Svn cDNA clones.

  5. eReported as present in the Arabidopsis sperm transcriptome (Borges et al., 2008).

Sua
 Ubiquitin ligaseAt5g42190e6e-04433Protein degradation
 Putative phosphataseAt1g17710e1e-06327Unclassified
 Ubiquitin-conjugating enzymeAt3g52560e6e-07112Protein degradation
 High mobility group proteinAt1g206935e-04211Chromatin modeling
 Histone H3At5g10980e7e-072 8Chromatin modeling
 Heat shock proteinAt5g597201e-055 7Cell rescue, defense and virulence
 Actin-depolymerizing factorAt1g017509e-063 5Protein binding/cytoskeleton
 ABC transporter family proteinAt3g106701e-054 3Cellular transport, facilitation, targeting
Svn
 Ubiquitin ligaseAt1g75950e3e-05034Protein degradation
 Putative phosphataseAt1g17710e4e-055 8Unclassified
 High mobility group proteinAt1g206931e-04213Chromatin modeling
 Histone H3At5g10980e2e-071 8Chromatin modeling
 Heat shock proteinAt4g276703e-033 6Cell rescue, defense and virulence
 Actin-depolymerizing factorAt4g255903e-052 6Protein binding/cytoskeleton
 F-box family proteinAt4g032205e-015 6Protein degradation
 PolyubiquitinAt5g20620e4e-079 4Protein degradation
 ExopolygalacturonaseAt3g140401e-021 4Carbohydrate metabolism
 Ubiquitin-conjugating enzymeAt3g52560e4e-056 3Protein degradation

Comparison of ESTs among gametes or gamete-related cells

Plumbago zeylanica sperm-generated ESTs were compared with ESTs from male and female germ lineages of rice (Gou et al., 2001), maize (Engel et al., 2003; Yang et al., 2006), Arabidopsis (Honys and Twell, 2004; Borges et al., 2008), lily (Okada et al., 2006a) and Triticum aestivum (wheat) (Sprunck et al., 2005) to examine potentially conserved gamete-expressed genes (Tables S4 and S5). Comparison with 61 rice sperm ESTs indicated high similarity for five Sua and ten Svn ESTs. Comparison with maize sperm cells indicated significant matches for 56 of 426 non-redundant Sua ESTs (13.4%) and 45 of 419 non-redundant Svn ESTs (10.7%). Comparison with 1355 genes expressed in Arabidopsis pollen revealed high similarity with 39 Sua (9.2%) and 41 Svn ESTs (9.8%), and 121 (28.4%) Sua ESTs and 97 (23.2%) Svn ESTs showed high similarity to Arabidopsis sperm cell-expressed genes. Comparison with 886 lily generative cell ESTs, indicated significant similarity for 31 (7.3%) Sua and 31 (7.4%) Svn ESTs. Male gamete-expressed chromatin-related genes included histones H2A, H2B, H3 and H4 (Tables S4 and S5). Genes involved in modulating protein turnover included polyubiquitin, ubiquitin ligase and ubiquitin-conjugating enzyme (Tables S4 and S5). These genes are up-regulated in generative and sperm cells of both monocots and dicots (Singh et al., 2002).

To examine possible conservation of germ lineage sequences, female gametophytic lineages were also compared. Maize embryo sac ESTs (Yang et al., 2006) shared high similarity with 73 (17.1%) Sua and 61 (14.6%) Svn ESTs, maize egg cells (Le et al., 2005) shared high similarity with 54 (12.7%) Sua and 38 (9.1%) Svn ESTs, and wheat egg and pro-embryo ESTs (Sprunck et al., 2005) shared high similarity with 48 (11.3%) Sua and 31 (7.4%) Svn ESTs. Common expression patterns found in these cells frequently included histone variants, polyubiquitin, ubiquitin ligase and ubiquitin-conjugating enzymes (Tables S4 and S5). Overall, 148 (34.7%) Sua and 122 (29.1%) Svn ESTs displayed high similarity to other gamete-related ESTs, whereas 278 (65.3%) Sua and 297 (70.9%) Svn ESTs did not display high similarity to examined gamete-related ESTs.

Expression profiles of Plumbago zeylanica sperm cell genes

To examine the expressional profiles of ESTs isolated from sperm cells, real-time RT-PCR was used to compare a spectrum of tissues, including sporophyte (roots, stems, leaves, sepals, petals, unpollinated mature ovules, ovules at 72 h post-pollination) and gametophyte (microspore, bicellular pollen, anthesis-stage pollen, Sua and Svn) (Figure 2). Examined genes and corresponding primer sequences are listed in Table S6. For each sample, 10 ng of pre-amplified cDNA was used as template for the real-time RT-PCR reactions. The expression level in the Sua was set as 1, as no examined gene was found that displayed consistent expression across the spectrum of cells examined.

image

Figure 2.  Real-time RT-PCR expression profiles for selected transcripts derived from Sua and Svn cDNA libraries. Transcripts are indicated by Genbank accession number and annotation. R, root; S, stem; L, leaf; Se, sepal; Pe, petals; O, mature ovule; E, fertilized ovule (72 h after pollination); Un, uni-nucleate microspore; Bi, bicellular pollen: P, anthesis-stage pollen; Su, Sua; Sv, Svn. The expression level in the Sua is set to 1. See Table S6 and Figure S3 for further data.

