Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements in the flowering plant male germ lineage

Authors

  • Scott D. Russell,

    1. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA
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  • Xiaoping Gou,

    1. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA
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  • Chui E. Wong,

    1. Plant Molecular Biology and Biotechnology Laboratory, Australian Research Council Centre of Excellence for Integrative Legume Research, Melbourne School of Land and Environment, University of Melbourne, Parkville, Victoria 3010, Australia
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  • Xinkun Wang,

    1. Higuchi Biosciences Center, University of Kansas, Lawrence, KS 66047, USA
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  • Tong Yuan,

    1. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA
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  • Xiaoping Wei,

    1. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA
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  • Prem L. Bhalla,

    1. Plant Molecular Biology and Biotechnology Laboratory, Australian Research Council Centre of Excellence for Integrative Legume Research, Melbourne School of Land and Environment, University of Melbourne, Parkville, Victoria 3010, Australia
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  • Mohan B. Singh

    1. Plant Molecular Biology and Biotechnology Laboratory, Australian Research Council Centre of Excellence for Integrative Legume Research, Melbourne School of Land and Environment, University of Melbourne, Parkville, Victoria 3010, Australia
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Author for correspondence:
Scott D. Russell
Tel: +1 405 325 4391
Email: srussell@ou.edu

Summary

  • Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization.
  • Sperm cells of rice (Oryza sativa) were isolated from field-grown, disease-free plants and RNA was processed for use with the full-genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles.
  • A total of 10 732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin-mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing.
  • Genome-wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self-regulating through highly up-regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post-fertilization expression of early embryo and (or) endosperm development.

Introduction

Sperm cells represent the male partner that fuses with the egg cell during fertilization in all multicellular eukaryotic organisms. In flowering plants, the male germ lineage is established during pollen mitosis I when the microspore divides to form the large pollen vegetative cell and the much smaller generative cell. This asymmetric division triggers establishment of the generative cell and the developmental divergence of the male germ lineage (Eady et al., 1995). The male germ lineage is contained within the pollen vegetative cell inside the pollen grain and later tube. An ensuing mitotic division results in two nonmotile sperm cells, which become progressively smaller than typical somatic cells during their maturation and passage, occupying < 0.1% of the pollen grain (Russell & Strout, 2005). Although outwardly appearing to be simple cells, these two sperm cells have the capacity to fuse with egg cells and central cells, triggering double fertilization and embryogenesis. Double fertilization initiates remodeling of the egg into a totipotent zygote giving rise to the embryo and remodels the central cell into the nutritive endosperm.

Despite the small size of sperm cells and their dependence on surrounding pollen cytoplasm for nutrition and transport, these cells are transcriptionally active, possessing translation, regulatory and control elements, and a diversity of transcripts (reviewed by Singh et al., 2008). An increasing number of sperm genes have proved to be essential for fertilization and normal embryo establishment, including, for example, HAPLESS2 (HAP2) and SHORT SUSPENSOR (SSP). HAP2 is known to encode a sperm-specific, surface-linked protein required for fertilization and is also implicated in directing pollen tubes to their female targets (von Besser et al., 2006; Frank & Johnson, 2009). SSP is transmitted as a sperm transcript into the egg cell during gamete fusion and encodes the protein SSP, which activates the developmentally critical asymmetrical division of the zygote, producing a polarized proembryo, which has been shown to establish embryogenesis (Bayer et al., 2009). Other sperm-originating transcripts have also been reported in zygotes and proembryos of tobacco (Nicotiana tabacum) (Ning et al., 2006). Thus, accumulating evidence indicates that transcribed sperm products may fulfill essential roles in the successful establishment of the next generation through untraditional nongenetic mechanisms (Gou et al., 2009; Russell et al., 2010). Sperm cells are also known to form products that may directly communicate with female gametes (Tian et al., 2005), even when outwardly these cells appear to be mere passengers within the elongating pollen tube.

Although the genes controlling fertilization are being identified (Berger, 2008; Russell & Dresselhaus, 2008), only a small proportion of those in the male germ lineage have been fully characterized. The attraction of genomic profiling of transcripts generated by sperm cells lies in the identification of gene candidates selectively upregulated in the male germ line and in understanding their evolutionary involvement in reproductive biology. In Arabidopsis, the first report of what may constitute a canonical sperm transcriptome revealed 5829 transcribed genes using an Affymetrix 24K microarray (Affymetrix, Santa Clara, CA, USA; http://www.affymetrix.com/) (Borges et al., 2008). That study confirmed over-representation of genes associated with DNA repair, ubiquitin-mediated proteolysis, epigenetic labeling and cell cycle progression which were also reported in prior expressed sequenced tag (EST) studies of sperm cells (Gou et al., 2001, 2009; Engel et al., 2003; Okada et al., 2006). However, the evolutionary context of transcription and expression in the male germline will require genomic level investigations in a range of plants (Paterson et al., 2010). This study is the first to extend this range to any monocot or crop plant. Only with expansion of these data will we gain genomic level understanding of the unique contribution of sperm cells to sexual reproduction and their role in fertility and crop productivity as founder cells with direct input into the fusion products of fertilization.

Oryza sativa L. (rice) is a useful model for studies of fertilization because genomic tools are available, and rice has an ideally short progamic phase—from pollination to fertilization—of 15–30 min. In the rice pollen tube, which contains the sperm, essentially no transcriptional changes were detected as compared with mature pollen (Wei et al., 2010). Unlike Arabidopsis, sperm cells of rice are mature; they do not require completion of DNA synthesis and protracted pollen tube growth to effect fertilization (Friedman, 1999). This study provides genomic evidence of highly up-regulated transcripts encoding genes involved in chromatin modification in the male germ lineage, up-regulated pathways regulating miRNA and siRNA processing, and distinct transcription factors and signaling molecules indicating a unique transcriptional profile in the male germline.