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Five expression patterns were observed in the examined transcripts: (i) essentially constitutive genes with up-regulated expression in sperm cells (Figure 2a,b), (ii) constitutive gene expression in multiple organs or cells, with down-regulated gametophytic expression (Figure 2c), (iii) highly expressed in both sperm cells with negligible expression in other tissues (Figure 2d–g), (iv) up-regulated expression in the Sua (Figure 2h–m), and (v) constitutive expression, detected in all organs or cells (probable ‘housekeeping genes’) (Figures 2n,o and S3a–f).

Changes in the transcriptional abundance of genes were observed during reproductive cell development, for example lower expression levels in early development (Figure S3a,d) or the converse (Figure S3e). Ten of 21 selected ESTs displayed negligible expression in somatic cells and early reproductive phases, consistent with male germ lineage expression involving some unique genes. Differential expression in the Sua and the Svn was also observed (Figure 2h–o). ESTs sampled from non-suppression subtractive hybridization cDNA libraries showed either similar or consistently higher expression in the Sua.

Expression of differentially up-regulated and down-regulated genes in Sua and Svn sperm cells

Suppression subtractive hybridization (SSH) was used to enhance differentially up-regulated transcripts in dimorphic sperm cells, using the Sua or the Svn as the tester or driver, respectively. Most recombinant insertions in SSH clones displayed sizes ranging from 200 bp to 1.5 kb. A custom microarray of 4608 clones was constructed to partially characterize the extent of expressional dimorphism and identify strongly differentially expressed genes (Figure 3a).

image

Figure 3.  Identification of differentially expressed genes in the Sua and the Svn. (a) Scatter plot of more than twofold up-regulated ESTs identified by SSH and microarray by sperm type, Sua and Svn. (b, c) Functional categorizations from SSH cDNA libraries indicate dissimilar functional profiles for the Sua (b) and the Svn (c). See Tables S7 and S8 for further annotation.

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Differentially expressed genes displaying more than twofold up- or down-regulation in the Sua and the Svn were identified, showing similar numbers of distinct products. Although most products displayed less than twofold expression differences between dimorphic populations, 13.2% of the Sua SSH sequences and 12.8% of the Svn SSH sequences assayed showed significant up-regulation (2 to 4-fold) and 187 Sua and 229 Svn clones showed expression levels greater than fourfold (Figure 3a); 106 Sua and 149 Svn ESTs from the latter group were successfully sequenced. Functional categories of differentially expressed transcripts are summarized in Figure 3(b,c) and Tables S7 and S8. Among SSH-derived Sua ESTs, 27% displayed no homology; this proportion was nearly tripled (76%) for the SSH-derived Svn ESTs. Unclassified proteins constituted 26% of unique Sua sequences and 4% of Svn sequences.

The most abundantly represented functional categories in the Sua include transcription, protein synthesis and protein fate (Figure 3b, Tables 2 and S7). As the Sua has over 50 times more plastids compared to the Svn, it is not surprising that chloroplast-encoded genes are found in Sua SSH ESTs. Plastid-related ESTs encode chloroplast 30S ribosomal protein, ATP-dependent Clp protease proteolytic subunit, the ATP synthase CF1 α chain and CF0 B subunit, photosystem I assembly proteins Ycf3 and Ycf4, photosystem II 47 kDa protein and the RNA polymerase α chain (Table 2), and were not recovered from Svn SSH sequences. The SSH-derived Sua library also contained arabinogalactan proteins (AGPs), a family of extensively glycosylated hydroxyproline-rich glycoproteins thought to play important roles in various aspects of plant growth and development (Schultz et al., 2000; Showalter, 2001). The identified Plumbago zeylanica AGP (PzAGP) sequences have >50% amino acid identity with Arabidopsis AGP16, AGP20 and AGP22. AGPs are present on male germ cell membranes of Brassica campestris and Lilium longiflorum (Southworth and Kwiatkowski, 1996). In situ hybridizations indicated that PzAGP is localized only in the Sua (Figure S4). SSH screening also revealed an EST that was up-regulated in the Sua and shows strong similarity to the calmodulin-binding receptor-like cytoplasmic kinase 1 (CRCK1) of Arabidopsis.

Table 2.   Up-regulated genes identified in Sua and Svn SSH cDNA libraries, listed by functional category
Accession numberaMean log2 ratiob Putative identitycNumber of ESTsd AGIe
  1. aGenBank accession numbers for Sua and Svn SSH ESTs.

  2. bMean log2 ratio value of Sua and Svn SSH probe from three repeats.

  3. cBased on sequence similarity to the genes in the non-redundant Genbank protein database.

  4. dNumber of cloned ESTs that show the same similarity in the database.

  5. eThe AGI codes of the most similar Arabidopsis genes.

  6. fReported as present in the Arabidopsis sperm transcriptome (Borges et al., 2008); f+ is >500, f− is <100 expression units.

  7. gNot represented on Affymetrix Arabidopsis ATH1 genome arrays.