Materials and Methods

Sperm and pollen isolation

Sperm cells were isolated from mature anthers of field-grown rice (Oryza sativa L. ssp. japonica, cultivar ‘Katy’), courtesy of the Dale Bumpers National Rice Research Center and University of Arkansas Extension Station near Stuttgart, AR, USA. A centrifugation-based separation method was used for isolating sperm cells (Gou et al., 1999). Collected anthers were immersed in cold 45% (w/v) sucrose solution and ground gently with a small glass rod to release pollen grains. This pollen mixture containing the sperm cells was filtered through 100-μm nylon mesh into a 15-ml conical tube and centrifuged at 300 g for 3 min. The pellet was resuspended in cold 45% (w/v) sucrose and washed once more, as above. Subsequent purifications were performed using autoclaved solutions on ice. Collected pollen grains were suspended in a 10× volume of 15% (w/v) sucrose solution and gently agitated for 20 min to allow pollen grains to burst, which releases sperm cells and pollen cytoplasm. The mixture was then filtered through 30-μm nylon mesh and the filtrate was layered on a 15%/40% discontinuous Percoll density gradient and centrifuged at 4000g for 40 min at 4°C. The sperm cell-enriched fraction forms a band at the interface between 15% and 40% from which c. 100 μl is collected using a pipette. This sample is then diluted with a 4× volume of 15% sucrose solution, and centrifuged at 900g for 10 min. The bottom 2 ml of the centrifuged volume was collected. The pellet was gently suspended and filtered though a 10-μm filter, with the filtrate layered on top of another 40% Percoll density gradient and centrifuged at 4000g for 20 min. The band between the sample solution and 40% Percoll was again collected. This mixture contained concentrated, purified sperm cells. Another 4× volume of 15% sucrose solution was added to the mixture and centrifuged at 900g for 10 min. Approximately 50 μl of purified sperm cells was collected from the bottom of the centrifuge tube and stored frozen at or below −80°C until use. Three biological replicates were isolated and retained for all samples during processing.

RNA preparation

Control tissues included mature rice pollen as a microgametophytic control and young seedlings as a sporophytic control for verifying sperm-enriched or -depleted probe matches. Anthesis rice pollen was isolated according to Russell et al. (2008) and frozen in liquid nitrogen. Seedlings grown from rice seed of Katy were germinated in soil, collected at developmental stage V3 (collar forming on leaf 3 of main stem; Counce et al., 2000) and frozen in liquid nitrogen. All samples were stored at or below −80°C until RNA isolation. Total RNA was purified using the RNeasy plant mini kit according to the manufacturer’s instructions (Qiagen; http://www.qiagen.com/). The RNA concentration and quality of pollen and seedlings were determined using routine spectrophotometric measurement and agarose gels. For sperm cell RNA, we did not determine concentration because of limited materials from the start. All accumulated isolated sperm RNA and 100 ng of total RNA of seedlings and mature pollen (calculated using spectrophotometric measurements at 260 and 280 nm on a Nanodrop ND-1000 spectrophotometer; NanoDrop Technologies, Wilmington, DE, USA; http://www.nanodrop.com/) were used for probe preparation for each of the three biological replicates performed.

RT-PCR analysis

Total RNA isolated was amplified using the SMART PCR cDNA synthesis kit according to the manufacturer’s instructions (Clontech; http://www.clontech.com/). For each sample, c. 10 ng of cDNA was used as the template in a 10-μl reaction volume for PCR amplification of target genes. For the PCR process, 25–30 cycles were used to ensure that the amount of amplified product remained in linear proportion to the initial template present in the reaction. The entire PCR reaction was separated on a 1% agarose gel containing 0.1 μg μl−1 ethidium bromide and visualized under UV light. Constitutive PROFILIN-2 (LOC_Os06g05880) was used as an internal control. Real-time PCR analysis for selected transcripts was carried out in triplicate using EXPRESS SYBR® GreenER™ qPCR Supermix Universal kit (Invitrogen) with 1 ng of cDNA template according to the manufacturer’s instructions. The starting concentration (expressed in arbitrary fluorescence units) of each transcript in a sample was calculated using LinRegPCR (Ramakers et al., 2003) using raw fluorescence data generated by a Stratagene MX3000P (Invitrogen, Melbourne, Australia). This was then expressed relative to the starting concentration of PROFILIN-2. Primers are listed in Supporting Information Table S5.

Oligonucleotide microarray hybridization and data collection

As the amount of starting total RNA was low (in the range of 10–100 ng per sperm cell sample), the Affymetrix GeneChip Two-Cycle cDNA Synthesis Kit was used for target preparation with signal amplification. The Affymetrix 57K Rice Genome GeneChip oligonucleotide microarray was hybridized with 15 μg of fragmented cRNA for 16 h at 45°C, washed, stained, scanned and processed strictly following the Affymetrix GeneChip Expression Analysis Technical Manual as in Russell et al. (2008). Microarray data generated from all chips met quality control criteria set by Affymetrix. All data are posted in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database, accession entry GSE17002 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17002) for sperm, pollen vegetative cells and seedlings.

Calls for transcripts being present (< 0.05), absent (> 0.065) and marginal (0.05 < < 0.065) were calculated based on the Affymetrix PM/MM method (mas 5.0 algorithm, using Affymetrix gcos version 1.1.1). Pathway level differential expression was determined using the Wilcoxon–Mann–Whitney test; a weighted average (Tukey’s biweight) was used to determine fold differences between tissues. Functional classes were calculated by mapping rice orthologs on Arabidopsis BINS of the mapman data-mining program (Thimm et al., 2004), with gene assignments and annotation based on Affymetrix data (Netaffx website http://www.affymetrix.com/analysis/index.affx), rice genomic annotations (Michigan State University (MSU) release 7 and the Rice Annotation Project (RAP) release 5), RiceCyc (http://www.gramene.org/pathway/), GeneBins (Goffard & Weiller, 2007) and DAVID to estimate GO categorical enrichment and depletion (Huang et al., 2007, 2009).

The current MSU annotation of the rice nuclear coding genome (release 7) recognizes 56 081 individual gene loci and 66 433 gene models, 103 loci in the plastid genome and 54 loci in the mitochondrial genome. Probes on the Affymetrix rice 57K GeneChip™ are 25-mer 3’-oligomeric sequences grouped into probe sets that consist of from eight to 16 exact match probes. BLASTn analysis of perfect matches to cDNAs, introns, untranslated regions (UTRs), short models, and intergenic models was used to assign probe sets to loci and to identify exact probe sequence matches and overlaps.

The number of expressed genes was estimated from probe sets by normalizing for multiple sampling by removing duplicate probe sets that map redundantly to a given locus, yielding 34 830 unigene probe sets (of 57 272 noncontrol probe sets). The RAP, which omitted transposable element motifs, reported c. 32 000 genes annotated (Itoh et al., 2007). If transposable elements were added to their estimate, the result would be nearly identical to ours. Unanimous present calls were used to provide the most conservative estimate of transcripts expressed. Majority counts (two of three calls from biological replicates) increased gene estimates in pollen from 8101 to 9531, those in sperm from 10 732 to 12 331 and those in seedlings from 15 449 to 16 821. Mature pollen grains were reported to have from 9372 to 12 340 represented probe sets (Wei et al., 2010; their additional file 2a), which generally agrees with our average of 11 830, and is well within the standard error. Wei et al. (2010) reported 5939 unigenes in pollen, their count including only genes annotated as ‘expressed’. This under estimated the number of genes as The Institute for Genomic Research (TIGR) release 5 and MSU release 6.1 annotations reported only 68.543% and 69.317% of non-transposable element (TE) genes as ‘expressed’. In the current MSU release 7 annotation, however, the percentage of genes reported as ‘expressed’ increased by over 31.2% (90.945%); this would raise their number of estimated expressed genes to c. 7793, which is not significantly different from our estimate of 8101 pollen genes.

Results

Sperm and pollen collected from disease-free, field-grown rice plants in three separate fields provided biological replicates for this study. Sperm cells and pollen isolated for this study were examined using interference contrast microscopy and fluorescein diacetate tests for intact, viable cells (Fig. 1a). RT-PCR of selected marker transcripts (Fig. 1b) verified that sperm isolates were strongly enriched and only a few of the most highly expressed pollen genes (Russell et al., 2008) are reported present. GENERATIVE CELL SPECIFIC 1 (GCS1) is a sperm cell marker and PROFILIN-2 is a loading control (Fig. 1b). Pearson’s coefficient of correlation confirmed the reproducibility of the data, with mean r values of 0.983 for sperm, 0.993 for pollen, and 0.992 for seedlings. Each of the samples displayed a low correlation between different tissues, including sperm and pollen at r = 0.250 (Fig. 1c), sperm and seedlings at r = 0.239, and pollen and seedlings at r = 0.178. Scatter plots of all data sets are available at http://bomi.ou.edu/russell/plots/. These data reflect high sample consistency, excellent growth conditions and uniformity in biological and technical preparation, as is reflected in correlation matrix results (Fig. 1d).

Figure 1.

(a) Differential interference contrast microscopy and fluorescein diacetate viability screen of isolated Oryza sativa sperm cells (left; bar, 5 μm) and pollen (right; bar, 50 μm). (b) RT-PCR validation for purity of cell isolations using marker genes for pollen (allergen Ory s 1), sperm GENERATIVE CELL SPECIFIC 1 (GCS1) and HAPLESS 2 (HAP2) and constitutive loading control (PROFILIN-2). (c) Scatter plot of pollen vs sperm cell probe set signal intensities. (d) Correlation matrix of respective probe set signal intensities in sperm, pollen and seedling.

Diversity of transcript profile in sperm cells

A Venn diagram shows relative gene expression in sperm, pollen and seedlings (Fig. 2a) based on unanimous present/absent (P/A) calls in triplicate samples. Seedlings expressed 15 449 genes using this method of estimation, followed by 10 732 for sperm cells and 8101 for pollen. Genes representing distinct, nonoverlapping sequences totaled 33 278 for the Affymetrix 57K rice GeneChip. Pollen-specific expression suggests unique transcription of at least 626 genes, representing 1.88% of genes represented on the Oryza 57K chip—similar to previous microarray-based reports for Arabidopsis pollen-specific genes, ranging from 737 (Pina et al., 2005) to c. 800 in mature pollen (Honys & Twell, 2003). Seedling-specific expression indicates transcription of 5947 genes or c. 17.9% of genes represented on the 57K chip. Loci expressed in mature sperm indicate transcription of 1668 products, or an unexpectedly large 5.01% of genes represented on the 57K chip.

Figure 2.

(a) Venn diagram of genes expressed in different tissues of rice (Oryza sativa), including seedling (top), pollen (left) and sperm (right), in circles proportionate to the number of genes. (Probe sets corresponding to 14 785 unigene sequences were reported absent on the 57K chip.) (b) Pie chart showing functional categorization of transcripts expressed in sperm.

Sperm, pollen and seedlings transcribed 5537 gene sequences that appear to be represented in nearly all reference data accessed at NCBI GEO (platform GPL2025). We therefore believe that these may represent universal, potential ‘housekeeping genes’ of rice, in that they are expressed in all sporophyte and gametophyte cells observed to date. Approximately 20% of such transcripts are more highly represented in sperm cells than in other tissues and c. 5% are also highly represented in other reproductive tissues (Supporting Information Fig. S1). Although this might not reflect fundamental changes in function between this subset of up-regulated genes, there appear to be different levels of transcript abundance associated with the initiation of gametophyte development.

Functional categorization of sperm cell transcripts

The most highly represented functional categories in sperm cells involve metabolism, transcription and cell signaling (Fig. 2b). Functional categories up-regulated in sperm cells as compared with other tissues include transcription factors, cell signaling, protein modification, cellular identity and receptor-like molecules; these categories may each include some key players in functions unique to sperm cells.

Despite the small volume of male gametes, their short lifespan, and presumably small metabolic contribution to pollen, microarray results (this study and Borges et al., 2008) suggest that sperm cells transcribe a diversity of genes. Table 1 lists a group of especially highly up-regulated sperm- or germline-selective transcripts that show limited expression in other tissues (Fig. 3, rows 1–38). Of the genes apparently restricted to sperm cells, c. 62% are unclassified, or 18% higher than the number of unclassified genes in seedlings. Some sperm transcripts share up-regulation with female germ lineages (Fig. 3, rows 39–45), supporting the theory that some genes may be conserved in the germline between pollen and embryo sac lineages. Selected genes highly expressed in the microarray were examined using qRT-PCR (Fig. 4) and RT-PCR (Fig. 5). These results confirm microarray results for selected genes, including sperm markers GCS1, GAMETE EXPRESSED genes 1-3 (GEX1), GEX2 and GEX3 (Figs 1, 3–5). Additional highly transcribed sperm genes include sequences encoding aquaporin, F-box motif proteins, ubiquitin pathway-related proteins, DnaK Hsp70-related proteins, receptor-like kinases, cell signaling related proteins, apoptosis inhibitor BAX1 (BINDS ARCHAEL XPB helicase), and proteins involved in DNA repair pathways (Table 1); these have been found in multiple previous studies and putatively represent highly conserved functional themes of the male germline from both bicellular and tricellular pollen in monocot and dicot plants. The Arabidopsis transcriptome displayed sperm expression in homologs of 19 of the 45 transcripts shown in Table 1. Fourteen probe sets showed no close homolog, three homologs were called absent, whereas nine related loci were not represented on the Arabidopsis 24K genomic microarray chip (Table S1). Functional categories reported in sperm cell microarrays using the Arabidopsis and those annotated for the rice 57K Affymetrix genomic microarray chips reflected subtle differences in percentage representation, but overall gene numbers were much higher in Oryza sativa (Figs S3, S4), presumably largely because of historical expansion of the genome compared with that of Arabidopsis.