Sua
 Biogenesis of cellular components
  EE297483−3.18Polypyrimidine tract-binding protein 11At5g53180f+
 Cell cycle and DNA processing
  EE297492−2.068Ribonucleotide reductase large subunit A2At2g21790f
  EE297557−3.47Origin recognition complex protein1At2g37560f+
 Cell rescue, defense and virulence
  EE297464−2.50Heat shock cognate protein HSP702At5g02500f
  EE297507−3.26Heat shock factor protein HSF81At4g17750f
 Cellular communication
  EE297550−2.50CRCK1 (calmodulin-binding receptor-like cytoplasmic kinase 1)1At5g58940
 Cellular transport
  E297498−3.84Adaptin family1At5g11490
  EE297511−2.54Importin α subunit2At3g06720
  EE297515−3.09NLS receptor1At4g16143
 Metabolism
  EE297477−2.3Glyoxalase family protein (lactoylglutathione lyase family protein)1At2g32090
  EE297482−2.19Putative l-asparaginase1At3g16150
  EE297518−2.86Trehalose phosphatase family1At1g23870f−
EE297521−2.64Transferase hexapeptide repeat family1At1g19580
  EE297545−2.34Inositol-3-phosphate synthase (Myo-inositol-1-phosphate synthase) (IPS)1At2g22240f+
  EE297552−2.96Isoamylase isoform 11At2g39930
 Protein fate
  EE297471−2.81DnaJ protein family1At5g42480
  EE297505−3.53Ubiquitin-specific protease 23 (UBP23)1At5g57990f−
  EE297532−2.26Ubiquitin-conjugating enzyme 9 (UBC9)3At4g27960g
 Protein synthesis
  EE297472−2.7640S ribosomal protein S101At5g52650
  EE297488−3.3Elongation factor 2 (EF-2)1At1g56070f−
  EE297538−2.55Translation initiation factor IF-11At4g11175
 Transcription
  EE297460−2Transducin/WD-40 repeat protein family1At5g49430f
  EE297501−2.12Putative transcription initiation factor1At4g12610g
  EE297509−2.95Helicase-like transcription factor-like protein1At5g05130f+
  EE297533−2.66Transducin/WD-40 repeat protein family2At5g14530
  EE297559−2.6Transcriptional activator, putative1At3g57300f+
 Chloroplast-encoded genes
  EE297455−3.07Photosystem I assembly protein Ycf41AtCg00520
  EE297467−3.06ATP synthase CF0 B subunit4AtCg00130
  EE297468−2.22Ribosomal protein S167AtCg00050
  EE297469−3.06RNA polymerase α chain1AtCg00740f
  EE297470−2.1Photosystem I assembly protein Ycf31AtCg00360f−
  EE297473−2.81ATP-dependent Clp protease proteolytic subunit6AtCg00670
  EE297499−3.07ATP synthase CF1 α chain1AtCg00120
  EE297548−2.28Beta vulgaris mitochondrion protein1No hit
  EE297549−2.27Photosystem II 47 kDa protein1AtCg00680
 Unclassified protein
  EE297457−2.46Unknown protein1At1g26110f
  EE297458−2.31Expressed protein, FHA domain-containing protein2At3g54350f+
  EE297459−2.18Putative RING zinc finger protein1At1g63900
  EE297478−2.5F-box protein family1At4g10925f+
  EE297485−3.15Hypothetical protein AGP163At2g46330f
  EE297487−3.39Coatomer β subunit (β-coat protein)2At4g31480g
  EE297489−3.2Hypothetical protein1At2g34040
  EE297502−2.15cis-Golgi SNARE protein, putative1At1g15880f+
  EE297508−3.12Glycine-rich RNA-binding protein (AtGRP2)2At4g13850
  EE297510−2.96Expressed protein1At1g16180
  EE297525−3.09Expressed protein2At5g54540f+
  EE297527−2.36PWWP domain protein1At3g09670
  EE297528−5.46Unknown protein3At1g48840
  EE297529−2.67Tonneau 2 protein (TON2)1At5g18580
  EE297535−2.37Tuftelin-interacting-related protein1At1g17070f
  EE297536−2.5Tetratricopeptide repeat (TPR)-containing protein1At1g76630
  EE297537−2.16Expressed protein1At1g71780f
  EE297544−2.33Expressed protein1At3g47490f
  EE297558−2.07Expressed protein1At1g66080g
Svn
 Cell cycle and DNA processing
  EE2975902.12pol polyprotein1No hit
 Cell rescue, defense and virulence
  EE2975782.04Wound-induced protein3At3g04720
  EE2977074.26LEA protein2At1g01470
 Cellular communication
  EE2976682.29Serine/threonine protein phosphatase PP12At3g05580f−
 Cellular transport
  EE2975672.27Potassium channel protein SKOR1At3g02850f+
  EE2976152.12Mitochondrial carrier protein family1At2g17270f−
 Metabolism
  EE2975692.05tRNA isopentenylpyrophosphate transferase2At2g27760g
  EE2975983.07Glutathione transferase, putative2At3g62760g
  EE2976132.75β-fructofuranosidase precursor, soluble1At1g12240f
  EE2976212.40Patatin, putative1At4g37050g
  EE2976312.11tRNA isopentenyl transferase-like protein5At3g63110g
  EE2976892.22tRNA isopentenyltransferase-related protein3At5g19040g
  EE2977002.20tRNA isopentenyl transferase-like protein1At3g23630g
 Protein fate
  EE2976912.13Skp11At5g42190f
 Transcription
  EE2976033.28WRKY family transcription factor1At2g23320f
 Unclassified protein
  EE2976062.27Transcript antisense to ribosomal RNA; Tar1p1No hit
  EE2976162.03AMMECR1 family1At2g38710
  EE2976472.25Hypothetical protein1At1g66235g
  EE2976512.34Hypothetical protein1No hit
  EE2976882.09Predicted protein8At5g54820g
 Mitochondria-encoded genes
  EE2976402.13NADH2 dehydrogenase (ubiquinone) chain 41AtMg00580f
  EE2976652.16ATPase subunit 99AtMg01080g