Table 1.   Highly sperm-enriched genes from the rice (Oryza sativa) genome rice microarray with log2 signal intensity based on dChip normalization
Probe set IDLocus1Annotation (MSU7, RAP5)Intensity log2Homology
  1. Homologies in Lilium longiflorum (Ll), Plumbago zeylanica (Pz), Zea mays (Zm) and Arabidopsis thaliana (At) are described in detail in Supporting Information Table S1.

  2. 1Five-numbered loci are those annotated at Michigan State University (MSU) ver. 7; seven-numbered loci are Rice Annotation Project (RAP) ver. 5.

  3. 2Also reported among Gou et al. (2001) rice sperm expressed sequence tags (ESTs).

Os.10737.1.S1_atLOC_Os05g18730.1Generative cell specific-1 (GCS1/HAP2)14.20At
Os.54874.1.S1_atLOC_Os09g27040.1GAMETE EXPRESSED 1 (GEX1)12.47Zm, At
OsAffx.17894.1.S1_atLOC_Os09g25650.1GAMETE EXPRESSED 2 (GEX2)14.02Zm, At
Os.41333.1.A1_atLOC_Os01g42060.1Expressed protein (similar to GAMETE EXPRESSED 3 (GEX3))13.24Zm, At
Os.53049.1.S1_atLOC_Os04g46490.1Aquaporin TIP5.114.61Ll, Pz, Zm, At
Os.26448.1.A1_atLOC_Os03g08070.1Copper-transporting ATPase PAA110.59Pz, Zm
Os.21018.1.S1_atOs09g0525700Generative cell specific-1; HAP2-GCS1 domain14.00 
OsAffx.26224.1.S1_s_atLOC_Os04g29090.1FAD-binding and arabino-lactone oxidase protein11.16At
Os.18560.1.S1_atLOC_Os05g01500.1Tubulin-specific chaperone E10.47 
OsAffx.3617.1.S1_atLOC_Os03g55890.1Ternary complex factor MIP110.34 
OsAffx.2553.1.S1_atLOC_Os02g09580.1OsFBX39 - F-box domain containing protein10.26Ll, Pz, Zm, At
Os.51974.1.S1_atLOC_Os06g20860.1Paramyosin10.99At
Os.50267.1.S1_atLOC_Os08g34640.1Receptor-like protein kinase precursor13.12Ll, Pz, Zm
Os.55267.1.S1_atLOC_Os03g44630.1Plastocyanin-like domain containing protein13.75Ll, Pz, Zm
Os.9559.1.S1_atLOC_Os03g37570.1Expressed protein12.80 
OsAffx.2680.1.S1_atLOC_Os02g19180.1ZOS2-06 - C2H2 zinc finger protein13.12 
Os.52171.1.S1_atLOC_Os06g38950.1ABC transporter, ATP-binding protein14.10 
Os.32737.1.S1_atLOC_Os11g08440.1DnaK family protein12.70Ll, Pz, Zm, At
Os.38984.1.S1_atLOC_Os01g23580.1Inorganic H+ pyrophosphatase12.65Zm
Os.23286.1.A1_atLOC_Os10g02920.1Cytochrome b56112.18Zm, At
OsAffx.26489.1.S1_atLOC_Os04g46760.1Trehalose phosphatase12.03 
Os.38283.1.S1_a_atLOC_Os05g41550.1Expressed protein11.89 
Os.53437.1.S1_atLOC_Os03g45980.1Expressed protein12.63 
Os.46544.1.A1_atLOC_Os10g25060.1Expressed protein12.95At
Os.9431.1.A1_a_atLOC_Os08g16610.1Rad21/Rec8-like protein14.01Zm, At
OsAffx.12136.1.S1_atLOC_Os02g20530.1Expressed protein13.62At
OsAffx.24724.1.S1_x_atLOC_Os02g44600.1Expressed protein12.27Pz, Zm, At
Os.4125.1.S1_atUniGene Os.4125 Os12g05728002Similar to RSSG8 (RNA recognition/ankyrin motifs)11.78 
Os.27370.1.S1_atUniGene Os.54572 Os01g03539002Conserved hypothetical protein12.51 
Os.54137.1.S1_atLOC_Os02g08080.1Expressed protein12.84Zm
OsAffx.31616.1.S1_atLOC_Os12g06480.1PHD-finger family protein13.35At
Os.52287.1.S1_atLOC_Os02g02800.1AGAP001222-PA protein(Anopheles gambiae-like methyl transferase)13.02 
Os.54774.1.S1_atOs07g0438300Conserved hypothetical protein10.68 
Os.52770.1.S1_atOs03g0193300Similar to nitrate transporter12.70 
OsAffx.14496.1.S1_atLOC_Os05g02030.1OB-fold nucleic acid binding domain protein10.18 
Os.18262.1.S1_atOs11g/intergenic-11.81 
Os.50552.1.S1_atLOC_Os08g35700.1Leucine-rich repeat family protein12.94Pz, Zm, At
OsAffx.28262.1.S1_atLOC_Os07g04520.1Protein kinase12.00 
Os.56612.1.A1_x_atLOC_Os05g11980.1Timeless protein10.39At
Os.34965.2.S1_s_atLOC_Os06g07130.1SHR5-receptor-like kinase11.25Zm, At
Os.54810.1.A1_atOs08g03680002Coatomer delta subunit (Delta-coat protein)12.54Ll
Os.52821.1.S1_atLOC_Os11g37200.1Transmembrane BAX inhibitor motif-containing protein13.62Ll, Zm, At
Os.54486.1.S1_atLOC_Os05g03320.1Expressed protein12.82 
Os.10491.1.S1_atLOC_Os03g04690.1Expressed protein9.84 
Os.23535.1.A1_atLOC_Os08g05820.1Monocopper oxidase14.07 
Figure 3.