Svn ESTs appear to reflect the fact that these cells possess more than six times more mitochondria than the Sua, with metabolism and energy categories highly up-regulated (Figure 3c). Svn-abundant ESTs (Tables 2 and S8) include nine clones encoding mitochondrial ATPase subunits and 11 clones encoding isopentenyltransferase – a key enzyme controlling the first and rate-limiting steps of cytokinin biosynthesis (Kakimoto, 2003). Sequences displaying no homology with NCBI databases are abundant in SSH-derived cDNA libraries of the Sua and particularly the Svn.

Expression profiles of selected SSH transcripts of Plumbago zeylanica sperm cells

The expression patterns of 24 SSH and microarray-identified genes were further examined by real-time RT-PCR (Figures 4 and S5). Real-time RT-PCR characterizations of differentially up-regulated SSH-derived genes showed five general patterns: (i) gene expression in multiple organs or cells to some degree, but higher in the Sua (Figure 4a–f), (ii) genes that are highly up-regulated in the Sua (Figure 4g), (iii) genes that are highly up-regulated in the Svn (Figure 4h–r), (iv) expression detected in multiple organs or cells at different abundance levels (Figure S5a–e), and (v) gene expression in many organs or cells, with low expression level in sperm cells (Figure S5f). Genes with much higher expression levels in the Svn than the Sua tended to have much lower expression level in other tissues (Figure 4h–r), with only one similar example in the Sua, suggesting more highly divergent expression in the Svn. These data clearly demonstrate the utility of SSH in identifying differentially expressed dimorphic sperm cell genes.

image

Figure 4.  Real-time RT-PCR expression profiles for selected transcripts derived from subtractive Sua and Svn cDNA libraries. Transcripts are indicated by Genbank accession number and annotation. The abbreviations for tissues sampled are defined in the legend to Figure 2. The expression level in the Sua is set to 1. See Table S6 and Figure S5 for further data.

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In situ hybridization

In situ hybridizations were performed as an additional stringent test to validate our observations on differential gene expression in sperm cell types. The results shown in Figure 5 confirm three expression patterns for representative and SSH-identified genes: (i) high expression in both sperm cells, as shown in Figure 5(a–f), (ii) higher expression in the Sua, as shown in Figure 5(g–l), and (iii) essentially exclusive expression in the Svn, as shown for a sperm-specific isopentenyltransferase in Figure 5(m–o). In control experiments, no signal was detected using sense RNA probes (Figure 5p–r). These data (including the absence of signal in vegetative cell cytoplasm) are consistent with real-time RT-PCR results (Figures 2, 4, S3 and S5).

image

Figure 5.  Whole-mount in situ hybridization of mature pollen of Plumbago zeylanica probed with different transcripts, listed by Genbank accession number and annotation. Three patterns of sperm cell gene expression are shown: (1) expressed in both sperm cells: (a–c) CB817206 (methyl-CpG binding domain-containing protein); (d–f) CB817765 (no similarity); (2) up-regulation in the Sua: (g–i) CB817223 (ubiquitin-conjugating enzyme); (j–l) CB817254 (predicted protein); and (3) up-regulation in the Svn: (m–o) FD800888 (PzIPT, isopentenyltransferase). The hybridization signal in sperm cells is clearly visible following digoxigenin labeling with antisense RNA, but not after labeling with a control sense probe (p–r). Bright-field (left), mixed epifluorescence/bright-field (center) and epifluorescence microscopic imaging (right) show the positions of nuclei. Scale bars = 10 μm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Diverse and abundant transcripts were recovered in both sperm cell EST libraries

Sampling of Plumbago zeylanica sperm cell ESTs yielded approximately 1000 high-quality sequences for each population, which at this level of coverage represent mostly transcripts of high and intermediate abundance (Sambrook and Russell, 2001). In Plumbago zeylanica, many ESTs were recovered only once, reflecting a high diversity of transcripts. Estimates using Poisson calculations indicate that additional EST sampling could increase the number of recovered ESTs to more than 2000 without reaching saturation (Wang et al., 2004, 2005), and, among these transcripts, additional male germ line-specific genes are likely to be recovered. The size of the transcriptome in Plumbago zeylanica is likely to be in excess of that of Arabidopsis, which transcribes in excess of 5829 genes in sperm cells at anthesis (Borges et al., 2008).

Although many Plumbago zeylanica ESTs encode known proteins, many do not – a feature consistent with male germ cells of rice, lily, maize and Arabidopsis (Gou et al., 2001; Engel et al., 2003; Okada et al., 2006a; Borges et al., 2008). Sequences without homologs may represent a combination of coding and non-coding RNA.

The most highly abundant ESTs reflect the dynamism of gene expression in sperm cells

Similar to male germ cells of rice, lily, maize and Arabidopsis, Plumbago zeylanica displays abundant transcripts associated with ubiquitin, ubiquitin-conjugating enzyme, ubiquitin ligase and F-box proteins (Table 1). If its associated proteolytic complex is altered, male gametophyte development is known to be impaired (Doelling et al., 2007; Gallois et al., 2009). Ubiquitin control of protein longevity therefore appears to be activated in sperm. Svn ubiquitin transcripts appear to be up-regulated relative to the Sua, suggesting differential control of gene products through changes in protein longevity (Singh et al., 2002).

Chromatin-altering proteins were abundant in Plumbago zeylanica in both sperm types (Table 1), suggesting the importance of chromatin condensation and transcription in the germ line. Male germ line-specific histone variants were first found in Lilium longiflorum (Ueda and Tanaka, 1995a,b; Xu et al., 1999a; Okada et al., 2005a, 2006b; Ueda et al., 2005), and AtMGH3 is regarded as a sperm marker gene in Arabidopsis (Okada et al., 2005b, 2006b; Ingouff et al., 2007). ESTs of Plumbago zeylanica histone H3 show close homology to sperm-expressed H3.3 variant histones in Arabidopsis (Okada et al., 2006b), and possess different sequences in the Sua and the Svn. Interestingly, the sperm cells of Plumbago zeylanica (T. Yuan and S.D. Russell, unpublished data) and Arabidopsis are in S phase at anthesis (Friedman, 1999), but appear to transcribe mainly replication-independent variant histone H3.

Functional and expressional categorization of sperm ESTs

The ESTs of the Plumbago Sua and Svn reflect a broad group of functional categories, especially transcription, protein synthesis, protein modification, metabolism, energy production, cellular transport, signal transduction and the cell cycle. This complement contrasts with prior descriptions of male germ cells as highly or wholly dependent on surrounding pollen cytoplasm (Mascarenhas, 1990). Nearly half of the real-time RT-PCR profiles of Plumbago zeylanica sperm cells reflect gene expression in other tissues as well.

The remaining profiles may indicate expression in sperm cells, with minor (approximately 15%) or negligible expression (approximately 35%) in other cells – presumably reflecting a gametophytic class of genes that may include male- and female-expressed sequences. As suggested by Engel et al. (2005), some sperm cell genes may not be represented in conventional EST libraries.

As predicted by Walbot and Evans (2003), transcripts in the male gametophyte may reflect an enhanced multi-genic rather than multi-allelic diversity, as haploids are subject to stringent screening, and potentially lethal genes are not masked as in a diploid. Functional overlap may be useful in overcoming some genetic defects. Interestingly, the ESTs recovered from the two representative sperm cell libraries showed similar profiles when grouped into functional categories, but comprised diverse male gamete genes. Of these ESTs, 17.63% were found in both sperm cells, 40.24% in the Svn only and 42.13% in the Sua only. There is a high degree of sequence diversity in sperm cells within represented functional categories (Borges et al., 2008), but the overall complement of genes may be somewhat restricted, as in pollen vegetative cells (Boavida et al., 2005).

Numerous genes appear to be highly up-regulated within sperm cells

The transcriptional activity of male germ cells in angiosperms appears to occur in the germ lineage itself (Singh et al., 2008), as corroborated by direct evidence on RNA synthesis and protein synthesis in sperm cells of maize (Zhang et al., 1993) and generative cells of lily (Blomstedt et al., 1996). Male germ lineage-specific promoters of lily, maize and Plumbago zeylanica have been isolated and successfully expressed in other species (Xu et al., 1999b; Singh et al., 2003; Engel et al., 2005; X. Gou and S.D. Russell, unpublished data), suggesting conservation of function in this context.

A remaining question regarding sperm transcripts is whether they are ultimately translated and functional. Mechanisms such as RNA editing or splicing (Zienkiewicz et al., 2008a,b,c), RNAi silencing (Slotkin et al., 2009) and epigenetic factors (Grant-Downton et al., 2009), as well as translational and post-translational modifications of gene products (Singh et al., 2003), may result in transcripts not being translated into proteins in the sperm cells.

RNA processing proteins such as mago nashi may be required to activate transcriptional messages, as in animals (Dadoune et al., 2004; Parma et al., 2007) and the aquatic fern Marsilea (van der Weele et al., 2007). In Plumbago, where different ESTs appear to occur in each sperm cell type, it is interesting to note that there are two distinct sequences encoding mago nashi – one in each sperm cell. Post-transcriptional modification is thus another possible mechanism for differential expression in target cells.