Expression profiles of 45 highly up-regulated sperm genes in 31 different tissues. (See Table 1 and Supporting Information Table S1 for details). Y_, young; SAM, shoot apical meristem; S_, stress-related.

Figure 4.

qPCR of Oryza sativa sperm genes corresponding to GENERATIVE CELL SPECIFIC 1 (GCS1), GAMETE EXPRESSED 2 & 3 (GEX2, GEX3), MALE GERMLINE HISTONE H3 (MGH3) and R2R3 MYB transcription factor DUO1 POLLEN1 (DUO1), and pollen gene Ory s 1; all are scaled to PROFILIN-2.

Figure 5.

RT-PCR analysis of selected transcripts from rice (Oryza sativa) tissues carried out under linear amplification conditions. The PROFILIN-2 gene (see Fig. 1b) was used as an internal control.

Divergent complements of expressed genes in sperm and other lineages

Principal components analysis (PCA) is a mathematically rigorous multivariate analysis method that reduces related variables to their component axes, allowing essentially n-dimensional data sets to be portrayed in typically three dimensions. Different rice tissues from GEO noncontrol probe sets depict the spatial relationship of the first three principal components of normalized expression data (Fig. 6). According to these results, pollen vegetative cells and sperm cells define the two most divergent cell types, setting limits on three axes. The classical vegetative sporophyte tissues, by contrast, are aggregated distantly from pollen and sperm. In rice, sperm were clearly closer to the cluster of sporophytic cell types than they were to pollen. Female gametophytic cells clustered closely together near the sporophyte cluster, suggesting a much closer transcriptional relationship than displayed by the male germline (Fig. 6). Such differences in transcription presumably set the initial condition of the gametes at double fertilization and indicate significant distinctions between the male and female germ lineages.

Figure 6.

Principal components analysis (PCA) of probe set signal intensity on the rice (Oryza sativa) GeneChip microarray reflecting gene expression patterns in 31 different tissues of rice. The relative distances of sperm and pollen profiles from those of other cell/tissue types (and each other) are indicative of particularly divergent patterns of overall gene expression in these two cell/tissue types.

Transcriptional profiles of sperm cells, pollen and seedlings suggest distinctly different patterns of gene expression

Signal intensities of sperm, pollen and seedling probe sets were compared to determine up- and down-regulated sequences that could be related to functional categories of genes (Table S2). The categories most markedly down-regulated between sperm cells and seedlings were those related to photosynthesis-associated pathways (e.g. redox-related genes, electron transport chain, nucleotide intermediates, and carbon-backbone synthetic pathways). By contrast, the most highly up-regulated sperm sequences compared with seedlings encoded proteins involved in ubiquitin pathways, DNA modification and repair, RNA transcription and regulation, modification of chromatin, protein degradation, signaling pathways, and a broad class of ‘unknown proteins’. Down-regulated pathways in sperm cells compared with pollen included cell wall metabolism, transport, synthesis, degradation, electron transport, and secondary and primary metabolites, whereas the most enriched were RNA-related control, chromatin modeling, DNA repair and ubiquitin-mediated proteolysis (Table S2). Activation of the ubiquitin pathway is inferred by the abundance of transcriptional products in each of the essential components of the ubiquitin pathway. Gametophyte development depends on the activation of proteosome Regulatory Particle 5 (RPT5) (Gallois et al., 2009), which is consistent with the importance of this pathway.

Among the most highly up-regulated sperm transcripts are those of genes that may facilitate or directly physically regulate sperm cell behavior (Tables 1, S1). One conspicuously abundant transcript encodes a Tonoplast Intrinsic Protein 5;1 (TIP5;1)-like aquaporin—which is the most abundant sperm-expressed transcript in rice (Table 1). Similar aquaporins are highly expressed in sperm of Arabidopsis (Borges et al., 2008), Zea mays (Engel et al., 2003) and Plumbago zeylanica Svn (Gou et al., 2009), suggesting a conserved and important role in sperm function. Such membrane-localized proteins as aquaporin, with the capacity to control cellular turgor, may regulate gametic membrane tensioning and contribute to sperm receptivity. Evidence of increased vacuolization of sperm cells during the final phases of pollen tube elongation in vivo has been reported in previous ultrastructure studies (Russell, 1992). Another highly transcribed gene, LOC_Os05g18730, is a presumed ortholog to GCS1 (Mori et al., 2006) and HAP2—a membrane protein of Arabidopsis sperm cells required for gamete fusion and involved in pollen tube guidance (von Besser et al., 2006). The HAP2 protein is also required for fusion in Chlamydomonas, indicating its highly conserved nature (Liu et al., 2008). Signal intensity and fold changes in all sperm-responsive Affymetrix probe sets are shown in Table S3.

Sperm transcription factors

Seventy sperm-enhanced transcription factors (TFs) were found among detected probe sets in rice, including a number having no previous EST support, as may be expected given limited EST sampling of sperm cells in the past (Gou et al., 1999). These sperm-enhanced TFs include Nin (Nodule Induction)-like and WRKY (WRKY motif zinc-finger-like) TFs (three of each), AP2/EREBP (APETALA2/ethylene-responsive element binding proteins), C2H2 (2 cysteine-2 histidine zinc-finger), CPP (cysteine-rich polycomb protein-like), MYB (myeloblastoma-like), PcG (Polycomb group), PHD (Plant HomeoDomain zinc finger) TFs (two each), and one each of bHLH (Helix-Loop-Helix), C3H (3 cysteine-1 histidine), ARR-B (type-B phospho-accepting response regulator), Homeobox, and NAC TFs. The transcription factor DUO1 (R2R3 MYB transcription factor DUO1 POLLEN1), which is not represented on the Affymetrix microarray, plays a key role in activating male germline genes of Arabidopsis (Borg et al., 2011); in the current study, DUO1 is transcribed selectively in sperm cells (Fig. 4). An examination of upstream promoter regions of some of the most highly expressed sperm genes bear MYB binding motifs and are also enriched in other TF-binding domains consistent with TFs reported here as sperm selective (Sharma et al., 2011). Some other highly sperm-enriched TFs are also implicated in modulating chromatin structure and controlling transcriptional repression of gene expression (Takeuchi et al., 2006), notably including 14 of 20 Jumonji (Jmj) genes in this study, of which six are highly transcribed in sperm cells.