Distinct complements of expressed transcripts distinguish structurally and functionally differentiated Sua and Svn sperm cells

Sperm dimorphism in Plumbago zeylanica arises from division of a highly polarized generative cell in which organelle patterns differ and cytokinesis forms distinctive Sua and Svn sperm cells (Russell et al., 1996). Despite the progressively diminishing cytoplasmic volume of male germ cells during maturation (Russell and Strout, 2005), a diversity of distinct products are present in the two cell types (Table 2). Prior ultrastructural studies indicate that the male gamete cytoplasm participates in plasmogamy (Russell, 1980), supporting the possibility that sperm transmit paternal transcripts into female target cells. Paternal expression has recently been confirmed by Bayer et al. (2009), who clearly demonstrated that sperm-transmitted transcripts present in the fertilized egg cell are translated, and the SHORT SUSPENSOR (SSP) protein controls the initial asymmetric zygotic division that establishes the embryo and suspensor lineages in the pro-embryo. Although only SSP transcripts have been shown to behave in this manner so far, additional paternal transcripts in fertilized fusion products have been reported (Ning et al., 2006).

Sperm transcripts delivered during fertilization in animal oocytes are believed to play an important developmental role during early embryogenesis (Ostermeier et al., 2004). When sperm nuclei lacking cytoplasm were implanted into oocytes during in vitro embryogenesis, severe developmental defects were often observed (Krawetz, 2005). Experiments involving implantation of activated sperm nuclei during in vitro fertilization in maize egg cells also similarly impaired embryogenesis (Matthys-Rochon et al., 1994).

In Plumbago zeylanica, the two sperm cell types express divergent, complex and unique transcriptional profiles. SSH ESTs and microarray analysis identified 80 highly up-regulated Sua and 97 up-regulated Svn non-redundant genes (Table S7 and S8). The existence of distinct patterns of gene expression in the Sua and Svn cell types was strongly confirmed by real-time RT-PCR and in situ hybridization. Based on these data, sperm dimorphism as displayed in Plumbago zeylanica appears to represent a highly apomorphic characteristic in the evolution of the male germ unit. Another conspicuous apomorphic characteristic of Plumbago zeylanica sexual reproduction is the structurally reduced female gametophyte. Lacking synergids, the embryo sac receives discharged sperm cells in an intercellular region located between the egg and central cell (Russell, 1982). Without synergids, a number of synergid-related sequences may be free for recruitment, providing an unusual opportunity for expressional plasticity.

The Sua, which fuses with the egg cell, displays a greater abundance of transcripts relating to transcription, translation and protein modification, and appears to share similarities with expected patterns of expression in the embryo. Some Sua-expressed genes are related to cellular signaling. For example, a sixfold up-regulated calmodulin-binding receptor-like cytoplasmic kinase (CRCK) homologous to AtCRCK1 was identified. Such CRCK proteins bind to calmodulin (CaM) in a calcium-dependent manner that stimulates the kinase activity of AtCRCK1 and plays a role in Ca2+/CaM-mediated signal transduction in plants (Yang et al., 2004). The calcium-dependent protein kinase (CDPK) identified in the Sua with homology to AtCPK1 belongs to the same family as AtCPK18 – a gene that is preferentially transcribed in Arabidopsis sperm cells (Borges et al., 2008). Ser/Thr protein kinases activated by calcium binding to a calmodulin-like domain function in a wide variety of physiological processes, including growth and development, hormone signaling and stress responses (Cheng et al., 2002; Dammann et al., 2003; Hrabak et al., 2003; Zhu et al., 2007). As a universal secondary messenger, calcium regulates plant growth, development and responses to environmental stimuli (Trewavas and Malho, 1998; Sanders et al., 2002). Calcium also plays important roles during fertilization in angiosperms (Dumas and Gaude, 2006; Ge et al., 2007). Calcium-regulated protein kinases may function during double fertilization in angiosperms to trigger myriad events during Sua–egg cell fusion.

Interestingly, many of the genes isolated from the SSH cDNA library that were up-regulated in the Svn lacked homologies in available databases and had few highly expressed homologs in the Arabidopsis sperm transcriptome (Table 2), suggesting that the Svn has a more strongly divergent gene expression program than the Sua. The Svn, which fuses with the central cell and forms the endosperm, displays a greater abundance of transcripts relating to metabolism and phytohormone biosynthesis – including multiple copies of cytokinin biosynthesis-related isopentenyltransferase. As cytokinin is a hormone that displays a negligible effect in pollen (Wu et al., 2008), its up-regulation is unexpected, although strongly increased cytokinin and isopentenyltransferase levels are found during endosperm induction and growth (Miyawaki et al., 2004; Day et al., 2008); this may represent a paternal effect in endosperm, similar to embryos (Bayer et al., 2009). Sperm transcripts or translated proteins activated in the newly fertilized central cell could contribute to the precocious development of the endosperm in comparison with the zygote.