Interestingly, one TF transcribed in rice sperm cells appears to encode an AP2/EREBP TF homolog of BABY BOOM (LOC_Os09g25600)—which in Arabidopsis is associated with somatic embryogenesis (Boutilier et al., 2002) and enhancement of plant regeneration (Srinivasan et al., 2007). Although BABY BOOM has not been found in unfertilized egg cells of Arabidopsis (Curtis & Grossniklaus, 2008), it is highly transcribed in their sperm cells (Borges et al., 2008).

Chromatin state and histone transcription in sperm cells

Modification of the chromatin state is an important requirement in the establishment of germ cells, in plants as well as in animals, and it is clear that the chromatin state of the gametes can precondition later patterns of imprinting and may strongly contribute to early expression (Luo et al., 2011). Proteins contributing to chromatin state and chromatin-based gene activity include histones, DNA- and RNA-binding proteins, and enzymes that alter DNA and associated proteins through methylation and demethylation, acetylation and deacetylation, and control of their turnover, through synthesis and degradation (ubiquitination). These complex and interlinked pathways contribute to controlling expression on a local or regional genomic level and are highly conserved among eukaryotes (http://www.chromdb.org/).

Histone composition is a foundational element of chromatin state, and rice sperm cells display a unique combination of up-regulated transcripts in each major histone category (H1, H2A, H2B, H3 and H4), as is indicated among the 28 tissue types shown in Fig. 7. Among histone types, particular diversity is evident in histones H2B and especially H3. In Arabidopsis, a number of substitution H3 genes have been reported in sperm cells (Okada et al., 2005) that are transcribed independently of replication and correspondingly lack an OCT promoter motif. Arabidopsis male-germline histone H3 (AtMGH3) is a sperm-specific histone H3 variant that has three rice histone H3 genes as close homologs in Oryza sativa, HRT704, HRT11, and HRT12 (Chrom DB database, http://www.chromdb.org/), all of which are abundantly transcribed in the germline, as are the most highly transcribed H3 variants, HRT707 and HRT709 (Fig. 7). Each of these male germline-transcribed histones appears to represent substitution histones, as they lack the OCT motif of histones transcribed during replication. Each of the histone subgroups has one or multiple sperm-enhanced representatives (Fig. 7) and each may be a target of further modification through chromatin modeling. Specialized histone proteins by themselves or in combination with other proteins may directly regulate transcription through changes in binding of chromatin to key gene regulatory elements.

Figure 7.

Expression profiles of probe sets corresponding to histone genes represented on the rice (Oryza sativa) genome microarray chip and listed according to their ChromDB gene identifiers. Items marked with ‘*’ have probe set sequences that overlap with more than one gene. Distinctive patterns of up- and down-regulation are particularly conspicuous in sperm and pollen. Y_, young; SAM, shoot apical meristem; S_, stress-related.

Transcriptional activation of genes relating to chromatin state in sperm cells

Chromatin-modifying proteins known to modify DNA and DNA-associated proteins may in turn alter the activation, deactivation and longevity of DNA-associated proteins. Fig. 8 displays expression profiles for eight chromatin state modifiers. DNA methyltransferases (DMTs) mediate the addition of methyl groups at the DNA level (Pavlopoulou & Kossida, 2007), of which a unique group appears selectively up-regulated in sperm cells. Methylation, reversible by DNA glycosylases (DNGs), facilitates epigenetic reprogramming and DNA repair (Gehring et al., 2009); a number of such genes are active in sperm cells and more types of DNG are active in pollen. Histone acetyltransferases (HATs) conjugate acetyl groups to histone proteins where these subgroups have the effect of loosening binding with DNA, thus facilitating transcription. By contrast, histone deacetylases (HDAs) tend to promote stronger bonding with DNA, thus inhibiting transcriptional activity. Sperm and pollen show distinct complements, as sperm cells reflect activation through enriched HAT transcript abundance, whereas the pollen reflects deactivation through enriched HDA transcript abundance.

Figure 8.

Expression of probe sets corresponding to proteins involved in DNA and chromatin methylation, histone acetylation and histone ubiquitination in rice (Oryza sativa). DMT, DNA methyltransferase; DNG, DNA glycosylases; HAT, histone acetyltransferases; HDA, histone deacetylases; SDG, SET domain group proteins; HDM, histone demethylases; HUP, histone ubiquitination proteins; PRM, protein arginine methyltransferases. Y_, young; SAM, shoot apical meristem; S_, stress-related.

Another group of histone-modifying proteins with complicated modes of controlling transcription are the SET domain group (SDG) proteins, which represent particularly conserved proteins that modify histone proteins through methylation of specific single or multiple lysine locations, with the consequence of activating or deactivating transcription. Expression profiles of SDG proteins involve H3K9 histone methylation, DNA-level CpG and CNG cytosine methylation, and other related chromatin modeling themes, indicating that SDG proteins may secondarily mediate a broad spectrum of protein- and DNA-level methylation (Ding et al., 2007). Sperm show up-regulation of two histone demethylases (HMDs), which reverse the actions of SDG proteins, thus allowing a resetting of their chromatin state (Shinkai, 2007). Sperm also display up-regulated sequences encoding histone ubiquitination proteins (HUPs), which are believed to mediate DNA repair and accelerate histone turnover. Interestingly, protein arginine methyltransferases (PRMs), which are implicated in signal transduction, nuclear transport and transcription regulation controlling floral timing (Schmitz et al., 2008), are down-regulated as a class in sperm cells.

RNAi pathway members in sperm cells

The expression of small RNAs acting through RNA-interference (RNAi) pathways is reflected in sperm cells through activation of an evolutionarily conserved family of proteins that include argonautes (AGO), dicer-like (DCL) and RNA-dependent RNA polymerases (RDRs).

Although early studies suggested transcriptional absence of RNAi pathways in mature pollen of Arabidopsis (Pina et al., 2005), RNAi pathway genes appear to be present in sperm cells, including AGO5, AGO6, AGO9, and DCL1 (Borges et al., 2008), and have been more extensively characterized in pollen (Grant-Downton et al., 2009). In rice sperm cells, transcripts encoding the PAZ (Piwi, Argonaut and Zwille) dsRNA recognition motif, characteristic of RNAi pathway genes, appear to be highly up-regulated compared with other tissues (Fig. S2). The seven argonautes represent each of the four major gene clades: AGO1, which functions in miRNA processing; AGO4, which cooperates with DCL3 in chromatin silencing; MEL1, which functions in maintaining germ cell identity; and ZIPPY, which is implicated in heterochronic development (Nonomura et al., 2007; Kapoor et al., 2008). Most highly up-regulated in sperm cells are nontraditional AGO genes that encode a modified rather than the canonical AGO1 catalytic site at the PIWI domain (Table 2). DCL1 is implicated in biogenesis of miRNA, producing small RNAs from endogenous inverted repeats. DCL3, in turn, produces the 24nt siRNAs that mediate de novo DNA methylation, gene silencing and chromatin modification (Henderson et al., 2006). Finding DCL3 transcripts absent from Arabidopsis sperm cells, Borges et al. (2008) suggested that novel small RNA pathways may be activated instead. In rice, however, DCL3 appears to be present in the sperm and egg, and thus siRNAs could be more conventionally involved in silencing transposable elements, a role that may involve pollen-produced elements being transported to sperm targets (Slotkin et al., 2009). Sperm cells transcribe RDR3, which is one of four genes in rice that regulate and potentially amplify components of the RNAi machinery (Sijen et al., 2001); these appear to be differentially enriched in the rice germline in general, as transcripts are present in both sperm and egg cells.

Table 2.   Differentially highly transcribed RNAi pathway genes in rice (Oryza sativa) sperm cells
Gene IDProbe IDLocusIntensity (log2)Fold over seedling
  1. Microarray intensity was normalized using dChip. Motifs below represent divergences from the normal PIWI catalytic domain of D760, D845, and H986/H798 of AGO1. Gene IDs and motifs are according to Kapoor et al. (2008).

  2. 1HDR/C motif.

  3. 2DDH/P motif.

  4. 3-D-/H motif.

  5. 4DDD/H motif.

Argonaute-related genes
 OsAGO1 group
  OsAGO1bOs.8293.1.S1_atLOC_Os09g2706014.032.25
  OsAGO171Os.54612.1.A1_atLOC_Os02g0731011.595.21
 OsAGO4 group
  OsAGO162Os.19635.1.S1_atLOC_Os07g1622411.813.20
 MEL1 group
  OsAGO12Os.50897.1.S1_atLOC_Os03g5756012.713.66
  OsAGO133OsAffx.25614.1.S1_atLOC_Os03g478208.992.79
  OsAGO142Os.53826.1.S1_atLOC_Os07g0902011.225.78
 ZIPPY group
  OsAGO24Os.50333.1.S1_atLOC_Os04g5254013.833.14
  OsAGO34Os.55120.1.S1_atLOC_Os04g525508.173.05
Dicer-like-related genes
 DCL1 group
  DCL1aOs.13488.1.S1_atLOC_Os03g0297012.062.71
 DCL3 group
  DCL3bOsAffx.8007.1.S1_atLOC_Os10g344306.573.63
RNA-dependent RNase-related genes
 RDR3 group
  RDR3Os.32212.1.A1_atLOC_Os01g101307.963.48
  RDR3Os.32212.2.S1_x_atLOC_Os01g101306.652.68

Differentially represented GO categories in sperm cell transcripts

The GeneBin analyses of O. sativa and Arabidopsis produced largely similar transcriptional profiles, with few exceptions with regard to percentage representation in categories (Fig. S3) but striking differences in the number of represented transcripts (Fig. S4). DAVID analysis found no categorical differences in the uniquely reported probe sets in sperm cells, as few have been functionally annotated. By contrast, significantly more probe sets were depleted in sperm (448) and in pollen (884) compared with sporophyte tissues. GO categories apparently depleted in sperm cells included metabolic cofactors, mitochondrial membrane proteins, redox-related pathways, auxin response pathways and protein binding motifs, whereas those depleted in the pollen vegetative cell included ribosome synthesis, hormone response, signal transduction-related histidine kinase pathways, tRNA acetylation processing, and helicase-related motifs (Table S4). In both cell types, these depletion motifs may reflect the short lifespan of the pollen tube and sperm cells such that metabolic needs can be met by surrounding cells.

Discussion

Compared with pollen, sperm cells contain numerous transcripts, consistent with their role as stem-cell-like founder cells. That such a distinct set of transcripts are present in sperm cells compared with pollen and other cells suggests that the regulatory role of the cells of the male germline is substantially autonomous from that of other tissues—this is all the more unexpected because the sperm cells are small, their volume diminishing progressively during maturation (Russell & Strout, 2005), and they are contained in a unique cell-within-a-cell relationship within the pollen. Sperm cells contain a large complement of transcripts that are apparently also expressed in other somatic tissues, presumably as consistently expressed transcripts with core metabolic functions. Although some transcripts in mature sperm cells are likely to persist from earlier developmental stages, others such as DUO1, GCS1, GEX2, GEX3, and MGH3, for example, have specific and potentially crucial roles in the biology of the male gamete.

The degree of transcriptomic complexity in sperm cells appears to exceed these diminutive cells’ own metabolic requirements. In fact, some sperm transcripts appear not to be translated into protein in sperm cells, but may display delayed expression (Bayer et al., 2009), although others are clearly transcribed and translated inside sperm cells (Ge et al., 2011). Some sperm proteins could also persist from previous stages, but these may be very restricted given the highly up-regulated ubiquitin pathways that are a common theme in male germline studies (Singh et al., 2008). Thus, both synthesis and degradation appear to have crucial roles in establishing the distinct transcriptional and expression profile of sperm cells, which reflects their own unique developmental niche.