The differences between the two sperm cells did not provide any insight into control of the origin of these two sperm cell types, but suggest that the expressional complement of each sperm cell type aligns with that of each sperm cell’s respective fusion target. A remaining question is whether male gametes in S phase [e.g. sperm isolated at anthesis from pollen of Arabidopsis (Friedman, 1999) and Plumbago zeylanica (T. Yuan and S.D. Russell, unpublished data)] express all genes required for successful fusion and development 5–9 h prior to pollen tube arrival, discharge and gamete fusion. As single cell-based techniques for capturing and sequencing transcriptome and proteome signals are perfected, the ultimate goal of describing and functionally testing the molecules that control sexual receptivity and gamete fusion may soon be realized.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The detailed experimental procedures used in this study are provided in the online version of this article (Appendix S1).

Plant materials

Greenhouse-grown Plumbago zeylanica L. plants were used. Microspores and bicellular pollen were staged as described by Russell et al. (1996). For comparison, seedling stems, roots, young leaves, petals, sepals, and mature unpollinated and early-embryogenesis (72 h post-pollination) ovules were collected.

For sperm isolation, anthesis-stage pollen was placed in isolation buffer (10 mm MOPS in 0.8 m mannitol, pH 4.6) for 5 min for sperm cell release, and 12 000 Sua and Svn were separated from male germ units (MGUs) according to size, shape, relative position and association with vegetative nucleus (Zhang et al., 1998). Sperm cells were washed twice in buffer, collected in 2 μl of mannitol solution, frozen in liquid nitrogen and stored at −80°C.

Total RNA isolation, cDNA synthesis and library construction

An RNeasy plant mini kit (Qiagen, http://www.qiagen.com/) was used to purify total RNA from tissues. The total RNA of sperm cells was isolated using an Absolutely RNA microprep kit (Stratagene, http://www.stratagene.com/), and precipitated with glycogen and ethanol. Double-stranded cDNA was synthesized using a SMART PCR cDNA synthesis kit (Clontech, http://www.clontech.com/) according to the manufacturer’s instructions.

Representative cDNA libraries were constructed using the Clontech SMART cDNA library construction kit according to the manufacturer’s instructions. The resulting cDNA libraries were amplified and stored in 7% DMSO at −80°C. Lambda phage clones were converted to plasmid clones using the Clontech Cre loxP system.

Subtractive cDNA libraries were constructed using a Clontech PCR-Select cDNA subtraction kit. Purified subtractive products were cloned into the pCR2.1-TOPO vector (Invitrogen, http://www.invitrogen.com/) and transformed into Escherichia coli XL10-Gold cells (Stratagene, http://www.stratagene.com/).

Microarray experiments and data analysis

For each of the subtracted Sua and Svn cDNA libraries, inserts from 2304 putative recombinant colonies were PCR-amplified, purified by ethanol precipitation, dissolved in 50% DMSO, and spotted onto Corning CMT-GAPS-coated glass slides. Targets were prepared using non-subtracted cDNAs of the Sua and the Svn labeled by indirect random primer extension using Cy3 and Cy5 dyes (Hegde et al., 2000).

Microarray hybridizations were performed at 42°C for 16 h. The signals were detected using an Axon GenePix 4000A microarray scanner, and data were analyzed using GenePix pro 4.0 software (Molecular Devices, http://www.moleculardevices.com/). A fourfold or greater difference in probe expression between the Sua and the Svn was used to identify differentially expressed genes in the two sperm cell types of Plumbago zeylanica in mature pollen.

Sequence analysis of Plumbago zeylanica sperm cell-generated ESTs

DNA sequencing reactions for clones of representative cDNA libraries were performed from both the 5′ and 3′ ends. For clones from subtracted cDNA libraries, the M13 reverse primer was employed. Sequences were edited to omit sequences of linkers and vectors. The accession numbers for the representative Sua ESTs are CB816827CB817719, those for the representative Svn ESTs are CB817720CB818348, those for the Sua SSH ESTs are EE297454EE297559 and FD800889, and those for the Svn SSH ESTs are EE297560EE297708 and FD800890. The accession number for PzIPT is FD800888. All EST sequences from representative sperm cDNA libraries and subtracted sperm cDNA libraries were deposited in GenBank.

To determine sequence identity, high-quality cDNA sequences were searched against the GenBank non-redundant protein database and protein sequences of Arabidopsis (TAIR 6.0 release) using BLASTX (Altschul et al., 1997) with a Seqtools software interface (http://www.seqtools.dk/). Transcripts were assigned as matches to a protein of known identity if the probability was <1.0 × e−10. Genes with an e-value >1.0 × e−10 were also assigned if nearly exact matches to sequences were observed. Clusters were assembled by Seqtools, omitting unassembled ESTs shorter than 150 bp. Sequence identity between Sua and Svn sperm cells was indicated by local BLASTN searches with an e-value <1.0 × e−100. To compare sequence similarities between ESTs of Plumbago zeylanica sperm cells and those of other gamete or gamete-related cells, local TBLASTX searches were performed using a cut-off value of 1.0 × e−10.

Real-time RT-PCR analysis

Quantitative expression differences of sperm-derived transcripts were estimated by real-time RT-PCR with double-stranded cDNA from all samples pre-amplified using the SMART PCR cDNA synthesis kit. After purification and measurement, 10 ng of double-stranded cDNA from each sample was used as template for real-time PCR analysis using an ABI Prism 7000 SDS system (Applied Biosystems, http://www.appliedbiosystems.com/). Data were analyzed using ABI Prism 7000 SDS software. For each examined gene, the ΔCt value between each tested sample and the Sua was calculated. The expression levels for each sample relative to the Sua are shown by the value of 2Ct. The accession numbers of analyzed ESTs and primers used are listed in Table S6.