Common and divergent themes in male germ lineage expression patterns

Among the most critically important conserved male germline genes are those that encode membrane proteins for fusion, such as GCS1, which apparently arose before the divergence of green plants (Liu et al., 2008). In the next tier are common functional themes with evident homologs that meet the needs of regulating sperm expression, as for instance DUO1 (Borg et al., 2011). By contrast, some expressed proteins appear to show relatively low conservation. For example, there are some highly transcribed sequences encoding proteins that have no obvious counterparts in other male germ lineages studied to date (Table S1), which suggests specialized functions and evolutionary divergence in the male germline potentially dating back to the divergence of monocots and dicots, some > 120 million years ago (Frohlich & Chase, 2007). Understanding both gene conservation and innovation in the context of germline evolution will require expanded genomic studies (Paterson et al., 2010). Rather than sperm transcripts merely serving their own specific metabolic needs, which are probably met largely by the pollen, this complex transcriptome appears to control its own unique expression pattern through extensive chromatin modeling both before and after fertilization (Grant-Downton & Dickinson, 2006; Ingouff et al., 2007, 2010).

A significant proportion of the transcription of sperm cells appears to be related to programming the male germline determinants—a developmental event that begins with the asymmetric division of the microspore into pollen and generative cells (Eady et al., 1995) and extends to sperm maturity (Twell, 2011). Sperm-expressed substitution histone H3 proteins and altered methylation state in the sperm nucleus represent examples of epigenetic chromatin modification (Okada et al., 2005). The current study indicates that up-regulation of chromatin-modifying transcripts in the male germline reflects activation of multiple genes across nearly all classes of chromatin-modifying genes. Heterochromatin formation in the male germ lineage, a commonly known and historically described phenomenon (Maheshwari, 1950), appears to represent a most conspicuous self-regulating aspect of male germline epigenesis that has a significant molecular impact.

Cytoplasmic determinants unique to sperm cells may include abundant noncoding, small RNAs such as those involved in gene silencing (Slotkin et al., 2009). Rice sperm cells are particularly enriched in transcripts encoding RNA-related processing proteins, and with highly up-regulated ubiquitin/proteosome pathways, are anticipated to result in dynamic shifts in proteome composition during maturation and the onset of receptivity. Numerous parallels are present between plants and animals in the establishment of their germlines (Dickinson & Grant-Downton, 2009).

In animals, germlines are established early in life and maintained by unique noncoding RNAs that are involved in germ identity through epigenetic marking and which play a crucial role in RNA silencing that prevents the expression of transpositional elements. In Drosophila, for example, piRNA and the protein Piwi are germline-specific subsets that are essential for spermatogenesis, and in mouse, similar orthologs, Miwi, Mili and their corresponding noncoding RNA, are also essential in the male germline (O’Donnell & Boeke, 2007). Regulation and suppression of transposable elements occur via up-regulation of an RNA-silencing mechanism as a frontline strategy for defending the germline genome from the uncontrolled insertion of transposable elements in plants, as well as animals. Pollen and sperm cells are known to contain 21nt miRNA sequences that are believed to be directed to the male germline (Slotkin et al., 2009); small RNAs associated with AGO9 are specific for the female germline (Olmedo-Monfil et al., 2010). Chromatin condensation driving the formation of heterochromatin in animals silences much of the male genome before fertilization and is augmented by polyamine binding which also inhibits transcription (Baulcombe, 2007). Although polyamines have not been found in nonmotile plant sperm cells, chromatin-silencing gene pathways are highly represented in rice sperm transcripts and could have a similar role in plants. Prevention of male germline transmission of viruses is critically required and is reflected in the diversity of highly up-regulated RNA silencing in animals (Ding & Voinnet, 2007). In rice, the up-regulation of a range of pathways for RNA silencing is noteworthy. Such defenses may aid in reducing the relatively rare occurrence of pollen-transmitted viral diseases (Mink, 2003).

Post-fertilization impact of paternally transcribed messages in development

An unexpectedly large complement of genes are enriched in sperm cells, compared with seedlings and the pollen vegetative cell; however, their role may not be evident before fertilization, nor are they necessarily translated, but they may influence post-fertilization development through transmission during plasmogamy. Recently, Bayer et al. (2009) demonstrated that, in Arabidopsis, SHORT SUSPENSOR (SSP) is an activator gene that is transcribed in the male germ lineage, transmitted into the egg cell and translated in the zygote, which initiates asymmetric division in the zygote. In P. zeylanica, which bears dimorphic sperm cells, similar transmission may occur; the dimorphic sperm cells of this plant are targeted to fuse specifically with either the egg or the central cell and display transcriptional profiles that appear to reflect the respective female cell with which the gamete will fuse (Gou et al., 2009). For example, the sperm cell type that normally fuses with the central cell contains numerous copies of isopentenyl transferase, a control enzyme for cytokinin synthase, which drives endosperm development, whereas the sperm cell that fuses with the egg has an embryo-like profile (Russell et al., 2010). Paternal transcripts have also been observed in tobacco zygotes using RT-PCR (Ning et al., 2006) and are selectively persistent after fertilization (Xin et al., 2011). Perhaps, as in animal systems, a complex repertoire of mRNAs may be delivered during fertilization (Ostermeier et al., 2004; Krawetz, 2005). In plants, a similar failure to successfully perform in vitro fertilization using extracted sperm nuclei in maize (Zea mays) (Matthys-Rochon et al., 1994) also suggests an essential role of the male cytoplasm in early post-fertilization development of plants.

Further insights into activated genes and pathways regulating flowering and male germline differentiation will advance not only our fundamental understanding of these reproductive cells but also cell–cell recognition, membrane fusion and fertilization and may aid in regulating these processes, which may be exploited in altering events from the earliest stages in seed development.

Acknowledgements

We thank Dr Yulin Jia (Dale Bumpers National Rice Research Center, Stuttgart, AR, USA) for providing field material and advice; Prof. Karen Moldenhauer (University of Arkansas Extension Station, Stuttgart, AR, USA) for seeds; and Cal Lemke (University of Oklahoma) for excellent technical assistance in growing greenhouse plants. We also thank Prof. Terry Speed and the software development staff of the Walter and Elisa Hall Institute of Medical Research (Melbourne, Australia) for advice on statistical data analysis, and Drs Peter Ades, Farzad Haerizadeh and Harald Ottenhof (University of Melbourne) for additional help and encouragement. This work was supported by the Australian Research Council discovery grant DP 1097262 and US National Science Foundation award # IOS-1128145.

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