Whole-mount in situ hybridization

Whole-mount in situ hybridizations were performed as described previously (Bouget et al., 1995, 1996; Torres et al., 1995). Selected genes representative of different expression patterns were cloned into a pBlueScript II SK(+) vector (Stratagene). After linearization of templates, both sense and antisense riboprobes were labeled with digoxigenin (DIG)-UTP using DIG RNA labeling mix (Roche Applied Science, http://www.roche.com) and purified using a Qiagen RNeasy kit. Hybridization signal was detected using a Roche alkaline phosphatase-conjugated anti-DIG antibody and a DIG nucleic acid detection kit, and counter-stained using 4′,6-diamidino-2-phenylindole to visualize nuclei (Singh et al., 2002).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Bruce Roe, Dr Doris Kupfer, Dr Yuhong Tang, Sunkyoung So and Hongshing Lai (Advanced Center for Genome Technology, University of Oklahoma, Norman, OK) for help with EST sequencing, Dr Tyrrell Conway and Dr Mary Beth Langer (University of Oklahoma Microarray Core Facility) for help with microarray screening, Drs Jia Li, Mohan Singh, Prem Bhalla and Takashi Okada (ARC Centre for Excellence in Integrative Legume Research, The University of Melbourne, Parkville, Victoria, Australia) for helpful advice and discussion during the course of this study, and Dr Yuanji Zhang (Samuel Roberts Noble Foundation, Ardmore, OK) for gene ontology analysis. This research was supported by a grant from the US Department of Agriculture National Research Initiative Competitive Grants Program (number 99-35304-8097), the University of Oklahoma and private funds.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Size distribution of cDNAs of Sua and Svn libraries. (a, b) Synthesized Sua and Svn sperm cell cDNA. Complementary DNAs from both types of sperm cells have similar size distributions between 0.1 to 6.0 kb, with higher abundance between 0.5-3.0 kb. (c, d) Insertions in randomly picked clones of Sua and Svn cDNA libraries. Insert size of examined cDNA clones in Sua cDNA library ranged from 0.6 to 2.5 kb, distributed around 1.5 kb. Insert size of cDNA clones in Svn cDNA library ranged from 0.4 to 1.5 kb, distributed around 1.0 kb. M, molecular weight markers (kb).

Figure S2. Functional categorization of ESTs derived from Sua (a) and Svn (b) sperm cell cDNA libraries. Representative cDNA libraries provide similar functional profiles; however, exact EST identifications often differ (Tables S1, S2, S3).

Figure S3. Real-time RT-PCR-generated expression profiles from Sua and Svn cDNA libraries showing significant expression in multiple tissues. (a–f) Six charts showing additional expression profiles for selected ESTs, by Genbank accession and annotation. Legend to tissues sampled: R, root; S, stem; L, leaf; Se, sepal; Pe, petals; O, mature ovule; E, fertilized [72 h pp] ovule; Un, uninucleate microspore; Bi, bicellular pollen: P, anthesis-stage pollen; Su, Sua sperm cell; Sv, Svn sperm cell. Expression level in Sua is set to 1.

Figure S4.In situ hybridization showing localization of transcripts of PzAGP (encoding an arabinogalactan protein), which is upregulated in the Sua. (a and b) Plumbago pollen grain probed by antisense PzAGP. (a) Brightfield image. (b) Mixed brightfield and epifluorescence image displaying DAPI-labeled nuclei; (c and d) Plumbago pollen grain probed by sense PzAGP. (c) Brightfield image. (d) Mixed brightfield and epifluorescence image displaying DAPI-labeled nuclei.

Figure S5. Real-time RT-PCR-generated expression profiles for representative transcripts derived from Sua and Svn SSH-cDNA libraries showing significant expression in multiple tissues. (a-f) Six charts showing additional expression profiles for selected ESTs generated using SSH, listed by Genbank accession and annotation. Legend to tissues sampled is in Supplementary Figure S3 legend. Expression level in Sua is set to 1.

Table S1. Detailed information on Sua sperm cell ESTs (Excel spreadsheet).

Table S2. Detailed information on Svn sperm cell ESTs (Excel spreadsheet).

Table S3. Functional categories of ESTs derived from Sua and Svn sperm cell cDNA libraries.

Table S4. Non-redundant Sua ESTs that have significant similarity to other gamete or gamete-related cells (Excel spreadsheet).

Table S5. Non-redundant Svn ESTs that have significant similarity to other gamete or gamete-related cells (Excel spreadsheet).

Table S6. Primers and annotation of selected cDNA clones used for real-time RT-PCR analysis.

Table S7. Identified transcripts with upregulated expression in Sua sperm cell by SSH and microarray analysis (Excel spreadsheet).

Table S8. Identified transcripts with upregulated expression in Svn sperm cell by SSH and microarray analysis (Excel spreadsheet).

Appendix S1. Additional details of experimental procedures used.

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